1 Research Article

2

3 A Population of Innate Myelolymphoblastoid Effector Cell

4 Expanded by Inactivation of mTOR Complex 1 in Mice

5 Fei Tang1, Peng Zhang1,2, Peiying Ye1, Christopher A. Lazarski1, Qi Wu4, Ingrid

6 L. Bergin5, Timothy P. Bender6, Michael N. Hall7, Ya Cui2, Liguo Zhang3, Taijiao

7 Jiang2, Yang Liu1,* & Pan Zheng1,*

8 9 1Center for Cancer and Immunology Research, Children's Research Institute, 10 Children’s National Medical Center, Washington, DC 20010, USA 11 12 2Key Laboratory of Protein and Peptide Pharmaceuticals, 3Key Laboratory of 13 Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 14 Beijing 100101, China 15 16 4Department of Neurology, 5ULAM In-Vivo Animal Core, University of Michigan 17 Medical School, Ann Arbor, MI 48109, USA 18 19 6Department of Microbiology, Immunology and Cancer Biology, University of 20 Virginia, Charlottesville, VA 22908, USA 21 22 7Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, 23 Switzerland 24 25 Correspondence should be addressed to Pan Zheng ([email protected]) or 26 Yang Liu ([email protected] );

1 27

28 Abstract 29

30 Adaptive autoimmunity is restrained by controlling population sizes and

31 pathogenicity of harmful clones, while innate destruction is controlled at

32 effector phase. We report here that deletion of Rptor in mouse

33 hematopoietic stem/progenitor cells causes self-destructive innate

34 immunity by massively increasing the population of previously

35 uncharacterized innate myelolymphoblastoid effector cells (IMLECs).

36 Mouse IMLECs are CD3-B220-NK1.1-Ter119- CD11clow/-CD115-F4/80low/-Gr-1-

37 CD11b+, but surprisingly express high levels of PD-L1. Although they

38 morphologically resemble lymphocytes and actively produce transcripts

39 from Immunoglobulin loci, IMLECs have non-rearranged Ig loci, are

40 phenotypically distinguishable from all known lymphocytes, and have a

41 gene signature that bridges lymphoid and myeloid leukocytes. Rptor

42 deletion unleashes differentiation of IMLECs from common myeloid

43 progenitor cells by reducing expression of Myb. Importantly, IMLECs

44 broadly overexpress pattern-recognition receptors and their expansion

45 causes systemic inflammation in response to Toll-like receptor ligands in

46 mice. Our data unveil a novel leukocyte population and an unrecognized

47 role of Raptor/mTORC1 in innate immune tolerance.

48

2 49 Introduction 50

51 Autoreactive T and B lymphocytes are controlled by regulation of population

52 sizes and pathogenicity through clonal deletion 1, clonal anergy 2 and regulatory

53 T cells 3. Through broadly-reactive pattern recognition receptors (PRRs) 4-5,

54 innate immunity protects host against infections by both direct effector function

55 and, indirectly, by induction of adaptive immunity 6-9. Since innate immune

56 responses triggered by host components can also cause fatal tissue damage10-11,

57 it must be properly regulated to protect hosts against self-destruction. Although a

58 number of mechanisms have been proposed to prevent self-destructive innate

59 effector functions 12-16, it is less clear if population sizes of innate effectors are

60 suppressed to limit self-destruction.

61

62 The significance of protective self-tolerance mechanisms in adaptive immunity

63 are revealed only when they have gone awry. For example, the significance of

64 clonal deletion was elucidated when it was prevented by blocking either

65 costimulation or antigen-expression in the thymus 17-18, while mice with Foxp3

66 mutation informed us of the consequence of defective regulatory T cells 19-20.

67 Since removal of the forbidden autoreactive T and B cells are achieved during T

68 and B cell development 21-23, it is intriguing whether certain parallel mechanisms

69 in innate immunity might also remain to be uncovered through genetic

70 inactivation of key regulators in development of innate effector cells.

71

3 72 The hematopoietic system is among the best characterized of all tissue/systems

73 in mammalians, with cell types and lineages clearly defined in the context of

74 developmental stages and localization 24-25. Hematopoiesis in bone marrow (BM)

75 is responsible for generation of major lineages of innate effectors, including NK

76 cells, granulocytes, monocytes and dendritic cells. While genetic switch in

77 generation of innate immune system has been identified 25, we are not aware of

78 defects that predispose host to innate immune attack through increasing

79 population sizes of self-destructive innate effectors.

80

81 The mammalian target of rapamycin (mTOR) pathway, which couples energy

82 and nutrient abundance to the execution of cell growth and division, has emerged

83 as a major regulator of hematopoiesis. Thus, activation of mTOR complex 1

84 (mTORC1) by deleting Tsc1, which encodes a negative regulator for mTORC1 26,

85 causes loss of hematopoietic stem cell (HSC) function and renders mice prone to

86 leukemiogenesis in conjunction with loss of tumor suppressor Pten27-29. More

87 recently, two groups reported that deletion of Rptor, which encodes a critical

88 component of mTORC1 30-31, dramatically perturbed hematopoiesis in mice 32-33,

89 as evidenced by defects in production of mature lymphoid and myeloid cells.

90 Remarkably, cells with CD11b+ Gr-1- surface markers massively accumulated in

91 BM following Rptor deletion in HSCs 32-33. The nature of this population and

92 consequences of their accumulation, however, remains a mystery.

93

4 94 Here we systematically analyzed the gene expression signature, cell surface

95 markers, morphology and functions of the CD11b+Gr-1- population in the Rptor-

96 deficient BM and other organs and sought for their physiological counterpart in

97 the normal mice. We found that these cells can be identified in both normal and

98 Rptor-deficient hosts by CD3-B220-NK1.1-Ter119- CD11clow/-CD115-F4/80low/-Gr-

99 1- CD11b+PD-L1+ markers, lymphoid morphology and actively transcribed Ig loci.

100 Interestingly, these cells broadly express essentially all TLRs along with many

101 other pattern recognition receptors and mounted a greatly exacerbated response

102 to all TLR ligands tested. We name this population IMLEC for innate

103 myelolymphoblastoid effector cell that can be derived from common myeloid

104 progenitors. Because their expansion and broad distribution render the host

105 vulnerable to TLR ligands, we suggest that mTORC1-mediated repression of

106 IMLEC expansion represents a new mechanism of immune tolerance in the

107 innate immunity. Our study also raises an intriguing perspective that while

108 repressing mTOR over-activation suppresses leukemia, a functional mTORC1

109 must be maintained to limit generation of IMLECs to avoid innate immune

110 destruction.

111

112 113 Results 114 115 Raptor suppresses accumulation of a previously uncharacterized subset of 116 leukocytes with features of both myeloid and lymphoid cells 117

5 118 As germline deletion of Rptor (which encode the Raptor protein) is embryonic-

119 lethal, we crossed mice harboring homozygous loxp-flanked Rptor exon 6 34 to

120 those with interferon-inducible Mx1-Cre recombinase transgene, which allows

121 inducible deletion of target genes effectively in the hematopoietic system upon

122 treatment of interferon or its inducers 35. We treated the 6-8 weeks old Rptor F/F

123 and Rptor F/F,Mx1-Cre mice with polyinosinic: polycytidylic acid (pIpC) every

124 other day for 2 weeks to induce the deletion of Rptor. Hereafter, we refer to the

125 pIpC-treated Rptor F/F mice as Ctrl (control) mice, while the Rptor F/F, Mx1-Cre

126 mice as cKO (conditional knockout) mice (Figure 1A and Figure 1—figure

127 supplement 1). As has been reported by others 32-33, Rptor deletion causes

128 broad defects in all lineages of hematopoietic cells (see also Figure 1—figure

129 supplement 1, 2, 3). However, the number of hematopoietic stem/progenitor

130 cells (HSPCs) increased (Figure 1—figure supplement 4). Most notably,

131 CD11b+ Gr-1- cells, which amount to nearly 50% of BM cells in our model,

132 emerge at the expense of CD11b+ Gr-1+ granulocytes from the cKO mice (Figure

133 1B, C). Importantly, we also observed the massive accumulation of CD11b+Gr-1-

134 cells in the BM of RptorF/F, Cre-ER mice after tamoxifen induced targeted

135 mutation of Rptor, which clearly excludes the role of pIpC in the generation of

136 these cells (Figure 1—figure supplement 5).

137

138 The CD11b+ Gr-1- cells were smaller and had reduced granularity when

139 compared to CD11b+ Gr-1+ granulocytes, but were larger and more granular

140 than the CD11b- Gr-1- cells (Figure 1—figure supplement 2E, F). Surprisingly,

6 141 despite the expression of myeloid marker CD11b on the expanded population of

142 BM cells, histological analysis of BM section revealed pervasive expansion of

143 lymphoblastoid cells (Figure 1D). The cKO BM contained markedly decreased

144 erythroid and myeloid lineage cells and markedly increased lymphocytes.

145 Lymphocytes were predominantly small-to-medium sized and had normal

146 cytological features. There was also an increased population of large blast-like

147 cells with prominent nucleoli and perinuclear clearing resembling lymphoblasts.

148 Plasma cells were present in small numbers. The myeloid: erythroid ratio was

149 within normal range (3.04) but the overall number of erythroid and myeloid cells

150 was very low. In particular, very few erythroid cells were present. In the myeloid

151 lineage there was also maturation disruption since immature ring form

152 neutrophils (neutrophilic metamyelocytes) predominate over mature neutrophils

153 (condensed chromatin) (Figure 1D). Giemsa staining of BM smear revealed a

154 massively increased lymphoblast population and severe depletion of both

155 immature erythroid cells and granulocytes in the cKO mice (Figure 1E). These

156 cells were replaced by cells with prominent nucleoli and perinuclear clearing

157 resembling lymphoblasts. To confirm that the lymphoblasts were the CD11b+ Gr-

158 1- cells identified by flow cytometry in the cKO BM (Figure 1B, C), we FACS-

159 sorted the subset based on CD11b+ Gr-1- surface markers and validated its

160 lymphoid morphology (Figure 1F).

161

162 The spleen was greatly enlarged due to expansion of the follicular centers and

163 periarteriolar sheaths within the splenic white pulp (lymphoid areas) of the cKO

7 164 mice (Figure 1—figure supplement 1C). The expanding white pulp populations

165 consisted of lightly stained, large cells that morphologically resembled germinal

166 center lymphocytes. In some areas, these populations expanded within the

167 marginal zones while in others, they involved the periarteriolar sheaths. The cells

168 had an increased amount of pale eosinophilic cytoplasm and mild pleomorphism

169 with both centroblast-like cells (larger cells with large ovoid nuclei and 1-2

170 prominent nucleoli per cell) and centrocyte-like cells (smaller cells with cleaved or

171 elongated nuclei and unapparent nucleoli)..

172

173 Since cells with such combination of morphology and surface markers had not

174 been identified previously, we FACS-sorted the CD11b+ Gr-1- cells from the Rptor

175 cKO BM and carried out next-generation RNA sequencing (RNA-seq). Using

176 principal component analysis (PCA), we compared gene expression profiles of

177 these CD11b+ Gr-1- cKO BM cells with other known subsets of hematopoietic

178 cells, including B cells, T cell subsets, NK cells, myeloid cell subsets, dendritic

179 cells and erythroid cells. This analysis demonstrates that the CD11b+Gr-1- cKO

180 BM cells were distinct from all known blood cell types, although they appear to be

181 closely related to B lymphocytes and macrophages (Figure 1G). We hereafter

182 refer these cells as innate myeloidlymphoblastoid effector cells (IMLECs).

183

184 We identified subset specific genes using a threshold of 4-fold changes and an

185 adjusted FDR-adjusted p-value <0.01. As shown in Figure 2A, by comparing

186 RNA-seq data-based gene expression signature of all known leukocyte subsets,

8 187 a unique gene expression signature was identified in the CD11b+ Gr-1- cKO BM

188 IMLECs. The signature consists of 48 genes that are up-regulated by more than

189 4-fold. The 48 genes over expressed in the CD11b+ Gr-1- cKO BM IMLECs are

190 listed in Figure 2B. Among their diverse functions, these genes are involved in

191 intracellular signaling cascades (such as Arhgap31, Rab20, Gna12, Mink1 and

192 Prkch) and metabolic processes (such as Naga, Atf3, Aoah, Chst14 and Gns).

193 The uniqueness of cKO BM IMLEC is also supported by pair-wise comparisons

194 between IMLEC and peritoneal macrophage or other closely related cell types

195 that are prominent in BM (Figure 2—figure supplement 1).

196

197 A defining feature of the B cell lineage is activation of Ig gene loci, as evidenced

198 by “sterile” transcripts transcribed from the unarranged loci of Ig heavy chain (Igh)

199 and light chains (Igk and Igl) 36. RNA-seq data revealed high levels of sterile

200 transcripts within the Igh locus (Figure 2C) and Igk and Igl loci (Figure 2—figure

201 supplement 2) from both B cell lineages and cKO BM IMLECs. This is a

202 significant difference from macrophage, which had no detectable expression of

203 the sterile transcripts, as expected. Another defining feature of developing B-

204 lymphocytes is Ig gene rearrangement, a unique mechanism of genetic

205 recombination that occurs only during the early stages of B cell maturation. This

206 process is strictly dependent on recombinases genes Rag1 37-38 and Rag2 39-40.

207 As shown in Figure 2D, no expression of Rag1 was detectable by quantitative

208 PCR (qPCR), although a detectable but extremely low level of Rag2 was

209 observed. Consistent with lack of Rag1 expression, no gene rearrangement was

9 210 found in the Ig loci (Figure 2E). Taken together, our data so far demonstrate that

211 Raptor suppresses accumulation of a previously uncharacterized leukocyte

212 subset with features of both myeloid and lymphoid cells.

213

214 Characterization of IMLECs in Rptor cKO and WT mice

215 IMLECs identified in the Rptor cKO mice did not express surface markers that

216 are used to define other lymphocytes, such as B220 for B cells, CD3 for T cells

217 and NK1.1 for NK cells (Figure 3A). The high levels of CD11b indicated that

218 these cells are distinct from the recently identified innate lymphoid cells (ILCs)

219 that are CD11b negative 41. In addition, they lack surface markers for progenitor

220 cells, such as c-Kit and Sca-1 (Figure 3A). Although the cKO IMLECs retained a

221 high level of myeloid marker CD11b, they expressed a very low level of F4/80

222 macrophage marker and lacked CD115 monocyte marker (Figure 3B). To

223 identify a positive marker for IMLEC, we searched our RNA-seq database for

224 overexpression of genes that encode cell surface CD (cluster of differentiation)

225 markers. Among 317 CD markers (Supplementary file 1), the most up-

226 regulated gene in the cKO BM IMLECs over Ctrl whole BM cells was Cd274 (also

227 called B7h1 or Pdl1). As shown in Figure 3B and Figure 3C, PD-L1 was

228 expressed on the vast majority of CD11b+ Gr-1- BM cells from Rptor cKO mice.

229

230 Based on the above data and availability of robust cell surface markers, we

231 define IMLECs by their expression of CD11b and PD-L1, but lack of major

232 lineage markers for T cells (CD3), B cells (B220), natural killer cells (NK1.1),

10 233 erythroid cells (Ter119), granulocytes (Gr-1), macrophages and monocytes

234 (F4/80 and CD115). These markers allowed us to search wild-type BM for

235 IMLEC. Interestingly, a clear although small fraction of the Lin- (CD3-B220-

236 NK1.1- Ter119- Gr-1- F4/80- CD115-) CD11b+ BM cells in the Ctrl mice also

237 expressed PD-L1 (Figure 3C, left panel), although the overall PD-L1 expression

238 level was not as high as that from cKO IMLECs. Following Rptor deletion, a

239 robust expansion (approximately 500-fold) of Lin- CD11b+ PD-L1+ BM IMLECs

240 was observed (Figure 3C, right panel).

241

242 It should be noted that although cKO IMLECs also over-express Cd11c gene

243 (Supplementary file 1), IMLEC gene expression profiles are distinct from

244 dendritic cell (DC) based on gene signature (Figure 1G and Figure 2—figure

245 supplement 1E, F). In cKO IMLECs, the CD11c levels were somewhat lower

246 than the PD-L1- DC (Figure 3D). In WT mice, greater than 90% of Lin-

247 CD11b+PD-L1+ IMLEC in BM, lung and peripheral blood mononuclear cells

248 expressed only low levels of CD11c, while those in spleen and mesenteric

249 lymph nodes consisted of two major subsets: CD11chigh and CD11clow/- (Figure

250 3E). IMLECs were also found among the leukocytes isolated from lung and in

251 peripheral lymphoid organ (Figure 3F, 3G, 3H), and this population was greatly

252 expanded in the cKO mice (Figure 3I).

