bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1

1 Dynamic expression of Id3 defines the stepwise differentiation of tissue-resident 2 regulatory T cells 3 Jenna M. Sullivan1,2, Barbara Höllbacher1 and Daniel J. Campbell1,2* 4 5 1Immunology Program, Benaroya Research Institute, Seattle, WA 98101 6 2Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195 7 8 *Corresponding Author: Daniel J. Campbell, Benaroya Research Institute, 1201 Ninth Avenue, 9 Seattle, WA 98101-2795. E-mail address: [email protected] 10 11 12 13 14 Running Title: Dynamic Id3 expression in development of tissue Tregs 15 16 17 Funding: This work was supported by grants to DJC from the National Institutes of Health 18 (AI067750, AI124693). JMS was supported by NIH-NIAID T32 (AI106677, UW Immunology). 19 20 bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2

21 Abstract

+ 22 Foxp3 regulatory T (TR) cells are phenotypically and functionally diverse, and broadly

23 distributed in lymphoid and non-lymphoid tissues. However, the pathways guiding the

24 differentiation of tissue-resident TR populations have not been well defined. By regulating E-

+ 25 protein function, Id3 controls the differentiation of CD8 effector T cells and is essential for TR

26 maintenance and function. We show that dynamic expression of Id3 helps define three distinct

+ hi lo + lo hi - 27 TR populations, Id3 CD62L CD44 central (c)TR, Id3 CD62L CD44 effector (e)TR and Id3 eTR.

28 Adoptive transfer experiments and transcriptome analyses support a stepwise model of

+ + - - 29 differentiation from Id3 cTR to Id3 eTR to Id3 eTR. Furthermore, Id3 eTR have high expression

30 of functional inhibitory markers and a transcriptional signature of tissue-resident TR. Accordingly,

- 31 Id3 eTR are highly enriched in non-lymphoid organs, but virtually absent from blood and lymph.

32 Thus, we propose that tissue-resident TR develop in a multi-step process associated with Id3

33 downregulation. bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3

34 Introduction

35 Several recent studies have highlighted the phenotypic and functional heterogeneity of

36 regulatory T (TR) cells during both steady state and inflammation (1-4). We and others have

37 shown that at steady state in lymphoid organs TR can be broadly divided by expression of CD44

38 and CD62L into distinct subsets which differ in their localization, dependence on IL-2, and

hi lo 39 extent of PI3K signaling (2, 5, 6). Moreover, CD44 CD62L effector (e)TR display diverse

40 expression of transcription factors and chemokine receptors that promote their migration to

41 inflamed tissues and their response to different types of inflammatory signals (1, 7). Accordingly,

42 TR found in nonlymphoid tissues have a distinct molecular profile that includes high expression

43 of Gata3 and ST2 (the IL-33R), and are functionally equipped to suppress inflammation at

44 barrier sites (8, 9). Although these data highlight the anatomical, functional and molecular

45 diversity of TR, the pathways by which these TR populations differentiate have not been

46 completely defined.

47 The inhibitors of DNA binding (Id) proteins have been extensively studied in lymphocyte

48 development (10, 11). Studies of CD8+ effector T cells revealed that Id2 and Id3 are powerful

49 transcriptional regulators of differentiation that are dynamically regulated during T cell activation

50 and effector/memory T cell differentiation (12, 13). Through their regulation of E protein function,

51 Id2 and Id3 help to control expression of genes essential for CD8+ effector cell differentiation

52 and survival such as Tcf7, Tbx21, Bcl2 and Klrg1 (14, 15). Although less well studied, Id

53 proteins have been shown to have essential roles in CD4+ T cell function. For instance, Id2 and

54 Id3 are essential for TR maintenance and function, with TR lacking both Id2 and Id3 having

55 impaired proliferation and survival (16). In TR, Id3 helps to stabilize Foxp3 through restriction of

56 the E protein E47 and its downstream targets Spi-B and SOCS3 (17). However, Id3 expression

+ - 57 is not uniform in TR, and distinct populations of Id3 and Id3 have been identified (16, 18). In

58 this study, we show that Id3 is dynamically regulated in TR, and that progressive loss of Id3 bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4

59 correlates with the stepwise differentiation of a highly-functional TR population localized primarily

60 in non-lymphoid tissues. bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5

