bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

1 Title

2 Inflammatory signals are sufficient to elicit expression in mouse and human CD8 T cells

3

4 Authors

5 Nicholas J Maurice1,2,*, Jacqueline Berner3,*, Alexis K Taber1, Dietmar Zehn3,°, Martin Prlic1,4,°

6

7 1 Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109

8 2 Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA 98195

9 3Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical

10 University of Munich, Freising, Germany

11 4 Department of Immunology, University of Washington, Seattle, WA 98109

12

13 * Authors equally contributed to this work

14 ° Co-corresponding authors

15

16 Correspondence should be addressed to: D.Z. [email protected] or M.P. [email protected]

17

18 Competing interests

19 The authors declare no competing interests.

20 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

21 Abstract

22 (TCR) stimulation leads to expression of the TOX. Prolonged TCR

23 signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX

24 expression, which induces a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in

25 specific pathogen-free laboratory mice typically do not express TOX, functional human Tmem show

26 heterogeneous TOX expression levels. Whether TCR-independent mechanisms can alter TOX

27 expression in human and murine Tmem has not been defined. We report that human and mouse Tmem

28 increase TOX expression following stimulation with inflammatory cytokines IL-12, IL-15, and IL-18. TOX

29 and PD-1 expression patterns often appear to be directly correlated, however, we found that TOX is not

30 necessary for cytokine-driven expression of PD-1. Together, these observations highlight that

31 inflammation is sufficient to alter TOX and PD-1 expression and that the signals regulating TOX

32 expression appear well conserved in human and murine Tmem.

33 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

34 Introduction

35 T cell exhaustion (i.e. dysfunction) is driven by chronic TCR stimulation with cognate antigen

36 (Ag)1, 2, 3. It describes a differentiation state in which T cells have diminished capacity to respond to

37 stimulatory inputs and limited effector capacity2, 3, 4. The purpose of T cell exhaustion during chronic

38 infections may be to limit tissue pathologies when pathogen cannot be immunologically eliminated5, 6.

39 Though exhaustion could be considered an immunologic concession during chronic infection, it also

40 occurs in tumors and causes an attenuated anti-tumor response7. Thus, mechanistically

41 understanding and therapeutically overcoming T cell exhaustion has been a major goal of tumor

42 immunotherapy. Chronic TCR stimulation elicits a program that leads to constitutively high expression of

43 programmed cell death 1 (PD-1) 8. PD-1 is an inhibitory receptor which is expressed by activated and

44 exhausted T cells and often used as a biomarker to infer T cell functionality9. When bound to its ligands,

45 PD-1 negatively regulates T cell function2. Therapeutic targeting of PD-1 with monoclonal antibodies,

46 also referred to as immune checkpoint inhibitors, can reinvigorate a subset of these PD-1 expressing T

47 cells2, 10, 11, 12.

48 A set of recent studies demonstrated that the transcription factor, thymocyte selection-associated

49 high mobility group box (TOX) , drives or stabilizes this TCR-mediated T cell dysfunction and PD-

50 1 upregulation6, 13, 14, 15, 16. When stably expressed, TOX drove Ag-specific T cell exhaustion in mouse

51 models of chronic lymphocytic choriomeningitis virus (LCMV) infection, transplantable B16 melanoma,

52 and inducible hepatocellular carcinoma6, 13, 14. Further, putative tumor Ag-specific CD8 T cells isolated

53 from primary human breast, ovarian, and skin cancer samples, as well as those specific for hepatitis C

54 virus (HCV), mirrored this phenotype, suggesting TOX dictates exhaustion programs in humans, too6, 13,

55 14. Of note, TOX expression by HCV-specific T cells was reduced following treatment and clearance of

56 the infection but still detectable at higher levels than in T cells from HCV infections that spontaneously

57 resolved and among T cells specific for influenza A virus (IAV)6. Mechanistic insight was provided by

58 targeted deletion of TOX in Ag-specific cytotoxic T cells, which diminished PD-1 expression and restored

59 functionality at the expense of cell survival6, 13. Therefore, TOX concedes activation and effector function

60 for exhaustion (i.e. PD-1 expression) and T cell survival during chronic TCR stimulation. In instances of bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

61 brief TCR engagement, TOX is transiently induced to a level lower than that of exhausted T cells, but

62 with limited known functional consequence6, 13, 14.