253

254 To further confirm that this subset is the IMLEC in normal BM, we FACS-sorted

255 the Lin- (CD3- B220- NK1.1- Ter119- Gr-1- F4/80- CD115-) CD11b+ PD-L1+ cells

11 256 from wild type (WT) BM and characterized their morphology and levels of sterile

257 Ig transcripts. As shown in Figure 4A, the sorted cells had a lymphoid

258 morphology as did the cKO IMLECs. They also displayed comparable size and

259 granularity as cKO IMLECs (Figure 4B). Moreover, Lin- CD11b+ PD-L1+ cells

260 from WT BM expressed sterile transcripts of Ig loci identified by RNA-seq (Figure

261 4C). Furthermore, subsequent validation of IMLECs in WT BM was undertaken

262 by comparing the expression of other top candidate markers (CD14, CD16) and

263 MHC-I/MHC-II (Figure 4—figure supplement 1A), as well as population-specific

264 transcription factors Mitf, Atf3 and Zdhhc1 (Figure 4 D, E). The largely

265 comparable expression levels of these surface markers and transcription factors

266 between WT and cKO IMLECs provide additional lines of evidence for these cells

267 to be naturally occurring IMLECs. Therefore, a small fraction of normal

268 leukocytes in lymphoid and non-lymphoid tissues have the IMLEC phenotype,

269 and this subset is massively expanded after Rptor deletion.

270

271

272 Altered differentiation is responsible for accumulation of IMLECs

273 Theoretically, expansion of IMLECs in cKO BM may be caused by increased

274 proliferation and/or reduced apoptosis. To test this possibility, we analyzed the

275 proliferation of IMLECs by Ki-67 staining and BrdU incorporation. Remarkably,

276 Lin- CD11b+ PD-L1+ IMLECs from both Raptor Ctrl and cKO BM had much fewer

277 Ki-67+ cells (Figure 4F,G) or BrdU+ cells (Figure 4—figure supplement 1B, C)

278 when compared with other lineages. The fact that IMLECs are not proliferating at

12 279 a higher rate than other BM cell types effectively rules out rapid proliferation as

280 an explanation for IMLECs accumulation in Raptor cKO BM. Likewise, the

281 massive increase of IMLEC in cKO mice over those in the Ctrl mice cannot be

282 due to proliferation, as the percentage of Ki-67+ or BrdU+ cells is not increased in

283 cKO mice. Furthermore, based on cell surface Annexin V staining, IMLECs from

284 cKO BM were more prone to apoptosis than total lineage+ population (Figure 4H,

285 I) and had apoptosis rate that was comparable to granulocytes, B cells and T

286 cells in BM (Figure 4—figure supplement 1D, E). The pronounced apoptosis

287 also rules out the possibility that increased survival may account for preferential

288 accumulation of IMLECs in cKO mice. The robust apoptosis detected among WT

289 IMLECs likely contributed to the reduced amount of IMLECs in normal BM

290 (Figure 3C). Consistent with the reduced proliferation and increased apoptosis

291 of IMLECs, our exhaustive efforts to demonstrate self-renewal of IMLEC through

292 transplantation of massive numbers of IMLEC have all been unsuccessful (data

293 not shown).

294

295 As an alternative hypothesis, we evaluated whether IMLECs accumulated

296 because of altered differentiation of hematopoietic stem and progenitors

297 (HSPCs). As the first step to test this hypothesis, we evaluated if IMLEC

298 accumulation in cKO BM was cell-intrinsic. Briefly, we mixed either RptorF/F or

299 RptorF/F, Mx1-Cre (both CD45.2+) BM cells with recipient type CD45.1+ WT BM

300 cells at a 2:1 ratio. At six weeks after BM transplantation, Rptor was deleted

301 from the RptorF/F, Mx1-Cre donor-derived cells by pIpC treatment (Figure 5A).

13 302 As shown in Figure 5B, the accumulation of CD11b+ Gr-1- IMLECs was intrinsic

303 to Rptor -/- BM cells. Since our earlier data suggested that IMLECs accumulated

304 at the expense of granulocytes (Figure 1B, C), we tested if granulocytes were

305 converted to IMLECs following Rptor deletion. We produced RptorF/F, Lyz2-

306 Cre+/+ mice that should have myeloid lineage-specific deletion of Rptor. However,

307 despite the effective deletion of the Rptor gene in the granulocytes (Figure 5C),

308 the percentages of CD11b+ Gr-1+ granulocytes and CD11b+ Gr-1- IMLECs were

309 unchanged (Figure 5D). These data suggest that accumulation of IMLEC in the

310 cKO mice was not due to trans-differentiation from granulocytes.

311

312 Next, we use both in vitro co-culture and in vivo BM transplantation to identify the

313 progenitor that may give rise to IMLECs. We co-cultured OP9 stromal cells with

314 FACS-sorted BM LSK (Lin- Sca-1+ c-Kit+), CMP (Lin- Sca-1- c-Kit+ CD34Medium

315 CD16/32Medium) and CLP (Lin-CD127+ Sca-1Mediumc-KitMedium) populations from Ctrl

316 and cKO mice that had been treated with pIpC (Figure 5E). As shown in Figure

317 5F, both LSK and CMP populations from Rptor-/- BM gave rise to CD11b+ Gr-1-

318 PD-L1+ IMLECs. As expected, Rptor-sufficient CLPs were not able to give rise to

319 CD11b+ myeloid cells. Interestingly, Rptor-deficient CLPs generated progenies

320 with a small portion exhibiting immunophenotypes of IMLECs. We also

321 transplanted sorted LSK and CMP populations and induced Rptor deletion in the

322 donor cells by treating recipients with pIpC (Figure 5G) to confirm their ability in

323 giving rise to IMLECs. Due to lack of self-renewal activity of progenitor cells and

324 rapid apoptosis of IMLEC, we used a much shorter timeline than the whole bone

14 325 marrow transplantation studies in order to capture progenitor-derived IMLEC. As

326 shown in Figure 5H, deletion of Rptor in either LSKs or CMPs was sufficient to

327 induce the generation of CD11b+ Gr-1-PD-L1+ cells in recipients BM. The shorter

328 timeline explained relative paucity of LSK-derived IMLEC when compared with

329 long-term bone marrow transplantation (Figure 5A, B). As expected, since

330 CMPs do not have self-renewal capability, only a small number of progeny cells

331 were produced. However, since IMLEC can be generated from CLP in vitro, their

332 potential to do so under physiological conditions cannot be ruled out. Taken

333 together, our data demonstrate that the massive accumulation of IMLECs in cKO

334 mice can be caused by altered differentiation of CMPs, although other

335 differentiation pathway cannot be ruled out.

336

337 Reduced c-Myb expression is responsible for accumulation of IMLECs

338 A previous study demonstrated that heterozygous Myb mutation leads to an

339 expansion of BM CD11b+Gr-1- cells 42. Although expression of PD-L1 was not

340 evaluated in the earlier study, we were intrigued by the possibility that down-

341 regulation of Myb may be the underlying mechanism for the massive production

342 of IMLECs. Since both LSKs and CMPs are able to give rise to CD11b+ Gr-1-

343 PD-L1+ IMLECs, we evaluated expression of c-Myb in both LSK and CMP

344 populations. Indeed, the Myb transcripts were significantly reduced in both LSK

345 and CMP populations sorted from Rptor cKO mice (Figure 6A). Moreover, our

346 intracellular staining also revealed reduced levels of c-Myb protein in both LSKs

347 and CMPs from cKO BM (Figure 6B). Interestingly, induced deletion of c-Myb in

15 348 mice with homozygous floxed c-Myb (MybF/F, Mx1-Cre), but not heterozygous

349 floxed c-Myb (MybF/+, Mx1-Cre), showed obvious increase of PD-L1 expression

350 in CD11b+Gr-1- BM cells (Figure 6C, D, E and Figure 6—figure supplement 1A,

351 B, C), despite of significant decrease of BM whole leukocytes due to Myb-

352 reduction induced cell apoptosis. To avoid excessive apoptosis and test if the

353 down-regulation of Myb was necessary and sufficient to cause accumulation of

354 IMLECs, we transplanted BM cells from either MybF/F, Rag2-/- or MybF/F, Rag2-/- ,

355 CreER mice to CD45.1+ recipients, which then received tamoxifen to achieve

356 deletion of Myb specifically in donor-derived hematopoietic cells after their full

357 reconstitution (Figure 6F). Consistent with essential role for c-Myb in

358 hematopoiesis, surviving leukocytes appeared heterozygous for Myb deletion

359 (Figure 6G). As early as 1 week after the first injection of tamoxifen, a significant

360 decrease in CD11b+ Gr-1+ granulocytes and an increase in CD11b+ Gr-1- PD-L1+

361 IMLECs were observed in Myb cKO BM (Figure 6H). Moreover, the provision of

362 heterologous c-Myb significantly diminished the generation of IMLECs from

363 Raptor-deficient LSK cells in our in vitro OP9 co-culturing experiments (Figure

364 6—figure supplement 1D, E, F, G). Therefore, down-regulation of Myb is

365 necessary for production of IMLECs.

366

367 We recently reported that deletion of Rptor caused up-regulation of miRNA

368 biogenesis in HSPCs 43. We searched our miRNA microarray database and

369 mirSVR score database44 for potential impact of miRNAs in down-regulation of

370 Myb expression. Using the stringent criteria of mirSVR score <-1.0, we identified

16 371 50 miRNAs that presented in the HSPCs (Supplementary file 2). Among them,

372 13 miRNAs showed >2.0 folds up-regulation in the Rptor-deficient HSPCs

373 (P<0.05), while none showed statistically significant down-regulation (Figure 6I).

374 Significant up-regulation of miR-150 (1.4 folds increase, P=0.05), which was

375 previously demonstrated to inhibit Myb expression 45-46, was also observed. Up-

376 regulation of Myb-targeting miRNAs provides a plausible mechanism for down-

377 regulation of Myb by Rptor deletion. However, the broad spectrum of the up-

378 regulated miRNAs suggests that it is unlikely that a single miRNA is responsible

379 for the overall reduction of Myb expression.

380

381 Expansion of IMLECs associated with lethal inflammatory response to TLR

382 ligands

383 RNA-seq data indicated that IMLECs broadly up-regulate pattern recognition

384 receptors (PRRs) genes. TLRs, the first family of PRRs identified, were broadly

385 over-expressed in cKO IMLECs as determined by RNA-seq (Figure 7A). RT-

386 PCR confirmed that IMLECs from both cKO and WT mice over-expressed

387 essentially all TLRs tested, particularly Tlr2, Tlr3, Tlr4, Tlr6, Tlr7, Tlr8 and Tlr9

388 (Figure 7B). In addition to TLRs, expression of other PRRs, including NLRs,

389 ALRs and RLRs, was also broadly elevated (Figure 7—figure supplement 1).

390 When the BM from Ctrl and Rptor-/- mice were compared for their responses to

391 TLRs agonists, including synthetic tripalmitoylated lipopeptide Pam3CysSerLys4

392 (Pam3CSK4, TLR1/2 agonist), heat-killed Listeria monocytogenes (HKLM, TLR2

393 agonist), synthetic analog of double-stranded RNA poly I:C (pIpC, TLR3 agonist),

17 394 lipopolysaccharide (LPS, TLR4 agonist), flagellin from Salmonella typhimurium

395 (FLA-ST, TLR5 agonist), synthetic lipoprotein derived from Mycoplasma

396 salivarium (FSL-1, TLR2/6 agonist), single-stranded RNA Double-Right

397 complexed with LyoVec™ (ssRNA-DR, TLR7 agonist) and synthetic

398 oligonucleotides containing unmethylated CpG dinucleotides (ODN1826, TLR9

399 agonist), it is clear that Rptor-/- BM increased production of TNF-α (Figure 7C)

400 and MCP-1(Figure 7D) in response to all TLR ligands tested. To determine the

401 cellular basis for the enhanced cytokine production, we used intracellular and cell

402 surface staining to identify cells that produced the inflammatory cytokines. As

403 shown in Figure 7E, IMLECs from both WT and Rptor cKO BM were potent

404 producers of TNF-α when stimulated by LPS.

405

406 Consistent with exacerbated responses to TLR ligands in BM cells, deletion of

407 Rptor in cKO mice resulted in massive increase in inflammatory cytokines in

408 serum (Figure 8A). Approximately 40% of cKO mice died within 2 months after

409 7 pIpC treatments (Figure 8B). Histological analyses revealed extensive

410 inflammation in the liver with associated tissue injuries (Figure 8C). A substantial

411 proportion of the leukocytes in the liver of cKO mice were IMLECs, as

412 demonstrated by cell surface markers CD11b+ Gr-1- PD-L1+ F4/80low/- (Figure 8D,

413 E). To test if the mice with expanded IMLECs were more sensitive to endotoxin,

414 we challenged the Ctrl and cKO mice with low doses of LPS (5mg/kg body

415 weight). While all Ctrl mice survived the LPS challenge, all cKO mice succumbed

416 within 36 hours (Figure 8F). The dramatically increased mortality due to

18 417 endotoxic shock was associated with remarkably elevated levels inflammatory

418 cytokines. As shown in Figure 8G and Figure 8H, a more than 500-fold

419 increase in TNF- and an approximately 10-fold increase of MCP-1 were

420 detected in the serum of cKO mice at 6 hours after LPS injection. It has been

421 demonstrated that acetaminophen-triggered liver necrosis induces HMGB-1-

422 mediated inflammatory responses to danger-associated molecular patterns

423 (DAMPs)11, 47. As shown in Figure 8I and Figure 8J, cKO mice mounted a

424 significantly elevated inflammation to challenge by low doses of acetaminophen.

425 Therefore, amplification of IMLEC also leads to elevated response to tissue

426 injuries.

427

428 Since Mx1-Cre was broadly activated after pIpC treatment, it is less certain

429 whether the increased sensitivity of the cKO mice to TLR ligands is due to

430 immunological abnormality. To address this issue, we produced chimeric mice in

431 which pIpC induces deletion of the targeted gene exclusively in hematopoietic

432 cells by transplanting RptorF/F, Mx1-Cre (CD45.2+) BM cells into lethally irradiated

433 CD45.1+ recipients. After hematopoietic reconstitution, the recipients were

434 treated with 3 doses of pIpC to induce deletion of Rptor exclusively in the

435 hematopoietic cells. After 10 days of pause, the Ctrl and cKO chimera mice were

436 challenged with new pIpC injection and monitored for survival (Figure 9A). As

437 shown in Figure 9B, while a large portion of the cKO chimera mice died

438 progressively starting within a week of the second round of pIpC treatment, all

439 Ctrl chimera mice survived the observation period of more than 45 days.

19 440 Massive leukocytes infiltration was observed in the liver of cKO chimeric mice

441 (Figure 9C). Cell surface phenotyping of the donor-type leukocytes in BM

442 (Figure 9D), spleen (Figure 9E) and liver (Figure 9F) revealed accumulation of

443 CD11b+ Gr-1- PD-L1+ F4/80low/- IMLECs. Collectively, the data in Figure 8 and

444 Figure 9 demonstrate that over-expansion of IMLECs, as a result of Rptor

445 deletion, renders the host highly vulnerable to TLR ligands.

446

447

448 Discussion

449

450 We have characterized a leukocyte population, which we called IMLECs, that has

451 a strong innate effector function but with features of both myeloid and lymphoid

452 cells. Furthermore, our data reveal an unexpected function of mTORC1 in

453 suppressing IMLEC expansion. The high vulnerability to TLR ligands after

454 IMLEC expansion highlights a new consequence of defective hematopoiesis and

455 a new mechanism of immune tolerance.

456

457 Two groups have previous reported that inactivation of mTORC1 by deletion of

458 Rptor leads to massive accumulation of CD11b+Gr-1- cells in the BM 32-33.

459 Similar results were obtained in mice with Mtor deletion in the hematopoietic cells

460 48. We have demonstrated here that despite expression of a myeloid cell marker

461 CD11b, the CD11b+Gr-1- BM cells have lymphoid morphology. The active

462 production of sterile transcripts at Ig loci suggests that these cells have partially

20 463 committed to the B-cell lineage, while lack of VDJ rearrangement and cell surface

464 B cell markers suggest that the differentiation toward the B cell lineage is limited.

465 Importantly, this cell population expresses high levels of PD-L1 but does not

466 express markers for other lymphoid cells including T cells (CD3) and NK cells

467 (NK1.1) as well as for myeloid cells including F4/80, Gr-1 and CD115.