61 Materials and Methods

62 Mice

63 C57BL/6, RAG1-deficient and Foxp3-mRFP mice were purchased from The Jackson

64 Laboratory. Id3-GFP mice were a gift from Ananda Goldrath (UCSD, La Jolla, California) and

65 have been previously described (12, 14). Mice were bred and housed under the approval of the

66 Institutional Animal Care and Use Committee of the Benaroya Research Institute.

67

68 Cell Isolation

69 Unless noted below, single cell suspensions isolated from tissues using manual disruption. PEC

70 isolated by injecting sterile PBS into peritoneal cavity of euthanized mice, gentle disruption to

71 dislodge cells and collection of injected PBS. IEL and LPL were isolated from pooled large and

72 small intestine as previous described (19). Lymphocytes further purified by resuspension in 44%

73 PercollTM (GE Healthcare) layered over 67% PercollTM and spun at 2,800rpm for 20 mins. Lung

74 and fat were finely minced, digested with 0.26U/mL Liberase TM (Roche) and 10U/mL DNAse

75 (Sigma) for 1 hr at 37°C and filtered. For skin tissue, ears were processed as above with

76 0.14U/mL Liberase TM and 10U/mL DNAse. For lymph collection, mice were fed 20mL/kg ‘Half

77 and Half’ by oral gavage and sacrificed 2-3 hrs later. Lymph collected from the cisterna chyli

78 was directly stained for flow cytometry.

79

80 Flow cytometry

81 Single cells suspensions were stained with fixable Viability Dye eFluor 780 (eBiosciences) in

82 PBS for 10 min at RT. Cells were stained with directly conjugated Abs in PBS with 0.5% BCS

83 for 20 min at 4°C. Abs purchased from BioLegend: CD4 (RM4-5), TCRβ (H57-597), CD44 (IM7),

84 CD62L (MEL-14), CD25 (PC61), ICOS (C398.4A), TIGIT (1G9), CTLA4 (UC10-4B9), GITR

85 (DTA.1), CD69 (53-7.3). Abs purchased from eBiosciences: KLRG1 (2F1) and CD103 (2E7). bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 6

86 Intracellular stains were performed using a FixPerm Kit (eBiosciences). Data acquired on an

87 LSR II (BD Biosciences) and analyzed using FlowJo software (TreeStar).

88

89 In vitro assays

90 CD4+ T cells isolated from spleen and LN using CD4 microbeads (Miltenyi). 1x106 T cells

91 cultured with platebound α-CD3 (2C11) and α-CD28 (37.51) from BioXcell at 1μg/mL each for

92 48 or 66 hrs. Inhibitors purchased and used as follows: ZSTK474 (1μM, Sigma), Rapamycin

93 (10nM, Selleckchem), NFAT inhibitor (10µM, Tocoris), Mek inhibitor PD0325901 (100nM,

94 Peprotech) and Erk inhibitor FR180204 (10µM, Tocoris). In vitro TR suppression assays were

95 performed as previous described (20). Chemotaxis assay performed as previously described

96 (21).

97

98 In vivo TR transfer

99 Sorted TR isolated from spleen and LN as described for RNA-seq. 100,000 sorted cells were

100 injected retro-orbitally into RAG1-deficient hosts. Spleen, LN and blood of recipient mice

101 collected two wks later and analyzed by flow cytometry.

102

103 Statistical Analysis

104 The p values were calculated by Prism software (GraphPad) using either an unpaired Student’s

105 t test or one way ANOVA as indicated. Values less than 0.05 were considered significant.

106

107 RNA-seq

108 CD4+ T cells were isolated using CD4 microbreads (Miltenyi) from either peripheral LNs or

109 spleens of 3 littermate Id3-GFP x Foxp3-mRFP mice. Cells were sorted based on viability, CD4,

110 CD44, CD62L, Id3-GFP and Foxp3-mRFP expression on a FACs Aria II (BD Biosciences). 500

111 cells were sorted directly into SMART-Seq v4 Ultra Low Input RNA Kit (Takara) lysis buffer and bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 7

112 protocol followed to produce cDNA. Library construction was performed using a modified

113 protocol of the NexteraXT DNA sample preparation kit (Illumina). Dual-index, single-read

114 sequencing of pooled libraries was run on a HiSeq2500 sequencer (Illumina) with 58-base

115 reads and an average depth of 8.6Mio reads per library. Base-calling and demultiplexing were

116 performed automatically on BaseSpace (Illumina) to generate FASTQ files. bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 8