63 While the requirement for TOX has been well defined in the context of TCR-mediated

64 dysfunction, there is nascent evidence that TOX expression by itself is not indicative of T cell exhaustion.

65 Recent studies illustrated that TOX expression is detected in some functional CD8 memory T cells

66 (Tmem), for instance in CD8 effector memory (TEM) and effector memory CD45RA-expressing (TEMRA)

17 67 subsets . CD8 Tmem specific for the latent viruses, cytomegalovirus (CMV) and Epstein-Barr virus (EBV),

68 had elevated TOX expression, compared to those specific for acute infections, which further suggests

69 that TCR signals are critical in regulating TOX expression17. In a second study, it was shown that a

70 fraction of the human Tmem population expresses TOX transcripts amongst other signature

71 typically associated with T cell exhaustion18. The observation that functional humam memory T cells

72 express TOX also led to questioning whether TOX is functionally conserved between mouse and human

19 73 T cells . Further complicating TOX and exhaustion, the murine tissue resident memory T cell (TRM)

74 transcriptome is characterized by concomitant expression of transcripts encoding Tox, exhaustion

75 markers, TCR signaling components, and cytotoxic molecules, well after initial priming events20, 21. While

76 the role of TOX in these TOX-expressing populations with and without signs of T cell exhaustion is not

77 fully understood, these data suggest that TOX expression by memory T cells cannot be reliably used to

78 extrapolate T cell function.

79 While the role of TCR signals in initiating and maintaining PD-1 and TOX expression has been

80 well established, relatively little remains knows about non-TCR signals that could regulate their

81 expression in T cells22. We considered that cytokine-mediated stimuli could also affect TOX expression

82 levels without promoting the induction of T cell exhaustion. First, pro-inflammatory cytokines, like IL-15,

83 can induce PD-1 without agonist TCR signals. Second, TRM that are likely not detecting cognate Ag still

84 upregulate PD-1 and other markers associated with exhaustion20, 21, 23, 24, yet rely on IL-15 signaling for

85 maintenance in some tissues25, 26. Thus, inflammatory signals could provide an explanation for some of

86 the seemingly disparate results of TOX expression and T cell function. Here, we show that pro-

87 inflammatory cytokines were sufficient to induce TOX expression in the absence of agonist TCR signals bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

88 in both mouse and human CD8 Tmem, while concurrently inducing expression of cytotoxic molecules.

89 Together, these data demonstrate that TOX expression per se does not indicate TCR-mediated

90 dysfunction or even a recent TCR signals. We also demonstrate that PD-1 expression is still upregulated

91 in TOX-deficient T cells indicating that TOX is not necessary for PD-1 expression. Overall, our data

92 reveal new TCR-independent mechanisms that shape TOX and PD-1 expression heterogeneity in Tmem

93 and indicate that these mechanisms are conserved in both mouse and human T cells. Though these

94 findings ultimately complicate the use of TOX exclusively as an exhaustion biomarker, they implicate

95 TOX in inflammation-driven programs of memory T cell activation.

96 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

97 Results

98 Cytokine stimulation induces TOX expression in murine CD8 Tmem

99 The pro-inflammatory cytokines IL-12, IL-15, and IL-18 elicit interferon-γ (IFNγ) and granzyme B

27, 28, 29 100 (GzmB) expression in mouse and human CD8 Tmem in the absence of agonist TCR signals . We

101 first sought to determine if these cytokines could also induce TOX expression in a TCR-independent

102 manner. To generate a well-defined population of CD8 Tmem, we transferred congenically-marked OT-I

103 CD8 T cells, which express a TCR specific for the SIINFEKL peptide of OVA, into wildtype C57BL/6J

104 animals followed by infection with OVA-expressing vesicular stomatitis virus (VSV-OVA) (Fig. 1a). We