468 Expression of CD11b also distinguishes IMLECs from ILCs 41, which are CD11b-.

469 PCA analysis demonstrated that IMLECs are distinct from but close to B cells

470 and macrophages in gene expression profiles. While at much lower frequencies,

471 cells with the same phenotypes and functional properties were also identified in

472 normal BM, peripheral lymphoid and non-lymphoid organs.

473

474 It is of interest to note that the hallmark of IMLECs is the high expression of cell

475 surface PD-L1. First identified as B7-H1 49, PD-L1 has been shown to be

476 involved in tumor evasion of T cell immunity, both by inducing exhaustion of

477 effector T cells and by shielding tumor cells from effector T cells 50-51. With the

478 induction by cytokines such as IFN- and hypoxic tumor microenvironment, PD-

479 L1 has been found on both tumor cells and host inflammatory cells such as

480 myeloid derived DCs 52-53, tumor-infiltrating myeloid derived suppressor cells 54.

481 Recent studies have demonstrated that PD-L1 is an important biomarker and

482 therapeutic target in cancer immunotherapy 55-56. Since IMLECs constitutively

483 express high levels of PD-L1, it will be of interest to investigate their function in

484 cancer immunity. It’s worth noting that IMLECs of Raptor-deficient BM have an

485 overall higher expression of PD-L1 than that of Raptor-sufficient BM, perhaps this

21 486 reflects the indirect consequence of reduced mTORC1-mediated translation of

487 PD-L1 negative regulators.

488

489 Since the IMLEC population expanded at the expense of granulocytes in the BM,

490 we tested if IMLECs were derived by trans-differentiation of the granulocytes.

491 Our genetic analyses demonstrated that inactivation of mTORC1 in the

492 granulocytes, using Lyz2-Cre, failed to produce this subset, suggesting that loss

493 of mTORC1 in granulocytes does not cause their trans-differentiation into

494 IMLECs. Furthermore, since inactivation of mTORC1 in CD11b+Gr-1+

495 granulocytes did not affect their abundance in BM, loss of granulocytes in the

496 mice with broad deletion of Rptor in all hematopoietic cells was not due to a cell-

497 intrinsic requirement for mTORC1 in survival of granulocytes. Since IMLECs

498 generally do not undergo active proliferation and are prone to apoptosis, their

499 massive accumulation is most likely due to continuous production rather than

500 self-renewal. Indeed, while LSKs and CMPs differentiate into IMLECs both in

501 vitro and in vivo, IMLECs are not able to propagate in vivo (data not shown).

502 Since Lyz2-Cre mediated Rptor deletion does not result in accumulation of

503 IMLECs, it is obvious that IMLEC differentiation pathway is initiated before Lyz2-

504 Cre expression which also occurs in some CMPs, and not all CMPs can give rise

505 to IMLEC. On the other hand, since CLPs give rise to IMLEC in vitro, the

506 possibility that other progenitor cells may give rise to IMLECs has not been ruled

507 out.

508

22 509 Since heterozygous mutation of Myb causes an increase of CD11b+Gr-1low/- cells

510 in BM 42, we tested if conditional deletion of Myb is sufficient to cause

511 accumulation of IMLEC. Our data demonstrate that cell-intrinsic reduction of

512 Myb in BM resulted in accumulation of CD11b+ Gr-1- PD-L1+ IMLECs. It is of

513 interest to note that mice with inactivation of Rptor share many hematopoietic

514 phenotypes with mice harboring targeted disruption of Myb, such as an increase

515 in HSPCs 57 and defective B-lymphopoiesis58-60. Moreover, consistent with the

516 proposed roles of c-Myb in regulating precise hematopoietic commitments, CMPs

517 with deletion of either Rptor or Myb favored differentiation to CD11b+ Gr-1- cells 61.

518 Since our data show that the majority of the CD11b+Gr-1- cells generated from

519 CMPs are IMLECs, these data support the notion that IMLEC accumulation in

520 Rptor cKO BM is caused by reduced Myb expression in CMPs.

521 While the mechanism by which mTORC1 down-regulates Myb remains to be fully

522 elucidated, we have found a general up-regulation of putative miRNAs targeting

523 Myb in HSPCs after Rptor deletion. Our data suggest that mTORC1 inactivation

524 in HSPCs expands IMLECs by down-regulation of Myb, perhaps through

525 increased miRNA biogenesis.

526

527 Previous studies by us and others have demonstrated a critical role for regulated

528 mTOR signaling in hematopoiesis 27-28, 62. Thus, while mTOR activation by

529 deletion of Tsc1 complex expands the numbers of hematopoietic stem cells, it

530 causes reduced hematopoietic stem cell function but induced leukemic stem cells

531 in conjunction with Pten deletion. This is consistent with the generally accepted

23 532 association between defective differentiation and leukemiogenesis, which forms

533 the foundation for treatment of leukemia through induction of differentiation 63.

534 The expansion of IMLECs caused by mTORC1 inactivation did not lead to

535 leukemiogenesis as IMLECs are non-dividing cells that undergo a high rate of

536 apoptosis. Instead, our data demonstrate a new consequence of defective

537 differentiation, namely generation of a new population of cells with distinct

538 effector function, as discussed below.

539

540 Our RNA-seq data suggest that IMLECs broadly over-express pattern recognition

541 receptors for innate immunity. Corresponding to a broad, although not universal

542 TLR elevation, IMLECs mount drastically exacerbated responses to all TLR

543 ligands tested in vitro. Intracellular cytokine staining revealed that both normal

544 and Rptor-cKO IMLECs were among the most active producers of inflammatory

545 cytokines. Surprisingly, although Rptor-/- BM cells have normal TLR5 levels, they

546 produce 10-20 fold more inflammatory cytokines, such as TNF- and MCP-1 in

547 response to TLR5 ligand. It is therefore likely that beyond TLRs, IMLECs have

548 acquired other features that enhance their response to TLR ligands.

549

550 Apart from BM, expanded IMLECs are broadly distributed in lymphoid and non-

551 lymphoid organs such as lung and liver. Our data demonstrate that an increase

552 in IMLEC numbers makes the host highly vulnerable to TLR ligands such as LPS

553 and polyI:C, as indicated by rapid demise of mice following pIpC and low doses

554 of LPS injections. Since necrosis of normal tissues leads to release of

24 555 endogenous TLR ligands, such as HMGB147 and HSP70 64, and since

556 unregulated host response to HMGB1 leads to fatal inflammation11, it is

557 conceivable that expansion of IMLECs may also make the host more vulnerable

558 to tissue injury- induced inflammation. This is evidenced by elevated

559 inflammation in acetaminophen-triggered liver necrosis model.

560

561 The severe pathological consequences may explain the paradoxical functions of

562 mTOR in regulation of inflammation. While mTOR signaling is known to be

563 activated by inflammatory cytokines 65 and rapamycin has been shown to inhibit

564 production of inflammatory cytokines 66, perhaps through its inhibition of mTOR-

565 mediated NFB activation 67, rapamycin has been shown to induce inflammation

566 in a small number of transplantation patients 68-70. Likewise, we have observed

567 that rapamycin increases production of inflammatory cytokines in autoimmune

568 Scurfy mice and in mice treated with high doses of endotoxin 65. It would be of

569 great interest to determine if rapamycin can expand IMLECs in transplantation

570 patients and in mice that either receive high doses of endotoxin or are genetically

571 predisposed to autoimmune disease. It is also reported that persistent mTORC1

572 inhibition can result in elevated inflammation, activation of STAT3 and enhanced

573 hepatocellular carcinoma development 71. Our findings focused on characterizing

574 IMLECs and inflammation support the provocative findings of this report, and

575 might provide alternative and complementary explanations. However, despite an

576 extensive effort, we have failed to induce IMLECs by mTOR kinase inhibitor

577 Torin2 or rapamycin in WT mice (data not shown). Therefore, under normal

25 578 circumstances, pharmaceutical inhibition of mTORC1 alone cannot achieve

579 comparable levels of inflammation as those achieved by genetic inactivation of

580 either Mtor or Rptor. Additional conditions must be met for mTOR inhibitors to

581 cause inflammation. While these unknown barriers have ensured safety of

582 mTOR inhibitors in most circumstances, their breakdown may explain

583 paradoxical induction of inflammation by rapamycin.

584

585 The concept of immune tolerance has traditionally been reserved for adaptive

586 immunity to avoid autoimmune diseases. A multitude of mechanisms, including

587 clonal deletion 22-23, 72, clonal anergy 2, 73 and dominant regulatory T cells 3, have

588 been described to reduce self-reactive T and B cell clone sizes to avoid

589 autoimmune diseases. On the other hand, innate immunity is known to be

590 regulated at levels of cellular activation 74 and cellular recruitment 75. However,

591 we and others have reported that innate immune effectors, especially NK cells,

592 have features of adaptive immunity as their immune protective function against

593 cancer and viruses is amplified through increased population sizes 76-77.

594 However, a regulatory mechanism to control innate effector population size for

595 the sake of preventing self-destruction has not been described. Our discovery of

596 a developmentally regulated mechanism to control the population size of IMLECs

597 to avoid unwanted self-destruction, as described herein, reveals a parallel

598 between adaptive and innate immunity to avoid potentially life-threatening

599 inflammation and tissue damage. It is therefore of interest to consider the

600 concept of immune tolerance in the area of innate immunity.

26 601

602 An important consideration is whether IMLEC is a normal population of

603 hematopoietic cells or a population that arises after pathogenic mutations. While

604 we have identified cells of similar phenotypes and functional properties in normal

605 mice, they are extremely rare and thus have no obvious physiological functions

606 unless they are substantially expanded. We have demonstrated that these cells

607 do not undergo proliferation and are prone to apoptosis, and that their expansion

608 depends on abnormal hematopoiesis. Therefore, the pathological consequence

609 observed herein is only known to manifest itself if the mTORC1-MyB pathway is

610 genetically inactivated, resulting in disruption of normal hematopoiesis. Further

611 studies are needed to identify conditions that can lead to accumulation of IMLEC

612 short of these known mutations.

613

614 In summary, our data demonstrate that inactivation of mTORC1 in hematopoietic

615 stem/progenitor cells leads to generation of IMLECs, a new cell population that

616 shares features with myeloid and lymphoid lineages. The greater than 500-fold

617 increase in population size of IMLECs in mTORC1- or MyB-defective BM

618 highlights the critical role for mTORC1 and MyB in repressing the development

619 of sufficient number of IMLECs to cause serious inflammation and tissue damage.

620 Our study reveals a new consequence of defective hematopoiesis and may help

621 to extend the concept of immune tolerance to innate immunity.

622

623 Materials and Methods

27 624 Key Resources Table

Reagent type (species) or Designation Source or Identifiers Additional resource reference information strain, strain background (Mus RptorF/F PMID: 19046572 musculus,C57BL/6) strain, strain background (Mus MybF/F; PMID: musculus,C57BL/6) MybF/F,Rag2-/- 15195090; ,CreER PMID: 16169500 strain, strain background (Mus Mx1-Cre The Jackson Stock No: 003556; musculus,C57BL/6) Laboratory RRID:IMSR_JAX:003556 strain, strain background (Mus CreER The Jackson Stock No: 007001; musculus,C57BL/6) Laboratory RRID:IMSR_JAX:007001 strain, strain background (Mus Lyz2-Cre The Jackson Stock No: 004781; musculus,C57BL/6) Laboratory RRID:IMSR_JAX:004781 625

626

627 Mice and induction of gene deletion

628 Rptor F/F mice 78 were crossed to the C57BL/6 background for more than 10

629 generations. Myb F/F and Myb F/F, Rag2-/- , CreER mice were reported

630 previously58-59, 79. The interferon-inducible Mx1-Cre transgenic mice

631 (RRID:IMSR_JAX:003556) 35, tamoxifen-inducible Cre-ERT2 transgenic mice

632 (RRID:IMSR_JAX:007001) 80 and Lyz2-Cre knock-in mice

633 (RRID:IMSR_JAX:004781) 81 with C57BL/6 background were purchased from the

634 Jackson Laboratory. Rptor F/F mice were crossed with Mx1-Cre mice, Lyz2-Cre

635 mice or CreER mice to produce Rptor F/F, Mx1-Cre (cKO) mice, Rptor F/F, Lyz2-

636 Cre+/+ mice or RptorF/F, CreER mice, respectively. Myb F/F mice were crossed

637 with Mx1-Cre mice to generate MybF/F (Ctrl) and MybF/F, Mx1-Cre (cKO) mice.

638 Offspring of these mice were genotyped by PCR-based assays with genomic

639 DNA from mouse tail snips. Mice were cared for in the Unit of Laboratory Animal

640 Medicine (ULAM) at the University of Michigan, where these studies were

641 initiated, or Research Animal Facility (RAF) of Children’s National Medical Center,

28 642 where the studies were completed. All procedures involving experimental

643 animals were approved by the University Committee on the Use and Care of

644 Animals (UCUCA) at the University of Michigan or Children’s National Medical

645 Center.

646

647 Raptor Ctrl and cKO mice used in each experiment were sex-matched littermates.

648 Mice were given 2 mg/kg body weight of pIpC (GE Healthcare Life Sciences) or

649 400µg pIpC (Sigma-Aldrich) every other day for consecutive 3 to 7 times as

650 specified by intra-peritoneal (i.p.) injection to induce Cre expression as in

651 previous study 82. Deletion of target genes were confirmed as previously

652 described78-79. Wild type C57BL/6 (CD45.2) mice and congenic C57BL/6 (CD45.1)

653 mice were purchased from the Charles River Laboratories. Tamoxifen (Sigma-

654 Aldrich) was dissolved in corn oil (Sigma-Aldrich) to 20 mg/ml and injected i.p. at

655 150 mg/kg/day for 5 consecutive days.

656

657 Histology, cytology and complete blood cell count

658 Ctrl and cKO mice were euthanized by CO2 inhalation on day 30 of last pIpC

659 treatment. For histology, tissues were fixed in 10% neutral buffered formalin for

660 24-48 hours and the sternums were then decalcified in Immunocal (formic acid-

661 based decalcifier, Decal, Tallman, NY) for 24 hours. Tissues were trimmed and

662 cassetted and processed to wax on an automated processor using standard

663 methods. Sections were cut at 5 m thickness and hematoxylin and eosin-

664 stained slides prepared on an automated stainer. For cytology, BM was collected

29 665 from the femoral marrow cavity with a fine diameter paintbrush dipped in sterile

666 PBS with 5% fetal bovine serum and cytology smears were prepared by gently

667 brushing the collected cells in parallel lines on a glass slide. Cytology slides of

668 BM smear and cytospins of FACS-sorted BM cells were stained using a

669 Romanowsky-based stain (Diff-Quik, Hema 3 Manual staining system, Fisher

670 Scientific).

671

672 Histological and cytological parameters were evaluated using an Olympus BX45

673 light microscope at total magnifications ranging from 40 X to 100 X (oil).

674 Histological alterations were descriptively identified. Cytological alterations were

675 descriptively identified and quantitative BM differential counts were made using a

676 manual differential counter and standard criteria for cell identification. Images

677 were taken using a 12.5 megapixel microscope-mounted Olympus DP72 digital

678 camera and accompanying software (Olympus). Complete blood cell count was

679 performed using the Hemavet 950 Hematology System (Drew Scientific Inc.) by

680 the Animal Diagnostic Laboratory of ULAM Pathology Cores for Animal Research

681 in the University of Michigan.

682

683 Cells preparation and bleeding

684 BM cells were flushed out from the long bones (tibiae and femurs) by a 25-gauge

685 needle with staining buffer (1XHanks Balanced Saline Solution without calcium or

686 magnesium, supplemented with 2% heat-inactivated fetal bovine serum). Single

687 cell suspensions of spleen, thymus, lung and lymph nodes were generated by

30 688 gently squashing with frosted slides in a small volume of staining buffer. Cells

689 from mouse peritoneal cavity were harvested as described before83. For isolation

690 of mouse liver mononuclear cells, liver fragments were pressed through 70μm

691 round cell strainer (Becton Dickinson). Single-cell suspensions in a 35% Percoll

692 solution (GE Healthcare) were centrifuged for 20 minutes at 800g with brake off

693 at room temperature. Pellet was collected and washed with staining buffer.

694 Peripheral blood was collected by retro-orbital bleeding with heparinized capillary

695 tubes or by submandibular bleeding with a lancet.