117 Results/Discussion

118 Id3 is dynamically expressed in TR and regulated by TCR signaling

119 To examine Id3 expression in TR we generated Id3-GFP x Foxp3-mRFP double reporter

120 mice. In agreement with previously published reports, most CD4+ T cells in spleen and lymph

+ - + 121 nodes (LNs) were Id3 (16, 18). However, we noted a subset of Id3 cells in both Foxp3 TR and

- + - 122 Foxp3 conventional CD4 T cell populations (Fig 1A). Within TR, the Id3 population fell

hi lo + 123 exclusively within the CD44 CD62L eTR compartment (5), whereas Id3 TR were found in both

lo hi 124 the CD44 CD62L central (c)TR and eTR compartments (Fig 1B). Thus, in secondary lymphoid

125 organs (SLOs) TR can be divided into three distinct subsets based on Id3, CD62L and CD44

+ + - + + 126 expression, Id3 cTR, Id3 eTR or Id3 eTR. Within the Id3 TR populations Id3 cTR had higher

+ 127 Id3 expression than Id3 eTR measured by GFP-MFI (Fig 1B), leading us to hypothesize that TR

128 may downregulate Id3 as they transition during their activation and differentiation from cTR into

129 eTR. Indeed, Id3 expression can be downregulated by TCR signaling (16), and we observed a

130 loss of Id3 expression in the TR compartment correlating with the strength of TCR stimulation

131 when cells were activated with different concentrations of platebound αCD3/28 (Sup Fig 1).

132 Furthermore, inhibiting either the MAP-kinase/Erk or PI3-kinase/mTOR signaling pathways

133 blocked Id3 downregulation in TR (Fig 1C), consistent with reports that eTR development

134 requires TCR stimulation, mTOR signaling and the PI3-kinase-dependent inactivation of the

135 transcription factor Foxo1 (5, 6, 22).

136 To more precisely define the developmental relationship between these TR subsets we

137 utilized an adaptive transfer model in which TR stimulation and expansion depends on

+ + - 138 TCR:MHCII interactions (23). For this we sorted Id3 cTR, Id3 eTR or Id3 eTR from spleen and

139 LNs of reporter mice and transferred individual TR populations into RAG1-deficient animals, and

140 evaluated the phenotype and expansion of transferred TR after 2 weeks. Importantly, we did not

141 observe any difference in the extent of Foxp3 expression between the TR populations upon their bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 9

142 recovery, which varied between ~30-80% in different experiments (not shown). Transferred Id3+

143 cTR gave rise to all three subsets, with some cells retaining Id3 and CD62L expression but the

- + 144 majority converting into Id3 eTR (Fig 1D). The bulk of Id3 eTR downregulated Id3, with no cells

- 145 regaining CD62L expression, whereas Id3 eTR did not give rise to either of the other

146 populations, indicating that these cells are likely a terminal differentiated population. Thus, Id3-

+ + 147 eTR appear to develop from Id3 cTR in a stepwise manner during activation, with Id3 eTR

148 acting as an intermediate population.

- 149 Id3 eTR express inhibitory markers and are highly suppressive

150 To explore the phenotypic and functional differences between these three subsets of TR,

151 we assessed expression of the TR-associated surface markers on each population of splenic TR.

152 In agreement with previously published data, cTR and eTR showed distinct phenotypes, with eTR

153 having lower expression of the high affinity IL-2 receptor component CD25, but higher levels of

154 the activation and functional surface markers ICOS, KLRG1, TIGIT, GITR and CTLA4 (Fig 2A)

- 155 (5, 6). Moreover, within the eTR compartment the Id3 TR had higher expression of activation and

+ 156 inhibitory molecules than their Id3 TR counterparts, but had the lowest expression of CD25. As

157 our lab previously described (5), elevated expression of ICOS and diminished expression of

- 158 CD25 suggests that Id3 eTR are less dependent on IL-2 for their homeostatic maintenance, and

159 instead likely rely on continued ICOS signaling. Additionally, increased expression of these TR

- 160 functional molecules correlated with enhanced in vitro suppressive activity of Id3 eTR compared

+ + 161 with either Id3 cTR or Id3 eTR (Fig 2B).