105 waited ≥60 days before using these mice for subsequent experiments (referred to as VSV-OVA OT-I

106 memory mice) (Fig. 1a). We isolated T cells from the spleens and LNs from VSV-OVA OT-I memory

107 mice using negative-selection magnet-activated cell sorting (MACS) prior to ex vivo stimulation

108 experiments (Fig. 1a). This was done to ensure that cytokines act directly on T cells30. As a negative

109 control, we cultured bulk T cells in media alone (mock) and as a positive control, we stimulated T cells

110 with anti-CD3/CD28 microbeads (Fig. 1a). We used a combination of rIL-12, rIL-15, and rIL-18 (IL-

111 12/15/18) to induce IFNγ and GzmB expression in a TCR-independent manner (Fig. 1a). We found that

112 IL-12/15/18 stimulation induced PD-1 expression in OT-I Tmem, but the increase in expression was

113 markedly higher after TCR ligation (Fig. 1b). PD-1 frequency and median fluorescence intensity (MedFI)

114 in OT-I Tmem increased throughout the duration of IL-12/15/18 stimulation (Fig. 1b). Similarly, TCR and

115 IL-12/15/18 stimulation induced TOX upregulation in OT-I Tmem (Fig. 1c). Next, we measured TCF1

116 expression, a transcription factor needed for memory T cell self renewal that is lost in terminally

31, 32, 33, 34 117 exhausted Tmem . Alongside increasing PD-1 and TOX levels, both TCR- and IL-12/15/18-

118 mediated stimulation led to significant loss of TCF1 expression in OT-I Tmem (Fig. 1d). In sum, these data

119 indicate that phenotypes often associated with exhaustion can be induced by TCR-independent,

120 cytokine-mediated Tmem activation. Finally, we sought to determine whether stimulation similarly affected

121 endogenous CD8 Tmem and CD8 Tnaïve. IL-12/15/18 stimulation significantly increased TOX expression in

122 endogenous CD8 Tmem but was not observed to the same degree in CD8 Tnaïve (Supplemental Fig. 1a,

123 b). This CD8 Tmem-specific response is, too, reflected in IL-12/15/18-mediated upregulation of PD-1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

124 (Supplemental Fig. 1c, d). This is likely, in some degree, to the different propensities of T cell subsets

125 (both major and memory) to become efficiently activated by cytokines35 and differences in cytokine

126 receptor expression (particularly naïve T cells, which require TCR-mediated activation to induce IL-12R

36, 37 127 and strongly increase IL-18R expression ). Much akin to OT-I Tmem, TCR stimulation dramatically

128 increased both TOX MedFI and PD-1 expression across endogenous subsets (Supplemental fig. 1a-d),

129 though the fold change in TOX staining intensity was most pronounced in CD8 Tmem (Supplemental fig.

130 1a). Though IL-12/15/18 stimulation increases TOX MedFI in transgenic and endogenous CD8 Tmem, it is

131 initially to a lower degree than that of TCR-stimulated cells (Fig. 1c, Supplemental fig. 1a). Since short-

132 term TCR- and IL-12/15/18-stimulation could dramatically augment TOX and PD-1 expression in CD8

133 Tmem from VSV-OVA OT-I memory mice, we next sought to test if TOX and PD-1 upregulation

134 compromises functionality.

135

136 Functional CD8 Tmem express TOX and PD-1 and effector

137 We isolated T cells from VSV-OVA OT-I memory mice as outlined for Fig. 1. We stimulated T

138 cells in the presence of Golgi inhibitors and found that OT-I Tmem produced substantial amounts of IFNγ

139 after IL-12/15/18 or TCR stimulation(Fig. 2a); yet IFNγ-expressing OT-I Tmem demonstrated higher TOX

140 and PD-1 expression than those that failed to make IFNγ (Fig. 2b, c). Similarly, OT-I Tmem that produced

141 GzmB post-stimulation also demonstrated increased TOX and PD-1 expression (Supplemental fig. 2a,

142 b). Together, these data indicate that TOX and PD-1 expression are elevated in activated, functional