696

697 Flow cytometry

698 For surface staining, cells were stained with the indicated antibodies (Abs) in

699 staining buffer for 20 minutes at 4 °C. In the characterization of surface markers

700 for CD11b+ Gr-1- cells, Fcγ receptors were pre-blocked by incubating cells with

701 culture medium from hybridoma 2.4G2 84 for 20 minutes at 4°C. For intracellular

702 staining, cells were first stained with the indicated surface markers Abs and then

703 fixed with Cytofix/Cytoperm buffer (BD Biosciences) for 1-2 hours at 4°C,

704 followed by incubation with Cytoperm Plus buffer (BD Biosciences) for 15

705 minutes at room temperature (R.T.). After refixing for 15 minutes at R.T., cells

706 were incubated with antibodies or isotype controls for 20 minutes (anti-TNF/IgG,

707 anti-Ki-67Abs) or overnight (anti-c-Myb/IgG Abs) and further stained with the

708 secondary Ab if necessary. BrdU labeling experiments were performed per the

709 manufacture’s instruction (BD Biosciences), as previously reported 29, 82.

710 Apoptosis assays by 7-AAD and Annexin V (BD Biosciences) were according to

31 711 manufacturer’s instructions. All FACS analyses were performed on a BD LSR II

712 or a Canto II Flow Cytometer, and data were analyzed with FlowJo software

713 (Tree Star, Inc.). The enrichment of Lin- BM cells was performed using MACS

714 beads from mouse Lineage Cell Depletion Kit (Miltenyi Biotec). CD11b+ Gr-1- BM

715 cells from Rptor F/F, Mx1-Cre mice, Lin- CD11b+ PD-L1+ IMLECs / LSK / CLP /

716 CMP populations from Raptor Ctrl/cKO or WT mice, CD11b+ Gr-1+ / CD11b- Gr-1-

717 BM populations from Rptor F/F, Lyz2-Cre+/+ mice, CD11bhigh Gr-1- F4/80+

718 peritoneal macrophages were sorted using FACSAria II or Influx™ cell sorter

719 (BD Biosciences). The detailed information on Abs used in this study is in

720 Supplementary file 3.

721

722 BM cells transplantation

723 C57BL/6 Ly5.2 (CD45.1+) recipient mice at the age of 6-12 weeks old were

724 lethally irradiated for total 900-1,100 rads with a Cs-137 γ-ray source or a RS

725 2000 X-ray irradiator (Rad Source Technologies, Inc.). Indicated donor BM cells

726 (whole BM or FACS-sorted BM cells) were transplanted into recipients through

727 the mice tail by intra-venous (i.v.) injection within 24 hours after irradiation 82. At

728 different time points post-transplantation, peripheral blood from the recipient mice

729 was analyzed by flow cytometry to test the reconstitutions.

730

731 Conventional PCR and quantitative PCR

732 Genomic DNA was isolated from BM cells by DNeasy Blood and Tissue Kit

733 (Qiagen) as per manufacturer’s instructions. Total RNA was isolated using TRIzol

32 734 (Invitrogen) or ReliaPrep™ RNA Cell Miniprep System (Promega). Reverse

735 transcription was carried out using random hemaxmer primers and SuperScript II

736 Reverse Transcriptase (Invitrogen). Conventional PCR was performed using

737 GoTaq Green Master Mix (Promega). Quantitative PCR (q-PCR) was performed

738 by the 7500 real-time PCR system using Power SYBR Green Master Mixture

739 (Applied Biosystems). Fold changes were calculated according to the ΔΔCT

740 method 85. The primers used for conventional PCR and q-PCR are listed in

741 Supplementary file 4.

742

743 Ig gene rearrangement test by V(D)J analysis

744 Immunoglobulin (Ig) gene recombination was determined using genomic DNA as

745 previously described 86. For Heavy chain, a semi-nested PCR strategy was

746 employed to amplify the framework regions of VH to specific sites of JH. First

747 round amplification of 25 cycles was performed with primers FR/JH1 (70°C

748 annealing/ 20 sec extension). Second round amplification of 35 cycles was with

749 primers FR/JH2 (65°C annealing/ 30 sec extension). Light chain (Igk and Igl)

750 recombination was tested by primers Vκ /Jκ5 and Vλ1 /Jλ1,3 following previous

751 report 87. Sequences of primers used are listed in Supplementary file 4.

752

753 OP9 cell co-culturing with HSPCs

754 OP9 stromal cell line (ATCC Cat# CRL-2749, RRID:CVCL_4398) was purchased

755 from American Type Culture Collection (ATCC, Manassas, USA). No cell lines

756 used in this study were listed in the database of cross-contaminated or

33 757 misidentified cell lines suggested by International Cell Line Authentication

758 Committee (ICLAC). All cell lines from ATCC were authenticated by the STR

759 profiling method and tested as mycoplasma contamination free by ATCC. OP9

760 cells were maintained in α-MEM medium (Life Technologies) supplemented with

761 20% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml of penicillin and

762 100 µg/ml of streptomycin (Gibco). The 6-well and 12-well flat-bottomed plates

763 were pre-coated with OP9 cells at approximate 100% confluence after overnight

764 growth. Subsequently 2X103 LSK (Lin- c-Kit+ Sca-1+) or 5X104 CMP (Lin- c-Kit+

765 Sca-1- CD34MediumCD16/32Medium) cells or 5X104 CLP (Lin- CD127+c-KitMedium Sca-

766 1Medium) cells FACS-sorted from Raptor Ctrl/cKO BM were seeded. The co-

767 culturing medium was additionally supplemented with 2ng/ml murine recombinant

768 IL-3, 2ng/ml murine recombinant IL-6, 20ng/ml murine recombinant SCF,

769 10ng/ml murine recombinant Flt3L and 5ng/ml murine recombinant IL-7 (all from

770 R&D Systems). Lenti viral particles were produced in HEK 293T cells (ATCC

771 Cat# CRL-3216, RRID:CVCL_0063) by transiently co-transfecting control vector

772 pWPI (Plasmid #12254, Addgene), or pWPI-Myb (cDNA of Myb was purchased

773 from DharmaconTM of GE Lifesciences, Catalog Number: MMM1013-202763262;

774 Clone ID: 3672769) together with helper plasmids pMD2.G (Plasmid #12259,

775 Addgene) and psPAX2 (Plasmid #12260, Addgene) using FuGENE® HD

776 Transfection Reagent (Promega). OP9 cells were replaced every 3-4 days by

777 transferring co-culturing cells to new plates pre-coated with fresh OP9 cells. The

778 hematopoietic cells in suspension were harvested on day 10-14 post seeding

779 and subjected to flow cytometric analyses.

34 780

781 TLR stimulation and inflammatory cytokine assay

782 For in vitro TLR stimulation, fresh BM cells were seeded in a 12-well plate with a

783 density of 4X106 cells/ well(1ml medium/well) or in a 48-well plate with a density

784 of 1X106 cells/ well (200μl medium/well). The culture medium was RPMI 1640

785 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum

786 (Hyclone), 100 units/ml of penicillin and 100 µg/ml of streptomycin (Gibco). BM

787 cells were stimulated with 1μg/ml LPS (Sigma-Aldrich, from O111:B4 E.coli) or a

788 panel of TLR agonists (InvivoGen) for 16 hours. For intracellular cytokine staining,

789 the protein transport inhibitor Brefeldin A (eBioscience) was added to the

790 culturing medium during the last 4 hours of incubation.

791

792 The concentrations of the TLR agonists for in vitro studies were as following:

793 TLR1/2-Pam3CSK4, 300ng/ml; TLR2-HKLM, 108 cells/ml; TLR3-pIpC(HMW),

794 10μg/ml; TLR4-LPS-EK, 1μg/ml; TLR5-FLA-ST, 1μg/ml; TLR6/2-FSL-1, 100ng/ml;

795 TLR7-ssRNA-DR/LyoVec, 1μg/ml; TLR9-ODN1826, 1μM. For in vivo tests, pIpC

796 (GE Healthcare, 2 mg/kg), LPS (from O55:B5 E.coli, Sigma-Aldrich, 5 mg/kg) and

797 Acetaminophen (Children’s TYLENOL, 3.2 mg/mouse) were injected i.p.

798 Supernatant from in vitro cultured BM cells and serum from in vivo treated mice

799 were assayed for inflammatory cytokines by BD Cytometric Bead Array (CBA)-

800 Mouse Inflammation Kit according to the manufacturer's protocols.

801

802 cDNA library preparation and RNA sequencing

35 803 FACS-sorted CD11b+ Gr-1- BM cells from Raptor cKO mice and whole BM cells

804 from Raptor Ctrl mice were used for RNA isolation with TRIzol Reagent (Life

805 Technologies) per manufacturer’s instructions. The cDNA libraries were

806 constructed following the standard Illumina protocols by TruSeq RNA and DNA

807 sample preparation kits (Illumina). Briefly, beads containing oligo (dT) were used

808 to isolate poly(A) mRNA from total RNA. Purified mRNA was then fragmented in

809 fragmentation buffer. Using these short fragments as templates, random

810 hexamer-primers were used to synthesize the first-strand cDNA. The second-

811 strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA

812 polymerase I. Short double-stranded cDNA fragments were purified for end

813 repair and the addition of an ‘A’ base. Next, the short fragments were ligated to

814 Illumina sequencing adaptors. DNA fragments of a selected size were gel-

815 purified and amplified by PCR. The amplified cDNA libraries were quality

816 validated and then subjected to 50 nt single-end sequencing on an Illumina

817 HiSeq 2000 at the University of Michigan DNA Sequencing Core.

818

819 RNA-seq gene expression analysis

820 The reference sequences used were genome and transcriptome sequences

821 downloaded from the UCSC website (version mm10). Clean reads were

822 respectively aligned to the reference genome and transcriptome using Tophat

823 (RRID:SCR_013035)88. No more than 2 mismatches were allowed in the

824 alignment for each read. Reads that could be uniquely mapped to a gene were

825 used to calculate the expression level. The gene expression level was measured

36 826 by the number of uniquely mapped reads per kilobase of exon region per million

827 mappable reads (RPKM) and was calculated by DEGseq (RRID:SCR_008480)89.

828 The formula was defined as below:

829 in which C was the number of reads uniquely mapped to the given gene; N was

830 the number of reads uniquely mapped to all genes; L was the total length of

831 exons from the given gene. For genes with more than one alternative transcript,

832 the longest transcript was selected to calculate the RPKM.

833

834 PCA, differential and cell-specific expression analysis

835 The RPKM method eliminates the influence of different gene lengths and

836 sequencing discrepancies on the gene expression calculation. Therefore, the

837 RPKM value can be used for comparing the differences in gene expression

838 among samples. The RPKM value of all RNA-seq raw data were calculated

839 according to the same workflow as stated above.

840

841 A function was implemented in the R software to perform principal component

842 analysis (PCA). This function computes the eigenvalues and eigenvectors of the

843 dataset (23498 genes) using the correlation matrix. The eigenvalues were then

844 ordered from highest to lowest, indicating their relative contribution to the

845 structure of the data. The projection of each sample defined by components was

846 represented as a dot plot to generate the PCA figures.

847

37 848 Selected samples were then pooled by subtypes and a two sided t-test with FDR

849 (False discovery rate) of 0.05 and fold change of 4 was performed to identify

850 differentially expressed genes between IMLECs and other subtypes (mean

851 RPKM values of genes in two subtypes both below 5 were deleted). For subtype-

852 specific genes identified, a one-sided t-test (null hypothesis is greater) was

853 performed with FDR of 0.01 and fold change of 4 contrasting each subtype in

854 turn versus all other subtypes pooled, and statistically significant genes were

855 assigned to the respective subtype signature.

856

857 RNA-seq datasets in this study have been deposited in the Gene Expression

858 Omnibus (GEO) database as accession number GSE67863. Other public RNA-

859 seq datasets used are as followings: peritoneal CD11b+F4/80+ macrophages

860 (GSM1103013, GSM1103014 in GEO Series GSE45358); normal BM CD11b+

861 Gr-1+ granulocytes (GSM1166354, GSM1166355, GSM1166356 in GEO Series

862 GSE48048); BM-derived dendritic cells (GSM1012795, GSM1012796 and

863 GSM1012797 in GEO Series GSE41265); BM erythroid cells (GSM1208164,

864 GSM1208165 and GSM1208166 in GEO Series GSE49843 ); BM pro B and pre

865 B cells (GSM978778 and GSM978779 in GEO Series GSE39756); Naïve B cells

866 (GSM1155172, GSM1155176, GSM1155180 and GSM1155184 in GEO Series

867 GSE47703); activated B cells (GSM1155170, GSM1155174, GSM1155178 and

868 GSM1155182 in GEO Series GSE47703); CD4 T cells, CD8 T cells and natural

869 regulatory T (nTreg) cells (GSM1169492, GSM1169501, GSM1169493,

870 GSM1169502, GSM1169499 and GSM1169508 in GEO Series GSE48138); .

38 871 Spleen NK cells (GSM1257953, GSM1257954, GSM1257955 and GSM 1257956

872 in GEO Series GSE52047); Blood neutrophils (GSM1340629, GSM1340630,

873 GSM1340631 and GSM1340632 in GEO Series GSE55633); Lung basophils and

874 eosinophils (GSM1358432, GSM1358433, GSM1358436 and GSM1358437 in

875 GEO Series GSE56292). RNA-seq data on ex vivo DC subsets are obtained

876 from GEO Series GSE62704: GSM1531794 (CDP), GSM1531795 (pDC),

877 GSM1531796 (preDC), GSM1531797 (DN DC), GSM1531798 (CD4+ DC),

878 GSM1531799 (CD8+ DC).

879 Genome browser display of immunoglobulin expression

880 To facilitate the global viewing of transcript structure and gene expression

881 quantity of immunoglobulin, an interface in which RNA-seq gene expression of

882 immunoglobulin can be viewed in Genome Browser display was constructed. For

883 viewing and analysis, the UCSC Genome Browser (http://www.genome.ucsc.edu,

884 RRID:SCR_005780) 90 with the mm10 version of the mouse genome was used.

885 For each base in each cell type, the normalized number of aligned reads count

886 was defined as below:

887

888

889 in which N was the number of reads uniquely mapped to the given base in each

890 cell type ; n was the number of replicates for each cell type samples; A was a

891 constant t in which takes the value of 3.5E+7. The normalized number of aligned

892 reads count for each base in each cell type was stored as WIG file and was

39 893 uploaded to UCSC genome browser. For each track, y-axis depicted the

894 normalized number of aligned reads count and x-axis depicts physical distance in

895 bases along the chromosome.

896

897 Statistical analysis

898 All the data are presented as mean ± SD. Unless otherwise indicated, two-tailed,

899 unpaired student’s t tests were used for comparison between two experimental

900 groups. In Figure 8I and Figure 8J, where the data do not follow normal

901 distribution, Mann-Whitney tests were used. The log-rank tests were used for the

902 Kaplan-Meier survival analysis. Statistical significance was determined as p<0.05

903 (*, p<0.05; **, p<0.01; ***, p<0.001).

904

905

906 Acknowledgements

907 We thank Dr. Hans Schreiber (University of Chicago) and all the colleagues in

908 our laboratory for their helpful discussions, and Ms. Morgan E. Daley for editorial

909 assistance. This work was supported by the grants from National Institute of

910 Health AG036690, AI64350, CA183030 and CA171972. This work was initiated

911 at the University of Michigan then completed at Children’s Research Institute of

912 Children’s National Medical Center.

913

40 914 References

915

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1133

45 1134 Figure Legends

1135 Figure 1 Targeted mutation of Rptor in hematopoiesis led to massive

1136 accumulation of IMLEC.