- 162 Transcriptional profiling highlights the stepwise differentiation of Id3 eTR

163 To identify and compare their unique transcriptional profiles, we performed RNA-seq on

+ + - 164 sorted Id3 cTR, Id3 eTR and Id3 eTR from spleen or LNs of Id3-GFP x Foxp3-mRFP reporter

165 mice. Although there was little difference between LN and spleen samples, principle component

166 analysis (PCA) showed that each of the three TR populations were transcriptionally distinct (Fig

167 3A), and accordingly we identified 1,672 significantly differentially expressed (DE) genes bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10

168 between the three TR populations (Fig 3B, Supplemental Table 1). The largest differences were

- + 169 found between Id3 eTR and Id3 cTR with 1,471 DE genes, whereas only 474 genes differed

+ - 170 between Id3 and Id3 eTR (Fig 3B). Interestingly among the DE genes we observed a reciprocal

171 increase in Id2 expression as TR lose Id3 (Fig 3C). This differential Id expression in TR is similar

+ 172 to what occurs in CD8 T cells, which upregulate Id2 and downregulate Id3 while becoming

173 activated and gaining effector function (12, 13). This suggests that as TR downregulate Id3, the

174 closely related Id2 may take over some of its functions while also driving a unique E-protein-

175 dependent signature promoting eTR development. Consistent with the stepwise differentiation

176 model we propose for these populations, examination of the 300 most DE genes across the

177 three TR subsets (based on highest F-value) showed that both up and downregulated genes

+ 178 were generally expressed in a gradient fashion, with expression in Id3 eTR falling between that

+ - 179 of Id3 cTR and Id3 eTR (Fig 3D-E). Moreover, in accordance with our prior phenotypic analysis

- 180 and their enhanced suppressive activity, Id3 eTR showed elevated expression of known TR

181 function genes, including Il10, Ctla4, Pdcd1, Tnfrsf4, Lag3 and Ebi3 (Fig 3F). To further validate

182 our RNA-seq results, we confirmed differential expression of several TR-associated genes by

183 flow cytometry (Supplemental Fig 2). Gene Ontology (GO) term enrichment analysis of genes

+ - 184 DE between Id3 eTR and Id3 eTR identified specific molecular pathways altered between these

185 closely related populations (Fig 3G). The top six enriched categories all related to cytokine and

- 186 chemokine receptor signaling, with Id3 eTR expressing high levels of receptors indicating that

187 they can tune their activity in response to key inflammatory cytokines such as IL-1, IL-18, IL-23,

- 188 and IL-25 (Fig 3H). Moreover, among the DE chemokine receptors, Id3 eTR had the highest

189 expression and subsequent responsiveness to chemokines that promote lymphocyte migration

- 190 to inflamed tissues, such as Ccr4 and Cxcr3 (Fig 3H-I) (1) . Thus, Id3 eTR have a unique

+ 191 molecular profile indicative of their development from Id3 TR precursors, their elevated

192 suppressive function and altered migratory capacity.

- 193 Id3 TR are enriched and resident in non-lymphoid tissues bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 11

- 194 In contrast to their elevated expression of inflammatory chemokine receptors, Id3 eTR

195 had the lowest expression of Ccr7 and S1pr1 which function together to promote TR

196 recirculation through SLOs (24) (Fig 3H). Additionally, expression of CD103 and CD69, which

197 together act to retain tissue-resident memory T(RM) cells in non-lymphoid sites, was strongly

- 198 enriched in Id3 eTR, suggesting these cells may be tissue-resident (Fig 4A) (25, 26). Indeed,

+ 199 utilizing published gene signatures of CD8 TRM or circulating memory T cells (27), we found

+ - + 200 that the CD8 TRM signature gene set was enriched in Id3 eTR compared to either Id3 cTR or

+ + 201 Id3 eTR, whereas the circulating memory gene set was enriched in the Id3 TR populations (Fig

202 4B). Several groups have recently identified the ST2 (IL-33R)-Gata3 axis as a key determinant

- 203 of TR residency and function in non-lymphoid tissues (8, 9, 28). Accordingly, Id3 eTR had the

204 highest expression of all positively associated tissue TR genes such as Il1rl1 (ST2), Gata3,

+ 205 Areg, Irf4 and Rora, whereas Id3 cTR had the lowest expression of these genes but high

+ 206 expression of negative regulators of tissue residence such as Tcf7, Klf2 and Lef1, and Id3 eTR

207 displayed intermediate expression for all of these genes (Fig 4C). Consistent with their TRM-like

- 208 transcriptional signature, Id3 eTR were a minority of TR in LNs and spleen, but their frequency

209 dramatically increased in nonlymphoid tissues, where they highly expressed the TRM surface

210 markers CD103 and CD69 (Fig 4D-F). Indeed, tissues such as the fat and skin contained almost

- - 211 exclusively Id3 eTR. Of particular note Id3 eTR were rarest in the lymph and blood, indicating

- 212 that Id3 eTR do not actively recirculate, but instead are retained as tissue-resident cells in non-

213 lymphoid organs.