1137 (A) Schematic of experimental design. Sex-matched 6-8 weeks old Ctrl (Rptor

1138 F/F) and cKO (Rptor F/F, Mx1-Cre) mice were treated with pIpC for 7 times. The

1139 phenotypes were analyzed on day 7 or day 30 after the complement of pIpC

1140 treatment. (B) Representative flow cytometric analysis of myeloid cells from mice

1141 BM by CD11b and Gr-1 on day 7 after pIpC treatment. Similar data were

1142 obtained on day 30. (C) Frequencies of various hematopoietic cell populations in

1143 BM based on CD11b and Gr-1 markers. n=7 for Ctrl mice; n=9 for cKO mice.

1144 Data are pooled from 3 independent experiments. (D) Expansion of lymphoblast

1145 population in cKO BM revealed by histology. Left, H&E staining showing

1146 prominence of cells with lymphoid morphology in the cKO BM; Right, normal BM

1147 histology from Ctrl mouse showing normal myeloid and erythroid lineages. Scale

1148 bar, 20 m. (E) Expansion of lymphoblast revealed by cytology. Left, cytological

1149 preparation of BM smear from cKO mouse showing preponderance of

1150 lymphocytes (short arrows) and severe depletion of myeloid and erythroid

1151 lineages. There were some promyelocytes and myelocytes present (larger cells)

1152 but no mature neutrophils. Right, normal cytological preparation of BM from Ctrl

1153 mouse. There were numerous erythroid precursors with intensely basophilic,

1154 condensed chromatin (arrowheads) and a few lymphocytes (short arrow) with

1155 less condensed chromatin. Additionally, in BM there were numerous mature

1156 neutrophils (ringed nucleus with constrictions) which were not severely depleted

46 1157 in the cKO mouse. Scale bar, 10 m. (F) Giemsa staining of FACS-sorted Rptor-

1158 deficient CD11b+Gr-1- BM cells. Scale bar, 10μm. Similar morphology was

1159 observed in three independent experiments. (G) Principal component analysis

1160 (PCA) of gene expression in CD11b+ Gr-1- BM cells (IMLECs) and other

1161 hematopoietic cells. Numbers along axes indicate relative scaling of the principal

1162 variables. RNA-seq data from IMLECs obtained in our study were compared

1163 with those deposited in public database by others. Datasets are from known

1164 lymphoid (Pro-B, Pre-B, Naïve B, CD4 T, CD8 T, Treg and NK) or myeloid

1165 (macrophage, dendritic cell, granulocyte, neutrophil, eosinophil, basophil and

1166 erythrocyte) subsets.

1167

1168 Figure 2 Unique gene signature of Rptor -/- CD11b+ Gr-1- BM IMLECs.

1169 (A) Heat map representing the relative expression levels for indicated population-

1170 specific genes. Genes up-regulated for at least 4-fold with FDR-adjusted p-value

1171 <0.01 were considered population-specific. Numbers on right indicate amounts of

1172 population-specific genes. (B) Heat map representing expression levels of 48

1173 CD11b+Gr-1- BM IMLEC-specific genes among indicated populations including

1174 macrophage, B precursor, naive B and granulocyte. (C) Genome browser display

1175 of transcript structure and gene expression quantity for immunoglobulin heavy

1176 chain (IgH) complex gene region from indicated populations. For each track, the

1177 normalized numbers of aligned reads count or Reads Per Kilobase of transcript

1178 per Million mapped RNA (RPKM) are shown in y-axis, while the gene positions

1179 for the sterile transcripts are shown in x-axis. Numbers on top indicate the start

47 1180 and end loci on bases along chromosome 12 for specified gene region. Similar

1181 profiles of cKO IMLEC were observed in two other samples analyzed. (D) mRNA

1182 levels of Rag1 and Rag2 in cKO CD11b+ Gr-1- BM IMLECs in comparison to Ctrl

1183 whole BM cells. n=6 for Ctrl-WBM; n=8 for IMLEC. Data are pooled from 3

1184 independent experiments. (E) IMLECs did not display immunoglobulin gene

1185 rearrangement. FACS-sorted CD11b+ Gr-1- cells (IMLECs) from cKO BM were

1186 dissected for Ig gene rearrangement patterns of heavy chain (JH), light chains Igk

1187 (JLκ) and Igl (JLλ) by PCR. WBM cells from Raptor Ctrl mice were used as positive

1188 control. These data have been repeated twice.

1189

1190 Figure 3 Identification of IMLEC by cell surface markers in both Raptor cKO

1191 and WT mice.

1192 (A) IMLECs do not express surface markers for conventional lymphocytes (CD3,

1193 B220 and NK1.1) and stem and progenitor cells (c-Kit and Sca-1). CD11b+ Gr-1-

1194 IMLECs from Raptor cKO BM were tested for their expression of lymphocytes

1195 and progenitor cell markers. These data have been repeated 3 times. (B)

1196 IMLECs do not express markers for monocytes and macrophages but

1197 surprisingly express high levels of PD-L1. Filled gray areas indicate distributions

1198 of fluorescence from stainings by control antibodies. One representative result of

1199 at least five experiments is shown. (C) Rptor deletion causes expansion of Lin-

1200 (B220- CD3- Ter119- NK1.1- Gr-1- F4/80- CD115- ) CD11b+ PD-L1+ IMLECs in BM.

1201 Representative flow staining profiles for BM IMLECs (left) and their abundance

1202 summary data (right) are presented. n=5 for Ctrl BM; n=5 for cKO BM. (D) Lin-

48 1203 CD11b+ PD-L1+ BM IMLECs are CD11c-/low. One representative result of two

1204 experiments is shown. (E) The Lin- CD11b+ PD-L1+ CD11c-/low IMLECs are found

1205 in various lymphoid and non-lymphoid organs from WT mice. mLN, mesenteric

1206 lymph nodes; c/i LN, cervical and inguinal lymph nodes. The numbers (Mean±SD)

1207 are summarized abundances of IMLECs among mononuclear cells (MNCs) from

1208 3 WT mice. (F-H) Rptor deletion caused a broad accumulation of IMLECs in lung

1209 (F), mesenteric lymph nodes (G) and peripheral (cervical and inguinal) lymph

1210 nodes (H). Results shown are representative of three mice in each group. (I)

1211 Summary data showing increased IMLECs in the peripheral blood, lymphoid and

1212 non-lymphoid organs. n=3 for both groups.

1213

1214 Figure 4 Comparison of IMLECs identified in both WT and Raptor-deficient

1215 BM.

1216 (A) Giemsa staining of Lin- CD11b+ PD-L1+ IMLECs from WT BM and Rptor-cKO

1217 BM. One representative result of 3 independent experiments is shown. Scale bar,

1218 10μm. (B) Size and granularity of IMLECs from Raptor Ctrl and cKo BM were

1219 evaluated by FSC and SSC, respectively. n=5 for each group. (C) IMLEC from

1220 WT BM expresses sterile transcripts from the Igh and Igl loci. Ig transcript levels

1221 in macrophage are artificially defined as 1. (D) Heat map representing the

1222 relative expression levels for indicated population specific transcription factor

1223 genes. Genes up-regulated for at least 4-fold with FDR-adjusted p-value<0.01

1224 were considered population-specific. (E) IMLECs from both WT and Raptor-cKO

1225 BM exhibited high levels of mRNA transcripts for the specified transcription

49 1226 factors (Mitf, Atf3 and Zdhhc1). n=5 for each group. (F, G) Ctrl and cKO IMLECs

1227 are non-cycling cells in contrast to Lin+ BM cells. (H, I) IMLECs are prone to

1228 apoptosis based on their binding to Annexin V. Results in (C), (G) and (I)

1229 represent one of 2 independent experiments with each involving 3 mice per

1230 group.

1231

1232 Figure 5 Raptor represses differentiation of IMLEC from CMP.

1233 (A, B) IMLEC accumulation in Rptor-/- BM is cell autonomous. (A) Diagram

1234 depicting generation of mixed BM chimera, induction of Rptor deletion and

1235 analyses for IMLEC. (B) In mixed BM chimera mice, IMLEC was accumulated

1236 only in the Raptor-deficient hematopoietic cells. Mice were sacrificed and

1237 analyzed at 4 weeks post pIpC treatment. CD45.1+ and CD45.2+ donor-derived

1238 BM cells were gated to show the myeloid subsets based on CD11b and Gr-1.

1239 Similar data were obtained in 3 independent experiments, each involving at least

1240 3 mice per group. (C, D) BM IMLECs are not converted from CD11b+ Gr-1-

1241 granulocytes. (C) Genotyping and deletion efficacy. CD11b+Gr-1+ cells (DP) and

1242 CD11b-Gr-1- cells (DN) were sorted from BM of KO (RptorF/F, Lyz2-Cre+/+) mice.

1243 Floxed and deleted Rptor alleles, as well as Lyz2-Cre wild type (WT) and

1244 mutated (MT) alleles were confirmed by PCR. (D) Efficient deletion of Rptor in

1245 the CD11b+Gr-1+ compartment failed to cause IMLEC accumulation. Data

1246 shown are representative BM flow profiles of Ctrl mice and those with Rptor

1247 deletion in the CD11b+Gr-1+ compartment, depicting distributions of CD11b and

1248 Gr-1 markers in adult mice. Similar data have been obtained in 3 experiments,

50 1249 involving a total of 5 mice per group. (E, F) Rptor-/- LSK, CLP and CMP cells

1250 differentiate into IMLECs in vitro. (E) Diagram of experimental design. 2X103 LSK

1251 cells or 5X104 CLP or 5X104 CMP cells were co-cultured with the OP9 cells for

1252 10 days. (F) Rptor deletion promoted generation of IMLEC. The CD45.2+

1253 leukocytes were gated and analyzed for their expression of CD11b, Gr-1 and PD-

1254 L1. Data shown are representative of three independent experiments. (G, H)

1255 Both LSK and CMP cells differentiate into IMLECs in vivo. (G) Diagram of BM

1256 cells transplantation. FACS-sorted, Ctrl or cKO LSK cells (5X104/mouse) or CMP

1257 cells (1.2 X105/mouse) were injected i.v. to CD45.1 recipient mice which were

1258 immediately administrated with 5 daily pIpC treatments. BM cells were harvested

1259 on day 7 for FACS analyses. (H) Rptor deletion promoted differentiation of

1260 progenitor cells into IMLEC. Donor-derived CD45.2+ BM cells were gated to

1261 analyze surface markers CD11b, Gr-1 and PD-L1. Data shown are

1262 representative flow profiles from one of 3 independent experiments.

1263

1264 Figure 6 Inactivation of Myb is an underlying cause for accumulation of

1265 IMLEC.

1266 (A) Reduction of Myb mRNA in the Rptor-deficient HSPCs. BM LSK and CMP

1267 populations were FACS-sorted from Ctrl and cKO mice at 1-3 weeks after pIpC

1268 treatment. Quantitation of Myb mRNA was performed by qPCR. n=5 for Ctrl-LSK;

1269 n=4 for cKO-LSK (left). n=4 for Ctrl-CMP; n=3 for cKO-CMP (right). (B) Detection

1270 of c-Myb protein by intracellular staining. Data represent one of three

1271 independent experiments with similar results. (C-E) Deletion of c-Myb in mice

51 1272 with homozygous floxed Myb resulted in enhanced generation of IMLECs. (C)

1273 Schematic of experimental design. Sex-matched 6-8 weeks old c-Myb Ctrl (Myb

1274 F/F) and cKO (Myb F/F, Mx1-Cre) mice were treated with pIpC for 7 times. The

1275 phenotypes were analyzed on day 7 after the complement of pIpC treatment.

1276 Inducible deletion of c-Myb showed obvious increase of PD-L1 expression on

1277 CD11b+Gr-1- BM cells (D) and production of IMLECs (E). n=3 for c-Myb Ctrl mice;

1278 n=3 for c-Myb cKO mice. (F-H) Deletion of Myb enhances generation of IMLEC.

1279 (F) Diagram of experimental design. Whole BM cells (2X106/mice) of given

1280 genotypes were used for transplantation. Once the chimera mice were

1281 established, deletion of Myb was induced by five daily injection of tamoxifen. (G)

1282 Detection of Myb deletion in the whole BM cells after tamoxifen treatments. Data

1283 are representative of two independent experiments. (H) Generation of IMLEC is

1284 promoted by inactivation of Myb. BM cells were harvested at 7 days after first

1285 tamoxifen treatment and analyzed for IMLECs based on surface markers CD11b,

1286 Gr-1 and PD-L1 within the donor-derived CD45.2+ BM cells. Data shown

1287 represent one of three experiments using either first or second generation of BM

1288 chimeras. (I) Rptor deletion broadly increases miRNAs targeting Myb. Lin- c-Kit+

1289 HSPCs were isolated from Raptor Ctrl and cKO mice at 10 days after pIpC

1290 treatment. miRNA levels were measured by miRNA microarray. The y-axis

1291 shows the log2 ratio of signal, while the x-axis shows the -log10P value. The doted

1292 boxes show the numbers of significantly (P<0.05) up-regulated (fold change>2)

1293 or down-regulated (fold change <0.5) miRNAs among 50 miRNAs with mirSVR

52 1294 score < -1.0. Each dot represents the mean value of a unique miRNA from 3

1295 independent samples.

1296

1297 Figure 7 IMLECs broadly express PPRs and produce large amounts of

1298 inflammatory cytokines upon stimulation by various TLR ligands.

1299 (A) Heat map showing the relative transcript levels of TLRs among the indicated

1300 populations. (B) Both WT and Raptor-cKO IMLECs have greatly elevated

1301 expression of multiple TLRs genes in comparison to Ctrl BM. q-PCR was

1302 performed to determine transcript levels of Tlr1-9 genes. After normalizing for

1303 cDNA input based on Hprt mRNA in each sample, the Tlr1-9 levels in the FACS-

1304 sorted cKO CD11b+ Gr-1- BM IMLEC were compared with Ctrl BM (artificially

1305 defined as 1.0). n=3 for Ctrl WBM; n=5 for cKO- IMLEC; n=5 for WT-IMLEC.

1306 Similar results were obtained using mice sacrificed at 2 weeks ~ 2 months after

1307 pIpC treatment. (C, D) In responses to various TLR ligands, Rptor cKO BM cells

1308 produced greatly elevated amounts of TNF-α (C) and MCP-1 (D) than the Ctrl

1309 BM. Data shown are from one experiment involving 3 repeats per group and

1310 have been reproduced in five independent experiments. (E) Lin- (B220-CD3-

1311 Ter119-Gr-1-NK1.1-F4/80-CD115- ) CD11b+ PD-L1+ IMLECs from both cKO and

1312 WT BM were robust TNF-α producers after stimulation with LPS. BM cells

1313 (1X107 cells / well) were stimulated with LPS (1μg/ml) for 16 hours with the

1314 presence of Golgi blocker in the last 4 hours. Data shown are representative

1315 profiles from one experiment and have been reproduced in 3 independent

1316 experiments.

53 1317 1318 1319 Figure 8 Rptor cKO mice are hyper sensitive to challenges by TLR ligands.

1320 (A) High blood levels of inflammatory cytokines in cKO mice that have received 7

1321 pIpC treatments. Serum samples collected at 7 days after the last pIpC treatment

1322 were tested for levels of IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10 and IL-6. Dot

1323 plots depict cytokine levels in individual cKO (red dots) or Ctrl (blue dots) mice,

1324 and line drawings depict means and SD. n=12 for Ctrl mice and n=11 for cKO

1325 mice. (B) High proportions of Rptor cKO mice that received 7 pIpC injections

1326 died within 7 weeks after first pIpC treatment. Data shown are Kaplan-Meier

1327 survival curve, and the statistical significance is determined by log-rank test.

1328 n=32 for Ctrl mice and n=31 for cKO mice. (C) Representative H&E staining of

1329 liver sections from Rptor Ctrl and cKO mice at 2 weeks post pIpC treatment.

1330 Scale bars represent 200μm (10X) and 50μm (40X), respectively. (D, E) Massive

1331 accumulation of IMLEC in cKO liver. (D) Accumulation of CD11b+ Gr-1- cells in

1332 the cKO liver. (E) CD11b+ Gr-1- cells in cKO livers demonstrated IMLEC surface

1333 markers, including PD-L1high and F4/80low/-. Gray lines indicate the staining for

1334 isotype control antibodies. Results are representative of at least five

1335 independent analyses. (F-H) cKO mice are vulnerable to low doses of LPS

1336 challenges. At 2-3 months after pIpC treatments, cKO and Ctrl mice were

1337 challenged with 5mg/kg body weight of LPS and observed survival. (F) Kaplan-

1338 Meier survival analysis. Data are pooled from 3 independent experiments. n=14

1339 for Ctrl mice; n=11 for cKO mice. Inflammatory cytokines TNF-α (G) and MCP-1

1340 (H) levels from mice serum before (0h) and at 6 hours after (6h) LPS injection are

54 1341 shown. n=10 for Ctrl mice; n=9 for cKO mice. Note greater than 500-fold increase

1342 in plasma TNF- levels. (I, J) cKO mice mounted enhanced inflammatory

1343 response to acetaminophen-triggered liver necrosis. Serum TNF- (I) and MCP-

1344 1(J) levels at 6 hours upon acetaminophen (3.2 mg/mouse) treatment are shown.

1345 n=4 for Ctrl mice; n=6 for cKO mice. Mann-Whitney test was used for statistics

1346 analysis, and lines indicate Mean ±SD. Similar trends were observed in another

1347 independent experiment.

1348

1349 Figure 9 Rptor deletion in hematopoietic cells greatly increased

1350 vulnerability of mice to pIpC.

1351 (A) Diagram of experimental design. WT CD45.1+ recipient mice were irradiated

1352 and transplanted with 5X106 BM cells from RptorF/F and RptorF/F, Mx1-Cre BM.

1353 After the fully reconstitution, recipients were treated with pIpC 3 times every other

1354 day to induce gene deletion in chimera mice with cKO BM. 10 days after the last

1355 injection of pIpC, recipients were challenged with another pIpC injection and the

1356 survival of mice were followed for 4 more weeks. Arrows denote the injections of

1357 pIpC at indicated time points. (B) Kaplan-Meier survival analysis after pIpC

1358 treatments. n= 12 for Ctrl chimera mice; n=15 for cKO chimera mice. (C)

1359 Histological analysis of liver sections after H&E staining. Note extensive

1360 inflammation and liver damage in cKO chimera mice. Scale bars represent

1361 200μm (10X), 100μm (20X) and 50μm (40X). (D-F) Identification of IMLEC in BM

1362 (D), spleen (E) and livers (F) by flow cytometry. Mice were euthanized 45 days

1363 after the first pIpC injection. The distinct CD11b+ Gr-1- PD-L1+ F4/80low/- IMLECs

55 1364 greatly enriched in BM, spleen and liver of recipients of Rptor cKO BM. FACS

1365 profiles shown represent one of five independent experiments.

1366

1367

1368 Figure Supplements and Supplementary Files

1369

1370 Figure 1—figure supplement 1 Conditional deletion of Rptor resulted in

1371 abnormal hematopoiesis.

1372 (A) Deletion of Rptor in BM cells. PCR were performed to check the deletion in

1373 BM from mice 2 weeks after pIpC treatment (for Ctrl and cKO mice, no treatment

1374 for WT mice). (B) Representative pictures of leg bones (tibiae and femurs),

1375 spleen, and thymus harvested from mice on day 30 post pIpC treatment. (C)

1376 Histology findings in the cKO spleen by H&E staining. Up left panel: a spleen

1377 histological section showing expanded white pulp areas (WP) and compressed

1378 intervening red pulp (RP). The white pulp contains an increased population of

1379 lightly staining cells that sometimes is situated in the marginal zones and

1380 follicular centers (B cell areas) and sometimes infiltrates the periarteriolar

1381 sheaths (T cell area, long arrow). Up right panel: normal splenic architecture from

1382 control mouse. Scale bar, 200 m. Down left panel: higher magnification of

1383 expanded cell population within the splenic white pulp. Morphologically these

1384 cells resemble germinal center lymphocytes. There are both smaller cells with

1385 cleaved nuclei (long arrow) resembling centrocytes and larger cells with 1-2

1386 prominent nucleoli resembling centroblasts (short arrow). There are small

56 1387 numbers of plasmacytoid cells (arrowhead). Down right panel: histological

1388 section of splenic red pulp Red pulp of the spleen showing foci of extramedullary

1389 hematopoiesis with decreased erythroid (short arrow) and myeloid (long arrow)

1390 progenitors. The erythroid lineage contains occasional foci of pyknotic or

1391 karyorrhectic (fragmented) nuclei consistent with cell death (asterisk). Scale bar,

1392 20 m. (D) The cellularities of splenocytes (left) and thymocytes (right) from mice

1393 at day 30 post pIpC treatment. n=6 for Ctrl mice; n=5 for cKO mice. (E) BM

1394 cellularities (left) and spleen/body weight ratios (right) for mice on day 7 and day

1395 30 after pIpC treatment. n=3~8 for Ctrl mice; n=5~6 for cKO mice.

1396

1397 Figure 1—figure supplement 2 Raptor deletion led to impaired

1398 developments of B lymphoid, erythroid and myeloid compartments in BM.

1399 (A) Representative FACS profiles for Pro B (CD127+B220+BP-1-IgM-IgD- cells),

1400 Pre B ( CD127+B220+BP-1+IgM-IgD- cells), Immature B (CD127-B220+BP-1-

1401 IgM+IgD- cells), Mature B (CD127-B220+BP-1-IgM+IgD+ cells) in mice BM on day

1402 7 after pIpC treatment. (B) Frequencies of BM B cells subsets as in (A). n=5 for

1403 Ctrl mice and n=6 for cKO mice. (C) Representative FACS analysis of erythroid

1404 populations in BM by Ter119 and CD71. Roman numerals and numbers indicate

1405 the identity and percentages of the developmentally defined subpopulations: I,

1406 proerythroblasts; II, basophilic erythroblasts; III, polychromatophilic erythroblasts;

1407 IV, orthochromatophilic erythroblasts. (D) Frequencies of erythroblast subsets in

1408 mice BM. n=5 for Ctrl mice and n=6 for cKO mice. (E, F) Size and granularity of

1409 BM CD11b+ Gr-1- IMLECs from Rptor cKO mice. (E) Flow cytometric assays for

57 1410 size and granularity of Ctrl and cKO BM cells by FSC and SSC, respectively.

1411 Erythroid cells with smaller size and granularity were excluded in the initial gating.

1412 DP (double positive) represents CD11b+ Gr-1+ granulocytes, and DN (double

1413 negative) represents (CD11b- Gr-1-) cells. (F) Relative size (FSC-A) and

1414 granularities (SSC-A) of DN, DP and IMLEC. The mean values in Ctrl mice are

1415 artificially defined as 100%. n=5 for Ctrl mice; n=3 for cKO mice.

1416

1417 Figure 1—figure supplement 3 Rptor cKO mice are pancytopenic.

1418 (A) Complete blood cell counts (CBC) data for Ctrl and cKO mice on day 30 after

1419 pIpC treatment. WBC, white blood cells; NE, neutrophils; LY, lymphocytes; MO,

1420 monocytes; RBC, red blood cells; Hb, hemoglobin; PLT, platelets. Means of the

1421 Ctrl mice in each experiment are artificially defined as 100%. (B) Representative

1422 FACS profiles showing the percentages of different leukocyte populations in

1423 peripheral blood from Ctrl and cKO mice on day 30 after pIpC treatment. (C, D)

1424 Reduction of leukocyte populations in the peripheral blood of cKO mice.

1425 Frequencies among CD45.2+ leukocytes (C) and absolute numbers (D) of

1426 various cell types in blood on day 30 after pIpC treatment are shown. n=7 for Ctrl

1427 mice; n=6 for cKO mice. Data represent one of three independent experiments

1428 with similar results.

1429

1430 Figure 1—figure supplement 4 Rptor deletion increased hematopoietic

1431 stem and progenitor cells in BM.

58 1432 (A) Representative FACS profiles for Lin- Sca-1+ c-Kit+ cells (LSK, identified as

1433 CD3- B220- Ter119- CD11b- Gr-1- Sca-1+ c-Kit+ cells), multipotent progenitor cells

1434 (MPP, identified as Lin- Sca-1+ c-Kit+ CD150- CD48+ cells), short-term HSC (ST-

1435 HSC, identified as Lin- Sca-1+ c-Kit+ CD150+ CD48+ cells) and long-term HSC (LT-

1436 HSC, identified as Lin- Sca-1+ c-Kit+ CD150+ CD48- cells) in the BM from Raptor

1437 Ctrl and cKO mice on day 7 after pIpC treatment. Numbers indicate the

1438 percentages of gated populations in total BM cells. (B) Frequencies (left) and

1439 absolute numbers (right) of stem and progenitor cells as in (A) are shown. n=6 for

1440 Ctrl mice; n=7 for cKO mice. (C) Representative FACS profiles for common

1441 myeloid progenitor (CMP, identified as Lin- Sca-1- c-Kit+ CD34MediumCD16/32Medium

1442 cell), granulocyte / macrophage progenitor (GMP, identified as Lin- Sca-1- c-Kit+

1443 CD34+ CD16/32 + cell) and megakaryocyte / erythroid progenitor (MEP, identified

1444 as Lin- Sca-1- c-Kit+ CD34- CD16/32- cell ) in BM. (D) Frequencies (left) and

1445 absolute numbers (right) of myeloid progenitors as in (C) are shown. n=5 for Ctrl

1446 mice; n=5 for cKO mice.

1447

1448 Figure 1—figure supplement 5 Tamoxifen induced conditional deletion of

1449 Rptor in hematopoiesis also led to massive accumulation of IMLECs.

1450 (A) Schematic of experimental design. Sex-matched 6-8 weeks old Ctrl (Rptor F/F)

1451 and cKO (Rptor F/F, CreER) mice were treated with Tamoxifen for 5 consecutive

1452 days. The phenotypes were analyzed 3 weeks after the first treatment. (B)

1453 Representative flow cytometric analysis of myeloid cells from Ctrl and cKO mice

1454 BM by CD11b, Gr-1 and PD-L1. (C) Rptor deletion caused expansion of Lin-

59 1455 (B220- CD3- Ter119- NK1.1- Gr-1- F4/80- CD115- ) CD11b+ PD-L1+ IMLECs in

1456 BM. (D) Schematic of experimental design. CD45.2+ Ctrl BM and cKO BM were

1457 transplanted into lethal dose irradiated CD45.1+ recipient mice. Tamoxifen

1458 treatment of recipient mice was started 6 weeks after fully reconstitution of donor-

1459 derived cells. The phenotypes were analyzed 3 weeks after the first treatment. (E,

1460 F) Representative flow cytometrical profiles of donor-derived CD45.2+ Myeloid

1461 cells (E) and IMLECs (F) in BM of recipient mice are shown as in (B) and (C),

1462 respectively.

1463

1464 Figure 2—figure supplement 1 In silico pair wise comparisons between

1465 IMLECs and other closely related leukocytes based on our RNA-seq data

1466 and the publically available RNA-seq data.

1467 (A-D)Based on the space proximity in the PCA data, macrophage (A), B

1468 precursor (B), naive B (C) and granulocyte (D) were selected for one-to-one

1469 comparison. Numbers in corners indicate amounts of genes significantly (FDR-

1470 adjusted p-value<0.05) up-regulated (right) or down-regulated (left) by at least 4-

1471 fold. (E, F) IMLECs in Raptor cKO BM are distinct from all DC subsets tested. (E)

1472 Principal component analysis (PCA) of gene expression in Raptor cKO CD11b+

1473 Gr-1- BM cell (IMLEC), macrophage, B precursors and various ex vivo DC

1474 subsets. Numbers along axes indicate relative scaling of the principal variables.

1475 (F) Clustering of IMLEC, macrophage, B precursors with other DC subsets based

1476 on their gene expression profiles. Hierarchical clustering with complete linkage

1477 was carried out using the R software. The public RNA-seq datasets for DC

60 1478 subsets used are as followings: CDP (common DC precursor, GSM1531794);

1479 pDC (GSM1531795); preDC (GSM1531796); DN DC (CD4 and CD8 double

1480 negative DC, GSM1531797); CD4+ DC (GSM1531798) and CD8+ DC

1481 (GSM1531799).

1482

1483 Figure 2—figure supplement 2 cKO IMLECs have a similar expression

1484 patterns as that of B-lymphoid subsets in the sterile transcripts from Igk

1485 and Igl loci.

1486 (A, B) Genome browser display of transcript expression of Igk locus in

1487 chromosome 6 (A) and Igl locus in chromosome 16 (B). For each track, the y-

1488 axis is the normalized number of aligned reads counts of gene region, whereas

1489 the x-axis depicts physical distance in base-pairs (bp). Numbers on top indicate

1490 the start and end positions of specified gene regions.

1491

1492 Figure 4—figure supplement 1 Comparisons of surface markers, viability

1493 and cellular proliferation among IMLECs and other defined lineages.

1494 (A) IMLECs from Raptor Ctrl and cKO BM displayed comparable expression

1495 levels of MHC-I, MHC-II and other surface markers (Ly6C, CD14 and CD68).

1496 Data shown are representative of 3 independent experiments. (B, C) IMLECs

1497 from Raptor Ctrl and cKO BM exhibited much slower proliferation than other BM

1498 lineages. Representative flow profile (B) and summary data (C) are from BrdU

1499 incorporation assays. (D, E) IMLECs from Raptor Ctrl and cKO BM were more

1500 prone to apoptosis than other BM lineages. Representative flow profile (D) and

61 1501 summary data (E) are from Annexin V and 7-AAD staining. B cells, B220+ BM

1502 cells; T cells, CD3+ BM cells; Granulocytes, CD11b+Gr-1+ BM cells. n=5 for Ctrl

1503 mice; n=5 for cKO mice.

1504

1505 Figure 6—figure supplement 1 c-Myb expression and accumulation of

1506 IMLECs. (A-C) Induced deletion of c-Myb in mice with heterozygous floxed c-

1507 Myb (Myb F/+, Mx1-Cre) did not show significant accumulation of IMLECS in

1508 mouse BM. (A) Schematic of experimental design. Sex-matched 6-8 weeks old

1509 Ctrl (Myb F/+) and heterozygous floxed c-Myb (Myb F/+, Mx1-Cre) mice were

1510 treated with pIpC for 7 times. The phenotypes were analyzed on day 7 after the

1511 complement of pIpC treatment. (B) Representative flow profile of BM IMLECs. (C)

1512 Detection of Myb deletion in the whole BM cells after pIpC treatment. Data in (B)

1513 and (C) are representative results of independent experiments with 3 groups of

1514 paired mice. (D-G)The provision of heterologous c-Myb significantly diminished

1515 the generation of IMLECs from Raptor-deficient LSK cells. (D, E) Myb-expression

1516 lenti-virus (pWPI-Myb) was validated by intracellular staining (D) and western blot

1517 (E) of CHO cells 36 hours after virus infection. Lenti-virus with GFP co-

1518 expression (pWPI) was used as control. AF568, Alexa Fluor 568 dye. (F)

1519 Diagram of experimental design. 1X104 LSK cells sorted from pIpC treated

1520 Raptor cKO mice were infected with indicated lenti-virus and subsequently co-

1521 cultured with the OP9 cells for 12 days. (G) GFP+ cells with heterologous c-Myb

1522 expression did not give rise to CD11b+ Gr-1- PD-L1+ IMLECs. Data represent the

1523 results of two independent experiments.

62 1524

1525

1526 Figure 7—figure supplement 1 IMLECs broadly over-express multiple

1527 families of pattern recognition receptors (PRRs) when compared with other

1528 blood cells.

1529 (A) Heat map showing the relative expression levels of genes encoding key

1530 PRRs. TLRs, Toll-like receptors; NLR, Nod-like receptors; RLRs, RIG-I-like

1531 receptors; ALRs, Aim2-like receptors. (B) mRNA levels of the indicated PPRs

1532 genes were measured by q-PCR in WBM cells from Ctrl mice and CD11b+ Gr-1-

1533 BM IMLECs from cKO mice. n=3 for Ctrl WBM; n=5 for cKO BM IMLECs.

1534

1535 Supplementary file 1 Transcript levels of CD markers analyzed from RNA-

1536 seq datasets. The FPKM values of 3 mice samples for cKO-IMLEC group (Rptor

1537 cKO CD11b+Gr-1- BM IMLECs) and Ctrl-WBM group (Ctrl whole BM cells) are

1538 shown. Fold change indicates the ratio of average FPKM values.

1539

1540 Supplementary file 2 Expression levels of miRNAs targeting Myb in Raptor

1541 Ctrl and cKO BM HSPCs. The normalized signal values of 3 mice samples for

1542 cKO-HSPCs group and Ctrl-HSPCs group are shown. The microarray data have

1543 been deposited to NCBI GEO database with accession number GSE64042 as

1544 previously reported43.