214

215 Despite significant interest in tissue-resident TR, the mechanisms regulating their

216 differentiation and distribution are still poorly defined. Our data show that TR can be subdivided

217 based on Id3 expression and known markers of cTR and eTR into distinct populations, and

218 together our phenotypic, functional, transcriptional and transfer analyses strongly support a

219 stepwise differentiation model in which TR downregulate Id3 as they progressively gain effector bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12

220 function, tissue homing capacity and residency in non-lymphoid organs. Similarly, Li et al.

221 recently proposed a stepwise model for the differentiation of TR in adipose tissue in which

222 activation in the spleen allowed TR to migrate into the adipose tissue, where IL-33 signaling

223 drove their terminal differentiation and functional specialization (28). High expression of co-

224 stimulatory receptors such as ICOS and receptors for inflammatory cytokines would also allow

- 225 Id3 eTR to respond to inflammatory signals that enhance Foxp3-mediated transcriptional

226 repression and promote eTR differentiation and function (29), in part through activation of

227 mTORC1 signaling (22). Although the direct role of Id3 downregulation in the functional

228 differentiation of TR is still not established, our data highlight the complexity of tissue TR

229 development, and identify novel molecular pathways that are modulated during their stepwise

230 differentiation. bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 13

231 Acknowledgements:

232 We thank A. Goldrath for Id3-GFP mice. K. Arumuganathan and T. Nguyen for help with flow

233 cytometry and cell sorting. V. Gersuk and the BRI Genomics Core for running RNA-seq

234 samples. And members of the Campbell lab for helpful discussions.

235 bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14

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281 18. Rauch, K. S., M. Hils, A. J. Menner, M. Sigvardsson, S. Minguet, P. Aichele, C. Schachtrup, and K. 282 Schachtrup. 2017. Regulatory T cells characterized by low Id3 expression are highly suppressive 283 and accumulate during chronic infection. Oncotarget 8: 102835-102851. 284 19. Couter, C. J., and N. K. Surana. 2016. Isolation and Flow Cytometric Characterization of Murine 285 Small Intestinal Lymphocytes. JoVE: e54114. 286 20. Srivastava, S., M. A. Koch, M. Pepper, and D. J. Campbell. 2014. Type I interferons directly inhibit 287 regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection. J Exp 288 Med 211: 961-974. 289 21. Koch, M. A., G. Tucker-Heard, N. R. Perdue, J. R. Killebrew, K. B. Urdahl, and D. J. Campbell. 290 2009. The transcription factor T-bet controls regulatory T cell homeostasis and function during 291 type 1 inflammation. Nat Immunol 10: 595-602. 292 22. Sun, I. H., M. H. Oh, L. Zhao, C. H. Patel, M. L. Arwood, W. Xu, A. J. Tam, R. L. Blosser, J. Wen, and 293 J. D. Powell. 2018. mTOR Complex 1 Signaling Regulates the Generation and Function of Central 294 and Effector Foxp3(+) Regulatory T Cells. J Immunol. 295 23. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, and A. Rudensky. 2002. Homeostasis and 296 anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol 3: 33-41. 297 24. Lee, J. H., S. G. Kang, and C. H. Kim. 2006. FoxP3+ T Cells Undergo Conventional First Switch to 298 Lymphoid Tissue Homing Receptors in Thymus but Accelerated Second Switch to Nonlymphoid 299 Tissue Homing Receptors in Secondary Lymphoid Tissues. The Journal of Immunology 178: 301- 300 311. 301 25. Mackay, L. K., A. Rahimpour, J. Z. Ma, N. Collins, A. T. Stock, M. L. Hafon, J. Vega-Ramos, P. 302 Lauzurica, S. N. Mueller, T. Stefanovic, D. C. Tscharke, W. R. Heath, M. Inouye, F. R. Carbone, and 303 T. Gebhardt. 2013. The developmental pathway for CD103(+)CD8+ tissue-resident memory T 304 cells of skin. Nat Immunol 14: 1294-1301. 305 26. Schenkel, J. M., and D. Masopust. 2014. Tissue-resident memory T cells. Immunity 41: 886-897. 306 27. Milner, J. J., C. Toma, B. Yu, K. Zhang, K. Omilusik, A. T. Phan, D. Wang, A. J. Getzler, T. Nguyen, S. 307 Crotty, W. Wang, M. E. Pipkin, and A. W. Goldrath. 2017. Runx3 programs CD8(+) T cell 308 residency in non-lymphoid tissues and tumours. Nature 552: 253-257. 309 28. Li, C., J. R. DiSpirito, D. Zemmour, R. G. Spallanzani, W. Kuswanto, C. Benoist, and D. Mathis. 310 2018. TCR Transgenic Mice Reveal Stepwise, Multi-site Acquisition of the Distinctive Fat-Treg 311 Phenotype. Cell. 312 29. Arvey, A., J. van der Veeken, R. M. Samstein, Y. Feng, J. A. Stamatoyannopoulos, and A. Y. 313 Rudensky. 2014. Inflammation-induced repression of chromatin bound by the transcription 314 factor Foxp3 in regulatory T cells. Nat Immunol 15: 580-587.