1545

1546 Supplementary file 3 The list of antibodies used for flow cytometry.

63 1547

1548 Supplementary file 4 Sequences of DNA primers used in PCR assays.

1549

1550

64 Figure 1 D cKO Ctrl A Ctrl ( Rptor F/F ) F Phenotypes analyzed on pIpC day 7 and day 30

Sex-matched littermates, 6-8 weeks old 2 wks 0 7 30 Days after pIpC E cKO Ctrl cKO ( Rptor F/F , Mx1-Cre ) B

Ctrl BM cKO BM 10 4 1.15 28.2 46.3 17.9

10 3

10 2 F -0 1 - .4 10 G p.3 -0 0. Com - .2 3 0. 68.0 2.64 30.5 5.37 0 1 10 0 0. CD11b 0 1 2 4 0 1 10 0 10 1 10 2 10 3 10 4 10 10 10 10 3 10 .2 Gr-1 G C -0.2 IMLEC 90 Macrophage Ctrl Pro-B 80 cKO 0 Dendritic cell 70 Pre-B Granulocyte 60 Naïve B *** Comp.1 50 *** CD4 T Neutrophil 0.2 % of BM 40 CD8 T Eosinophil 30 * Treg Basophil 20 0.4 NK Erythrocyte 10 0 CD11b + CD11b + CD11b - CD11b - -0.2 Gr-1- Gr-1+ Gr-1+ Gr-1- -0.1 0 C om 0.1 p.2 0.2 0.3 Figure 1—figure supplement 1

B Ctrl cKO A WT Ctrl cKO 324 bp Il-2 Leg Cre 100 bp Cre Bones

228 bp F1-R1 141 bp Spleen 1042 bp floxed allele F1-Del 868 bp WT allele 204 bp deleted allele Thymus

Gapdh 200 bp D Splenocytes C Thymocytes 700 *** 120 600 )

-/- ) Rptor (cKO) +/+ 6 100

Rptor (Ctrl) 6 Overall Splenic Architecture 500 Overall Splenic Architecture 80 400 60 300 *** WP 200 40 Cells Cells No. ( X10 100 Cells No.( X10 20 WP 0 0 RP Ctrl cKO Ctrl cKO RP E BM Cells Weight 25 Ctrl -/- Rptor -/- Splenic White Pulp Rptor Splenic Red Pulp 90 Ctrl cKO *** 80 cKO 20 70 per ) leg 6 60 *** 15 50 ** *** 40 10 30

Spleen / (Body mg/g ) 5 20 Cells No. Cells ( X10 10 0 0 day 7 day 30 day 7 day 30 Figure 1—figure supplement 2

A B Ctrl 4 cKO 10 4

12.4 3 10 3 71.7 26.6 Ctrl 10 2 Pro B Pre B 4.58 2 BM % of BM 10 1 Immat. B Mat. B 1 ** 0 24.1 67.9 19.8 10 ** *** ** 10 4 0 ProB PreB Immat B Mat B 3 0.42 10 14.7 85.3 C cKO 10 2 0.19 Ctrl BM cKO BM BM 10 4 10 4 10 1 I II 0.99 10 3 28.6 10 3 10 0 33.3 61.6 2.05 IgM IgM IgM CD127 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 2 2 0.36 10 0.77 III 10 0.64 BP-1 B220 BP-1 IgD 1.03 10 1 10 1 IV 4.42 8.09 0 0

CD71 10 10 E 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Ter119 10 4 1000 DP 100 DP 80 DN 800 10 3 IMLEC 600 60 Ctrl 10 2 BM 40 D 400 F *** 10 1 FSC-A 200 20 1.2 *** SSC-A 0 DN 0 10 35 0 1 *** Ctrl cKO FSC-A *** 30 1000 10 4 100 0.8 25 80 800 10 3 0.6 20 cKO 600 60 10 2 % of BM 15 BM 400 IMLEC 40 0.4

10 1

20 Relative Mean Values 10 200 0.2 0 *** 10 0 0 5 % Max % Max CD11b SSC-A 0 200 400 600 800 1000 0 200 400 600 800 1000 10 0 10 1 10 2 10 3 10 4 ** *** 0 Gr-1 SSC-A 0 FSC-A DP DN IMLEC I II III IV Figure 1—figure supplement 3

A B

10 5 0.97 36.4 7.01 16.9

10 4 160 Ctrl Ctrl 3 cKO Blood 10 140 10 2 120 0 40.6 66.8 9.30

100

*** 5 *** 10 0.19 62.1 7.03 14.8 80 10 4

CBC ( CBC % of Ctrl ) 60 *** *** cKO *** *** 10 3 40 *** Blood 10 2 20 0 16.5 59.7 18.5 CD11b CD3 CD45.2 0 0 50K 100K 150K 200K 250K 010 2 10 3 10 4 10 5 010 2 10 3 10 4 10 5 WBC NE LY MO RBC Hb PLT FSC B220 Gr-1

C D

7 Ctrl Ctrl 70 ** cKO 6 cKO 60 5 50 4

Leukocytes 40 ** + 3 30 *** ** Cells Cells No.( K/ul ) 2 *** 20 *** % of CD45.2 1 *** *** ** 10 ** ** 0 0 - - + + + + + + + + 1 + + + + 3 1 -1 3 4 8 -1 1 -1 D 4 8 D D D + Gr- Gr- - Gr D D + Gr Gr- - Gr B220 C C C + B220 C C C + + + + + 3 3 3 3 11b 11b D D 11b 11b 11b D D 11b D D D C C D D D C C C C C C C C Figure 1—figure supplement 4

A B

10 4 Lin- LT-HSC ST-HSC 0.0048 0.0019 10 3 0.45 Ctrl Ctrl 2.61 *** *** Ctrl 2 10 LSK cKO 16 cKO BM 0.35 *** 10 1 0.027 MPP ***

0.012 leg) per 12 0 10 0.25 4 10 4

% of BM of % 8 0.0068 0.0297 10 3 0.15 cKO 5.24 ** ** 2 4 BM 10 0.05 10 1 0.229

0 No.(X10 Cells 0 0.159 Sca-1 c-Kit 10 0 CD150 LSK MPP ST-HSC LT-HSC LSK MPP ST-HSC LT-HSC 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Lin c-Kit CD48

C D 7 - - + 0.20 Ctrl Gated on Lin Sca-1 c-Kit population Ctrl * 6 cKO Ctrl BM cKO BM 0.16 cKO 10 4 leg) per 5 4

10 3 0.12 *** 4

2 GMP 3 10 0.041 0.098 % of BM of % 0.08 0.043 0.053 CMP 2 10 1 0.082 0.135 MEP 0.04 10 0 1 Cells No. (X 10 (X No. Cells CD16/32 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 CD34 0 CMP GMP MEP CMP GMP MEP Figure 1—figure supplement 5

B C A 100 10 5 2.28 10 5 6000 2.31 63.6 80 10 4 63.4 10 4 4000 60 10 3 3 5.29 Ctrl ( Rptor F/F ) 10 Ctrl Phenotypes analyzed Ctrl 40 2000 10 2 Tamoxifen on day 21 10 2 20 0 0 7.37 26.7 0 0 100 Sex-matched littermates, 600 10 5 40.8 39.2 10 5 28.3 6-8 weeks old 80 0 5 21 75.4 10 4 10 4 400 60 Days after Tamoxifen 10 3 cKO 10 3 62.1 cKO 40 200 2 cKO ( Rptor F/F , CreER ) 10 2 20 10 0 CD11b

Count 0 6.13 of Max % CD11b 13.9 2 3 4 5 0 0 CD45.2 2 3 4 5 0 10 10 10 10 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 10 10 10 PD-L1 CD45.2 Gr-1 PD-L1 Lin

DE F

100 10 5 1.35 15000 10 5 1.23 67.8 80 10 4 1X10 6 CD45.2 66.4 10 4 Ctrl ( Rptor F/F ) 10000 60 CD45.2 10 3 10 3 Ctrl 1.86 WBM Ctrl 40 5000 10 2 2 Donor Donor 10 20 0 0 5.33 25.7 0 CD45.2 0 cKO (Rptor F/F , CreER ) 100 10 5 10 5 58.8 400 69.7 20.3 80 10 4 10 4 300 83.5 60 10 3 cKO 10 3 cKO 200 40 86.6 Tamoxifen Donor 2 CD45.1 WT Donor 100 10 2 20 10 0 CD11b

Count 0 2.45 of Max %

CD11b 7.55

0 0 CD45.2 2 3 4 5 Analyses 2 3 4 5 0 10 10 10 10 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 10 10 10 6 wks 1 wk 2 wks Lin PD-L1 BMT CD45.2 Gr-1 PD-L1 e r e r A e g o e g o Figure 2 yt a s B yt a s c h r c h r o p B u o p B u l o C c l o C c u r E e e u r E e e n c L ïv r n c L ïv r ra a a P ra a a P G M IM N B G M IM N B 2610305D13Rik 3110043O21Rik Acer3 Adrbk2 109 Anxa9 Aoah Arhgap31 Atf3 Atp13a2 Bcl2a1b Bcl2l1 C030046E11Rik Chst14 177 Cxcl16 Dram2 Firre Fzd7 -2 Gabarapl1 Galnt6

-2 RowZ-Score

RowZ-Score Gm6194 -1 48 -1 Gm6377 Gna12 Gns 0 0 Hnrnpll Hyi 1 Itga5 1 Lacc1 2 Lamp1 2 Lrp1 Mink1 Mir7091 Mitf 288 Naga Nav2 Osgin1 Plin2 Prkch Rab20 Rgag4 Sash1 Sbf2 Slc23a2 Slc36a4 Smyd2 Snx24 88 Tspan4 Vasn Zdhhc1

C Igh 113,258,768 116,009,954 Chr 12: JH JLκ JLλ 113,500 kb D E 113,500 kb 114,000 kb 114,500 kb 115,000 kb 115,500 kb 116,000 kb 600 Macrophage 2500 IMLEC IMLEC 0 0 1.4 Ctrl-WBM IMLEC Ctrl WBM Ctrl 2500 600 WBM Ctrl WBM Ctrl IMLEC IMLEC 0 0 1.2 1500 bp 9674 1000 bp Pro-B 2500 600 1.0 0 0 0.8 26657 500 bp Pre-B 2500 600 0.6 *** 0 0 10212 Relative Level 0.4 300 bp Naïve B 2500 600 0.2 *** 0 0 100 bp 15607 0 Activated B Rag1 Rag2 2500 600 β-actin

0 0 RPKM Figure 2—figure supplement 1

E A B IMLEC vs Macrophage IMLEC vs B Precursor

2.5 2.5 -0.20 IMLEC 66 81 134 728 Macrophage 2.0 2.0 -0.10 Pre-B 1.5 1.5 0 (P Value)

(P Value) Pro-B

2

1.0 . 1.0 10 10 + p 0.10 CD4 DC

m + 0.5 0.5 o CD8 DC - Log - Log C 0.20 0 0 CDP 0.30 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 DN DC

Log 2 (fold change) Log 2 (fold change) 0.40 -0.34 pDC Pre-DC 0 -0.30 .20 0 -0.26 . 1 -0. p Co 20 om C m -0 C D p. .40 -0.22 3 -0 IMLEC vs Naïve B IMLEC vs Granulocyte F .60 6 6 486 488 484 333 5 5

4 4 (P Value) (P Value) 3 3 10 10 2 2 - Log - Log 1 1

0 0 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 CD4 CDP Macrophage 2Macrophage IMLEC 3 Pre-B Pro-B CD8 DN DCDN pDC Pre-DC Macrophage 1Macrophage IMLEC 1 IMLEC 2 Log 2 (fold change) Log 2 (fold change) + + DC DC Figure 2—figure supplement 2 A 67,555,636 Igk 70,726,754

68,000 kb mm10 Chr 6 70,500 kb 2000 1Mb Macrophage 0 2000 IMLEC 0 5065 Pro-B 2000

0 5008 Pre-B 2000

0 9214 Naïve B 2000

0 10552 Activated B 2000

0

B 19,026,858 Igl 19,260,844

19,050 kb 100 kb mm10 Chr 16 19,250 kb 600 Macrophage 0 600 IMLEC 0 3000 Pro-B 0 3000 Pre-B 0 5862 3000 Naïve B 0 3000 Activated B 0 Figure 3 Mesenteric Lymph Nodes A D G 100 Isotype 80 10 5 control 0.638 60 - + 10 4 40 Surface Lin CD11b 10 3 20 marker Ctrl BM cKO BM Ctrl 26.2 0 10 2 5 5 c-Kit Sca-1 10 9.20 10 79.6 0

100 10 4 10 4 80 10 3 10 3 10 5 60 7.42

4 40 2 10 10 10 2 20 0 0 cKO 10 3 2 3 4 5 0 0 10 10 10 10 0 10 2 10 3 10 4 10 5 CD11c 0 1 2 3 4 0 1 2 3 4 88.4 10 10 10 10 10 10 10 10 10 10 10 0 10 1 10 2 10 3 10 4 10 2 % of Max PD-L1 B220 CD3 NK1.1 0 CD11b B CD45.2 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Lin PD-L1 F4/80 CD115 PD-L1 E WT Lin - CD11b + leukocytes 100 H Cervical / Inguinal high - 80 Spleen BM Blood CD11b Gr-1 Lymph Nodes 60 Peritoneal 10 5 0.25 ±0.06 0.07 ±0.02 0.23 ±0.06 40 Macrophage 10 4 20 10 5 0.32 3 0 10 10 4 2 100 10 0 10 3 80 Ctrl 10.4 + - 10 2 CD11b Gr-1 60 mLN c/i LN Lung 0 Ctrl BM 40 10 5 0.10 ± 0.03 0.18 ± 0.04 0.12 ± 0.03

20 10 4

5 0 10 10 3 3.76 100 10 4 10 2 80 cKO CD11c 0 + - 10 3 CD11b Gr-1 60 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 86.6 10 2 cKO BM 40 PD-L1

0 CD11b

20 CD45.2 2 3 4 5 2 3 4 5

% of Max 0 10 10 10 10 0 10 10 10 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Lin PD-L1 Lung I C F

10 5 1.00 14 10 4 ** 1.61 3.42 10 4 12 Ctrl 10 3 100 *** 10 3 Ctrl cKO Ctrl 10 2 27.8 2 10

10 Cells

1 BM 0 + 10 10 8 0 10 10 5 1.14 10 4 6 54.2 86.7 1 10 4 % of BM 10 3 ** cKO 10 3 4 cKO 2 10 0.1 44.9 10 2 BM % of CD45.2 10 1 0 2 *

0.01 CD45.2 0 10 2 10 3 10 4 10 5 CD11b 0 10 2 10 3 10 4 10 5 0 CD45.2 10 CD11b 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 Ctrl cKO Lin PD-L1 Lin PD-L1 IMLEC IMLEC Lung mLN c/i LN (B220 /CD3 /Ter119 /Gr-1 /NK1.1 /F4/80 /CD115) Figure 4 A C D WT IMLEC cKO IMLEC 70 Igh 60 * Granulocyte-3 Granulocyte-1 Macrophage-1 Macrophage-2 IMLEC-1 IMLEC-2 IMLEC-3 B-1 Naïve B-2 Naïve B-3 Naïve B-4 Naïve Pre-B Pro-B Granulocyte-2 Cebpa 50 Cebpb Nupr1 Rai14 40 Rxra Abt1 30 Arid3a Cited2 Dach1 Fosl2 20 Foxm1

Relative Relative Level Gfi1 10 Hmgb2 Hmgn2 B Lmo4 1.2 0 Mapk3 FSC-A Mxi1 WT-IMLEC Macro. Nfe2 1 SSC-A Nfil3 Pbx1 Ppp1r12b Rogdi 0.8 Tfdp1 400 Igl Tob1 * Ugp2 0.6 350 Zfp277 Atf3 Mitf 0.4 300 Zdhhc1 Atm 250 Bach2 0.2 Bcl11a

Relative Relative Mean Value Ebf1 200 Egr3 Elk4 0 Ets1 150 Foxo1 Granul. B T IMLEC IMLEC Foxp1 100 Hdac10 cKO Relative Level Hes1 WT Hivep2 50 ** Lbh Mef2c 0 Mllt6 Nfkb2 + Lin WT-IMLEC Macro. Pax5 IMLEC Pou2f1 80K Pou2f2 150 G F Ppp1r13b Ppp1r16b 60K Rel 86.3 100 11.6 100 Lin + Rfx5 Ctrl 40K Spib Srebf2 BM 50 IMLEC Stat4 20K 80 Zfp36l1 Cdkn2c 0 0 Mcm3 60 Mcm5 Mcm7 250 Polr3h 400 Cells) % Trip13

200 + 40 Uhrf1 300 cKO 150 70.8 7.98 200 20 *** Row Z-Score BM 100 100 (Ki-67 *** 50 0 0 0

Count 2 3 4 5 2 3 4 5 -2 -1 0 1 2 010 10 10 10 010 10 10 10 Ctrl cKO E Ki-67 120 Ctrl-WBM H Lin + IMLEC ** I ** cKO-IMLEC 10 5 1.36 1.72 4.07 10.6 100 WT-IMLEC 10 4 Ctrl Lin + 10 3 *** 80 BM *** 10 2 30 IMLEC 0 94.4 2.50 66.1 19.3 60

10 5 20 1.173.22 0.32 0.98 Cells) % 40 + Relative Relative Level 10 4 cKO *** ** 3 10 BM 10 20 * 10 2 ** 0 91.1 4.51 75.2 23.5 7-AAD 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 0 Annexin V Annexin V ( Ctrl cKO Mitf Atf3 Zdhhc1 Figure 4—figure supplement 1

A Isotype Control B C Surface Marker

cKO Ctrl 100 T cell 80 IMLEC Granulocyte B cell 200 b 60 H2D 80 1500 100 40 15000 150 Ctrl 60 66.8 3.47 20 10000 51.2 1000 7.70 100 80 cKO 0 Ctrl 40 500 50 20 5000 60

0 0 Cells) % 100 0 0 + 40 80 300 20000 1500 b 60 300

(BrdU 20 H2K 15000 40 1000 67.9 200 5.07 200 78.7 3.90 20 cKO 10000 0 0 5000 500 100 100 IMLEC Granulocyte B cell T cell Count 0 0 0 0 4 5 100 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 10 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 I-A/I-E 80 BrdU MHC-II 60 40 20 0

100 D E 80 Ly6C 60 40 IMLEC Granulocyte B cell T cell 20 5 1.50 5.30 0.44 11.3 0.126 2.04 0 10 1.14 4.39 30 10 4 Ctrl 10 3 25 100 Ctrl cKO 2 10 20 80 80.8 13.6 84.4 8.85 79.8 8.45 90.3 7.51 0 Cells % )

CD14 60 + 15 40 10 5 1.83 1.02 1.01 6.73 0.88 14.2 0.19 2.57 10 20 4 0 10 5 10 3 cKO (Annexin V 0 100 10 2 IMLEC Granulocyte B cell T cell

80 7-AAD 0 81.5 15.7 81.2 11.1 81.4 3.47 90.2 6.95 CD68 60 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 40 Annexin V 20

% of Max % 0 010 2 10 3 10 4 10 5 010 2 10 3 10 4 10 5 FITC Figure 5 6 A CD45.2 1X10 E CD45.2 Sorted BM F/F F/F pIpC Ctrl ( Rptor ) or CD45.2 Ctrl ( Rptor ) LSK/CMP/CLP cKO ( Rptor F/F , Mx1-Cre ) WBM Co-culturing with Analyses CD45.1 WT or pIpC 2 weeks OP9 cells cKO Day 1 Day 10 Analyses (Rptor F/F , Mx1-Cre ) 6 wks 2 wks 4 wks BMT F CD45.1 WT 0.5X10 6 LSK CMP CLP CD45.1 WBM

5 250K 5 250K 10 27.6 10 55.5 5 250K B CD45.2 + CD45.1 + 42.2 20.4 14.8 74.4 10 0.01 0.00 0.00 200K 200K 10 4 4 200K 10 10 4 150K 150K 150K 10 3 3 100K 10 10 3 10 4 10 4 Ctrl 100K 100K 10 2 50K 10 2 50K 0.89 47.8 1.45 66.0 10 2 50K 3 22.7 3 10 10 0 37.0 0.41 0 9.23 1.57 0.00 0 0 0 99.9 0

10 2 10 2 250K 250K 10 5 5 250K Ctrl+CD45.1 98.5 10 98.8 10 5 0.06 94.7 94.4 1.98 200K 61.1 30.4 200K 14.1 200K 10 1 10 1 10 4 4 10 150K 10 4 cKO 150K 150K 3 3 10 10 3 10 0 46.1 10 0 49.4 1.87 27.2 5.39 100K 100K 10 100K 10 2 50K 2 50K 4 4 10 10 2 50K 10 10 0 SSC SSC SSC CD11b 83.8 2.04 3.46 0.19 CD11b 0 7.46 1.07 0 CD11b 0 56.8 4.65 2.67 59.6 0 0 2 3 4 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 10 10 10 10 3 40.7 10 3 Gr-1 PD-L1 Gr-1 PD-L1 Gr-1 PD-L1 cKO+CD45.1 10 2 10 2

10 1 10 1

CD45.1 21.8 CD11b 36.1 2.50 32.9 4.82 10 0 10 0 G 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD45.2 CD45.2 Gr-1 Ctrl ( Rptor F/F ) Sorted BM pIpC LSK/CMP CD45.1 WT or Analyses C Ctrl KO cKO Day 0 Day 1-5 Day 7 WBM DP DN (Rptor F/F , Mx1-Cre ) Rptor Floxed 1042 bp H 10 5 22.0 73.2 250K 3.40 200K 10 4 204 bp Ctrl 76.4 150K Rptor Deleted 10 3 LSK 100K 10 2 50K 0 4.11 0.672 0 Lyz2-Cre WT 350 bp 10 5 11.3 72.9 250K 59.3 200K 700 bp cKO 10 4 71.5 Lyz2-Cre MT 150K 10 3 LSK 100K 10 2 50K 0 9.98 5.80 β-actin 230 bp 0 10 5 1.40 75.9 250K 18.2 Ctrl 10 4 3.27 200K 150K D F/F F/F 10 3 Ctrl (Rptor , KO ( Rptor , CMP 100K -/- +/+ 10 2 50K Lyz2-Cre ) Lyz2-Cre ) 0 10.8 11.9 0 10 4

0.87 25.7 0.86 25.2 5 10 11.6 19.4 250K 10 3 50.9 200K 10 4 cKO 0.76 150K 2 10 3 10 CMP 100K 10 2 50K 10 1 0 54.8 14.3 SSC CD45.1 CD11b 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 70.7 2.76 71.4 2.54

CD11b 10 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD45.2 Gr-1 PD-L1 Gr-1 Figure 6 A E 5 10 1.41 3.5 1.4 10 4 *** Ctrl-LSK 1.4 Ctrl-CMP Myb Ctrl 3.0 *** cKO-LSK cKO-CMP 10 3 3.89 1.2 BM 1.2 10 2 2.5 + 1.0 1.0 10 0 2.0 0.8 0.8 1.5 *** ** 10 5 0.26 0.6 0.6 10 4 1.0 0.4 Myb cKO

0.4 3 % of CD45.2 Relative Relative Level Relative Relative Level 10 35.2 0.5

0.2 2 0.2 10 0.0 *** *** 0 0 0 CD45.2 10 CD11b 0 10 2 10 3 10 4 10 5 10 0 10 2 10 3 10 4 5 Myb Ctrl Myb cKO Myb Rptor Myb Rptor 10 10 Lin PD-L1

LSK CMP F CD45.1 B CD45.2 Recipients 100 IgG-Ctrl Ctrl 2X 10 6 F/F -/- Tamoxifen 80 IgG-cKO (Myb , Rag2 ) WBM 60 c-Myb-Ctrl or Analyses 40 c-Myb-cKO 6 wks Day 1-5 Day 8 20 cKO BMT 0

% of Max of Max % F/F -/- 010 2 10 3 10 4 10 5 010 2 10 3 10 4 10 5 (Myb , Rag2 , CreER ) c-Myb G I WT Ctrl cKO 3 Myb Deleted 267 bp No.=13 C Ctrl ( Myb F/F ) Phenotypes Myb Floxed 2 pIpC analyzed on day 7 194 bp Wild type 161 bp 1 Sex-matched 0 (cKO/Ctrl)

littermates, 6-8 weeks 2 2wks Il2 324 bp 1 2 3 4 old 0 7 -1 Days after pIpC Log Cre 100 bp -2 cKO ( Myb F/F , Mx1-Cre ) No.=0 -3 β-actin 230 bp -Log 10 (P Value) H D 100 12k 10 5 2.58 62.1 10 5 250K 80 7.23 9k 10 4 200K 67.1 4 60 10 150K Myb Ctrl 6k 3 Myb Ctrl 10 40 10 3 8.04 Chimera 100K 3k 10 2 10 2 20 50K 10 0 31.3 4.06 0 71.0 0 0

10 5 21.4 21.8 100 10 5 250K 10k 79.6 80 200K 4 8k 10 Myb cKO 10 4 Myb cKO 9.65 60 150K 6k 10 3 10 3 16.6 40 Chimera 100K 4k 10 2 10 2 50K 20 0 50.5

Count 2k CD45.1 SSC 0 CD11b CD11b 10 43.3 13.5 2 3 4 5 0 % of Max 0 10 10 10 10 2 3 4 5 2 3 4 5 0 0 10 10 10 10 0 10 10 10 10 0 2 3 4 5 10 0 10 2 10 3 10 4 10 5 0 2 3 4 5 10 10 10 10 10 10 10 10 10 10 CD45.2 Gr-1 PD-L1 CD45.2 Gr-1 PD-L1 Figure 6—figure supplement 1

A B C

10 5 1.76

10 4 Myb F/+ 10 3 1.65 Myb F/+ Myb Deleted 267 bp pIpC Analyses 10 2 Myb Floxed 194 bp 0 Wild type 161 bp Sex-matched littermates, 6-8 weeks old Il2 324 bp 10 5 2.33 2 wks 0 7 10 4 Days after pIpC Myb F/+ , Mx1-Cre Cre 100 bp Myb F/+ , Mx1-Cre 10 3 1.57 10 2 β-actin 230 bp 0 CD45.2 0 10 2 10 3 10 4 10 5 CD11b 0 10 2 10 3 10 4 10 5 D pWPI pWPI-Myb Lin Virus Virus PD-L1

10 5 27.9 0.12 27.1 0.09

4 Rabbit 10 F 3 Sorted BM LSK Control 10 pIpC cKO ( Lenti-virus infection) mAb 10 2 (Rptor F/F , Mx1-Cre ) 0 71.8 0.16 72.7 0.14 Co-culturing with Analyses 2 weeks OP9 cells 10 5 27.6 0.58 8.46 18.1 Day 1 Day 12

Rabbit 10 4 anti-c-Myb G 10 3 10 5 mAb 23.2 16.7 10 2 1.01 4 81.7 0 70.8 1.02 70.9 2.69 10

GFP Control pWPI 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 10 3 virus infection AF568 anti-Rabbit IgG 10 2 0 51.7 8.4 E 10 5 0 0 0.244 0 c-Myb- 10 4 expressing M.W.(kD) 3 pWPI-Myb 10 80 c-Myb virus infection 10 2 99.2 0.83 GFP 0 SSC CD11b 2 3 4 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 10 10 10 40 Actin CD45.2 Gr-1 PD-L1 Figure 7 A B Ctrl-WBM Tlr13 Tlr3 Tlr8 Tlr7 Tlr11 Tlr12 Tlr1 Tlr9 Tlr4 Tlr2 Tlr6 Tlr5 cKO-IMLEC DC WT-IMLEC Erythrocyte 60 Basophil ** * Eosinophil 40 ** * ** Granulocyte **

-4 Z-ScoreRow 20 Neutrophil -2 20 *** Macrophage 0 IMLEC 15 2 * Naive B 4 ** * Pre B/Pro B 10 * * * Relative Expression * NK 5 CD4 T CD8 T 0 Treg 1 3 4 6 8 lr lr2 r lr lr lr lr9 T T Tl T Tlr5 T Tlr7 T T C TNF-α Ctrl-WBM 20000 *** 15000 *** cKO-WBM 10000 E cKO BM WT BM *** 10 4 10 4 7000 49.6 0.72 *** 10 3 10 3 5000 *** ** (pg/ml) 10 2 10 2 α 3000 *** 10 1 10 1 TNF- 1000 10 0 10 0 CD11b CD11b 500 *** 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Lin Lin 250 * 0 - + - + T 6 cKO Lin CD11b WT Lin CD11b am PS S L1 P pIpC L 182 HKLM FS Medium FLA- Medium LPS Medium LPS ODN 10 4 10 4 D ssRNA-DR 0.01 0.04 0.010.23 0.24 1.74 0.02 0.19 10 3 10 3 MCP-1 10 2 10 2 4000 Ctrl-WBM 10 1 10 1 cKO-WBM 0.33 31.6 68.1 3000 *** 10 0 99.6 0.32 99.4 10 0 34.6 63.5

*** *** IgG 2000 *** 10 4 10 4 0.01 2.17 0.11 58.5 0.10 1.42 2.09 13.8 10 3 10 3 1000 1000 10 2 10 2 α 750 10 1 10 1 *** MCP-1 (pg/ml) MCP-1 ** 10 0 0.2297.6 0.21 41.2 10 0 32.066.5 28.5 55.7 500 *** TNF- 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 *** 250 * PD-L1 0 m Pam pIpC LPS HKLM FSL1 A-DR Mediu FLA-ST N sR s ODN1826 Figure 7—figure supplement 1

A B Pre-B/Pro-B Naïve B T CD4 Treg IMLEC NK DC Basophil Eosinophil Neutrophil Macrophage T CD8 Erythrocyte Granulocyte

Nod1 16 Nod2 14 Ctrl-WBM Nlrc3 IMLEC 12 Nlrc4 *

Nlrc5 10 * * Nlrp1a 8 **

Nlrp2 *** 6 ** Nlrp3 * 4 Relative Level Relative Ddx58 ** 2 Ifih1

Dhx58 0

Aim2 Nod1 Nod2 Nlrc3 Nlrc4 Nlrc5 Nlrp2 Nlrp3 Ifih1 Aim2 Tlr11 Tlr12 Tlr13 Nlrp1a Ddx58 Dhx58 Ifi204 Ifi204 NLR RLR ALR TLR Row Z-Score

-4 -2 0 2 4 Figure 8 D Liver Leukocytes E Liver CD11b + Gr-1- Cells Ctrl cKO Ctrl cKO A 10 4 10 4 100 Ctrl 4.43 19.5 40.3 15.8 400 80 cKO *** 10 3 10 3 60

10 2 10 2 300 40 10 1 10 1 20

58.1 18.0 31.5 12.5 0 200 * 10 0 10 0 CD11b * 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 F4/80 100 Gr-1 Cytokine(pg/ml ) 100 *** 80

60 0 F 0 α γ 1 40 7 - 10 6 100 20 IFN- IL- IL- TNF- MCP 0 IL-12p of Max % P<0.0001 0 1 2 3 4 0 1 2 3 4 B 80 10 10 10 10 10 10 10 10 10 10 PD-L1 100 60 Ctrl (n=14) P<0.0001 80 40 cKO (n=11) I 1200

Percent survival Percent ** 60 20 900 Ctrl (n=32) 0 40 0 12 24 36 48 60 72

cKO (n=31) pg/ml( ) Percent survival Percent 600 20 Hours after LPS α

0 TNF- 300 0 10 20 30 40 50 Days after first pIpC injection GH 6 0 6 *** 10 10 Ctrl *** Ctrl cKO Ctrl C 10 5 cKO Ctrl liver cKO liver 10 5 cKO J 5000 * 10 4 10 4 4000 10X 10 3 10 3 ( pg/ml) 3000

α ***

2 2 10 MCP-1 ( pg/ml) 10 2000

TNF- * MCP-1 ( pg/mlMCP-1 ) 40X 10 1 10 1 1000 0 10 0 10 0 0h 6h 0h 6h Ctrl cKO Figure 9 A CD45.2 Ctrl (Rptor F/F ) CD45.1 WT 1st pIpC 2nd pIpC 12 Weeks or Analyses 0 10 31 CD45.2 cKO 5X10 6 WBM BMT Days after 1 st pIpC (Rptor F/F , Mx1-Cre ) cells B C 10X 20X 40X 100

80 P=0.0002 Ctrl 60 Donor

40 Ctrl Donor cKO Donor Percent survival Percent 20 cKO 0 Donor 0 10 20 30 40 50 Days after first pIpC injection

D CD45.2 + BM Cells EFCD45.2 + Spleen Cells CD45.2 + Liver MNCs Ctrl Donor cKO Donor Ctrl Donor cKO Donor Ctrl Donor cKO Donor 10 4 10 4 10 4 4.15 58.2 72.0 14.1 2.19 2.45 35.9 19.2 7.75 19.1 42.5 22.1

10 3 10 3 10 3

10 2 10 2 10 2

10 1 10 1 10 1 35.3 2.34 10.9 3.05 91.8 3.53 37.5 7.42 68.1 5.13 32.6 2.78 10 0 0 0 CD11b 10 10 CD11b 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD11b 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Gr-1 Gr-1 Gr-1

100 100 100

80 80 80

60 60 60 cells - 40 40 40

20 20 20 Gr-1

+ + 0 0 0 F4/80 F4/80 F4/80

100 100 100

80 80 80

60 60 60

40 40 40 Gated on CD11b on Gated 20 20 20

0 % Max % Max

% Max % Max 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 10 4 10 10 10 10 10 10 10 10 10 10 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 10 10 10 10 PD-L1 PD-L1 PD-L1