315 316 317 318 319 320 321 322 323 324 bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16

325 Figure Legends

326 Figure 1: Id3 is dynamically expressed in TR and regulated by TCR signaling

327 A) Representative flow cytometry plots of Id3-GFP and Foxp3-RFP expression by gated splenic

328 TCRβ+ CD4+ T cells. B) Representative flow cytometry analysis of Id3-GFP expression by

lo hi hi lo 329 splenic CD44 CD62L cTR and CD44 CD62L eTR gated as indicated. (Bottom left) Graphical

330 analysis of gMFI of Id3-GFP in each of the three gated TR populations. C) Representative flow

+ + + 331 cytometry plots of Id3-GFP expression by gated splenic TCRβ CD4 Foxp3 TR 66 hrs after

332 stimulation of purified CD4+ T cells in the presence or absence of the idicated inhibitors,

333 representative of 2 independent expts. D) Representative flow cytometry plots and graphical

+ + + 334 analysis of CD44 and Id3-GFP expression by gated TCRβ CD4 Foxp3 TR recovered from the

335 LNs of RAG1-deficient mice 2 weeks after transfer of the indicated TR population, summary of 3

336 independent expts, 3-5 mice per group total. Significance determined by one way ANOVA with

337 Tukey’s post-test for pairwise comparisons, *p <0.05, **p < 0.01, ****p < 0.001

- 338 Figure 2: Id3 eTR express inhibitory markers and are highly suppressive

339 A) (Top) Representative flow cytometry histograms. (Bottom) Graphical analysis of expression

340 of the indicated markers by gated splenic TR populations. B) (Top) Representative flow

341 cytometry analysis of CPD dilution by CD4+Foxp3- effector T cells stimulated with or without the

342 indicated TR populations. (Bottom) Graphical analysis of suppression by each of the indicated

343 populations, n=3. Significance determined by one way ANOVA with Tukey’s post-test for

344 pairwise comparisons, *p <0.05, **p < 0.01, ****p < 0.001

- 345 Figure 3: Transcriptional profiling highlights the stepwise differentiation of Id3 eTR

+ + - 346 A) PCA of RNA-seq data from LN and splenic Id3 cTR, Id3 eTR and Id3 eTR populations sorted

347 from three individual mice. B) Bar graphs showing the number of differentially expressed genes

348 (adj.p.value < 0.05 and log2FC >1) for each of the indicated pairwise comparisons. C) Graphical

349 analysis of normalized transcript reads for Id2 or Id3 from RNA-seq data. D) The 300 most bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 17

350 differentially expressed genes (determined by F value) were split into the upregulated (red) and

- + 351 downregulated (blue) fractions based expression in Id3 eTR vs Id3 cTR. Graph shows the mean

+ 352 log2FC compared to Id3 cTR for both eTR populations. Error bars and shaded area represent 1x

353 SD. E) Heatmap and hierarchical clustering of splenic RNA-seq samples based on the 300 most

354 variably expressed genes. F) Heatmap and hierarchical clustering of splenic RNA-seq samples

355 based on TR signature genes identified in reference (8). G) GO term enrichment analysis for DE

- + 356 genes of Id3 eTR vs. Id3 eTR. Dashed lines represent adjusted p values of 0.05 and 0.01. H)

357 Heatmap and hierarchical clustering of splenic RNA-seq samples based on DE genes found in

+ - 358 the top 6 GO functional categories enriched in the comparison of Id3 and Id3 eTR. I) Graphical

359 analysis of chemotaxis assay.

- 360 Figure 4: Id3 eTR are enriched and resident in non-lymphoid tissues

361 A) Representative flow cytometry histograms and graphical analysis of expression of the CD69

+ 362 and CD103 by gated splenic TR populations. B) Enrichment of CD8 TRM (red) or circulating T

363 cell (blue) gene sets along ranked lists of the indicated pairwise comparisons. C) Heatmap and

+ 364 hierarchical clustering of splenic RNA-seq samples based on ST2 tissue TR associated genes.

365 D) Representative flow cytometry plots of CD103 and CD69 expression by gated TCRβ+ CD4+

+ - 366 Foxp3 TR in the indicated tissues. E) Graphical analysis of Id3 eTR frequency among total TR in

367 various tissues. F) Representative histograms of GFP expression from various tissues of Id3-

+ + + 368 GFP x Foxp3-mRFP mice, gated on TCRβ CD4 Foxp3 TR.

369 Supplementary Figure 1: Loss of Id3 expression correlates with strength of TCR signaling

370 Representative flow cytometry plots of Id3-GFP expression by gated splenic TCRβ+ CD4+

+ + 371 Foxp3 TR 48 hours after stimulation of purified CD4 T cells with 1μg/mL α-CD28 and indicated

372 titration of α-CD3, summary of 2 independent expts.

373 Supplementary Figure 2: Flow cytometry validation of RNA-seq targets bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 18

374 A) Representative flow cytometry histograms. B) Graphical analysis of expression of the

375 indicated markers by gated splenic TR populations. Significance determined by one way ANOVA

376 with Tukey’s post-test for pairwise comparisons, *p <0.05, **p < 0.01, ****p < 0.001

377 bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Sullivan et al. Figure 1

Ctrl Id3-GFP 2250 **** A 10.7 0 1.55 12.5 B cT R **** 2000 ** 0.25 99.5 16.3 80.9 1750 1500

cT eT gMFI Id3-GFP 250 89.2 0.05 19.2 66.8 CD4 eTR R R CD62L Foxp3-mRFP Id3-GFP CD44 Id3-GFP 0 R R R T - T + c + eT e C d3 d3 Id3 FR 180204 (Erk) D Transferred cells: I I + + - Id3-eT Id3+eT Id3+cT PD 0325901 (Mek) Id3 cTR Id3 eTR Id3 eTR R R R Rapamycin (mTor) 7.36 16.2 0.17 15.3 0 1.64 Id3-eT R ZSTK474 (PI3K) Id3+eT VIVIT peptide (NFAT) R stimulated Id3+cT unstimulated R 0.71 75.8 0.50 84.1 0 98.4

Ctrl Id3-GFP 0 25 50 75 100 CD44 Id3-GFP % of recovered TR

Id3 is dynamically expressed in TR and regulated by TCR signaling bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It Sullivanis made available et underal. Figure 2 aCC-BY-NC-ND 4.0 International license.

A CD25 ICOS KLRG1 B 1:2 Ratio

Eff+APC+αCD3

TIGIT GITR CLTA4 + +Id3 cTR

+ +Id3 eTR

- +Id3 eTR Id3+cT Id3+eT Id3-eT R R R CPD CD25 ICOS KLRG1 Id3+cT 3000 * 5000 **** 2500 *** R **** + 2500 * 4000 2000 *** Id3 eTR 2000 Id3-eT 3000 1500 R gMFI 1500 100 2000 1000 1000 1000 500 500 75 0 0 0 TIGIT GITR CTLA4 1200 50000 **** 1500 * 50 **** *** * 1000 **** 40000 1250

800 *** 1000 % Suppression 30000 25 600 750 20000 gMFI 400 500 200 10000 250 0 1:2 1:8 0 0 0 T : Effector Ratio + + - R Id3 cTR Id3 eTR Id3 eTR

- Id3 eTR express inhibitory markers and are highly suppressive bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Sullivan et al. Figure 3

Top 300 F value genes A B up down C D E 1500 1056 Id2 Id3 up down tissue 400 6 population Lymph node

1000 R 4 Spleen 300 2 520 population 500 269 200 0 + 415 Id3 cTR # DE genes + PC2 (8.9%) 205 −2 Id3 eTR 100 Id3-eT 89 log2FC from cT R 0 normalized expression −4 PC1 (28.9%) - + - + + - + + - Id3 eTR Id3 eTR Id3 eTR Id3 cTR Id3 eTR Id3 eTR Id3 cTR Id3 eTR Id3 eTR vs. vs. vs. + + + Id3 cTR Id3 cTR Id3 eTR F G H TR function DE genes in top 6 GO categories GO enrichment analysis

cytokine rec. act. Cd69 Gpr83 Tnfrsf18 chemokine rec. act. Ccr7 Cxcr5 + G−protein coupl. chemoattr. rec. act. Id3 cTR Il2ra Il17re + Id3 eTR Tgfb1 G−protein coupled peptide rec. act. Ccr10 −2 0 2 - Id3 eTR Itgae peptide rec. act. Il23r Row Z−Score Entpd1 Cxcr6 Id3pos_cTR C−C chemokine rec. act. Ltb4r1 I Id3pos_eTR Lag3 Th1 type immune resp. Ccr5 Id3neg_eTR Tnfrsf1b Ccr2 100 regulation of leukocyte migration Tnfrsf9 Ccr1 75 Nmur1 Ebi3 G−protein coupled rec. act. 50 leukocyte chemotaxis Il1r1 Tigit F2rl2 25 Nrp1 external side of plasma membrane Ccrl2 Pdcd1 chemokine binding Il18r1 −2Il1rl1 010 1 2 Nt5e positive reg. of leukocyte migration Cxcr3 Tnfrsf4 cell chemotaxis RowCcr4 Z−Score Gzmb Il13ra1 5 myeloid leukocyte migration Icos F2r C−C chemokine binding Il12rb1 Il10 Fold change over media Il18rap 0 Ctla4 reg. of Th1 type immune response Ccr3 + 0246 + Id3 cTR Id3 cT + R CCL20 CCL21 CCL22 Id3 eT + CXCL10CXCL12CXCL13 R −log10(adj.p.value) Id3 eTR −2 0 2 Id3-eT −1 0 1 - R Id3 eTR Row Z−Score Row Z−Score Id3pos_cTR Id3pos_cTR Id3pos_eTR Id3pos_eTR Id3neg_eTR Id3neg_eTR - Transcriptional profiling highlights the stepwise differentiation of Id3 eTR bioRxiv preprint doi: https://doi.org/10.1101/359687; this version posted October 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Sullivan et al. Figure 4

TRM signature (red) and A CD69 CD103 B circulating memory signature (blue) C D Tissue TR signature CD69 p = 1 x 10-5 p = 1 x 10-5 0 2.6 R R Enrichment cT

eT Lmna - + Irf4 Id3 Id3 Gata3 Areg + + - Id3 cT Id3 eT Id3 eT 2.7 0 Rora R R R CD103

Enrichment Klrg1 0.1 0.3 0.6 1.3 9.3 CD69 CD103 -8.1 -0.8 -0.4 -0.2 -0.1 -0.0 Il10 2000 800 **** **** log2FC log2FC Osbpl3 ** **** p = 0.001 p = 1 x 10-5 Tigit 1500 **** 600 **** Il1rl1

0 2 S1pr1 R R

Enrichment Lef1

1000 400 eT eT gMFI - + Tcf7

Id3 + Id3 Klf2 500 200 Spln Id3 cTR - + Id3 cT Spln Id3 eTR R - 1.8 0 + Lung Id3 eTR 0 0 -202 Id3 eTR - - Enrichment Skin Id3 eT + + - Row Z−Score Id3 eTR R 0.1 0.2 0.4 0.8 8.2 Id3 cTR Id3 eTR Id3 eTR -6.4 -0.7 -0.4 -0.2 -0.1 -0.0

E 100 F Lymph pLN Blood Spleen Thymus MLN 5.38 7.21 7.70 10.6 6.90 13.7 80 −2 0 1 2 )

R Row Z−Score 60 (of T R eT

- 40 Peyers Lung LPL IEL Skin Fat 22.2 Patches 21.4 48.6 43.6 72.5 83.3

% Id3 20

0

IEL Fat PLN Spln MLNPEC LungLPL Skin Lymph Blood Thymus

Peyer’s Patch Id3-GFP

- Id3 eTR are enriched and resident in non-lymphoid tissues