143 CD8 Tmem and suggest that TOX expression is also part of a cytokine-driven T cell activation program.

144

145 Induction of TOX and PD-1 is heterogeneous in CD8 Tmem

146 To ensure that our data were not solely reliant on OT-I T cells, we also generated gBT-I memory

147 mice using gBT-I TCR transgenic cells (specific for an epitope of the HSV2 gB protein) and a

148 recombinant, gB epitope-expressing LM strain (LM-gB) (Supplemental Fig. 3b). After stable contraction

149 of TCR transgenic Tmem (≥60d), we conducted stimulation assays as previously outlined (Fig. 1a). IL-

150 12/15/18- or TCR-mediated stimulation led to comparable TOX upregulation in OT-I and gBT-I Tmem (Fig bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

151 3a, b). Similarly, PD-1 expression was comparable in OT-I and gBT-I Tmem after stimulation

152 (Supplemental Fig 3c, d), with a concurrent loss of TCF1 expression (Supplemental Fig. 3e, f). We

153 next asked if altering the nature of the priming infection could affect the ability to express TOX in

154 response to cytokine-mediated activation at the memory stage. We adoptively transferred P14 transgenic

155 T cells, a TCR transgenic specific for lymphocytic choriomeningitis virus (LCMV) gp33, followed by

156 infection with LCMV Armstrong or Docile (Supplemental Fig. 3g, h). These LCMV strains elicit acute

157 and chronic infections, respectively (the latter causing T cell dysfunction). We then stimulated (same

158 culture set-up as outlined in Fig. 1a) T cells from these P14 memory mice. P14 Tmem from LCMV

159 Armstrong infected mice readily upregulated PD-1 after TCR- or IL-12/15/18 stimulation (Supplemental

160 Fig. 3i). The exhausted P14 Tmem from LCMV Docile-infected mice already uniformly expressed PD-1

161 prior to stimulation; but IL-12/15/18 or TCR stimulation further increased surface PD-1 expression (via

162 increased MedFI) (Supplemental Fig. 3j). P14 Tmem from LCMV Armstrong infected mice increased TOX

163 expression after TCR or IL-12/15/18 stimulation (Fig. 3c). However, exhausted P14 Tmem from LCMV

164 Docile-infected mice only significantly increased TOX expression after TCR stimulation (Fig. 3d) and

165 showed significantly lower fold changes in TOX MedFI compared to P14 Tmem from LCMV Armstrong-

166 infected mice. While differences between CD8 Tmem from acute and chronic infected are expected, the

167 differences between gBT-I and OT-I (~3 to 4-fold increase in TOX expression) compared to P14 (up to

168 ~2-fold) need to be interpreted with caution since the gBT-I, OT-I and P14 experiments used different

169 TOX antibody clones (REA473 and TXRX10, respectively). Overall, our data indicate that Tmem that were

170 generated by different acute infections increase TOX expression in response to pro-inflammatory

171 cytokines suggesting that this a broadly applicable mechanism of TOX induction in the memory T cell

172 compartment. We next sought to determine if PD-1 and TOX upregulation in response to stimulation was

173 similarly recapitulated in human CD8 T cells.

174

175 Cytokine stimulation induces TOX and PD-1 in human Tmem

176 Using cryopreserved PBMCs from healthy, HIV-seronegative donors, we interrogated TOX and

177 PD-1 expression by flow cytometry. We specifically gated CD8 T cells by a memory and naïve binary, bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

38 178 delineating CD8 Tnaïve as CD45RO-negative CCR7-positive, with remaining cells as CD8 Tmem (Fig. 4a),

179 and interrogated basal TOX and PD-1 expression between these two subsets (Fig. 4a). Since PD-1

180 expression is heterogeneous in humans39, 40, we measured TOX MedFI across PD-1 low-, medium-, and

181 high-expressing events. We found that CD8 Tmem with the highest PD-1 expression also demonstrated

182 significantly elevated TOX MedFI (Fig 4b), mirroring correlations of TOX and PD-1 expression in our

183 mouse model as well as human HCV infections6. We next tested whether IL-12/15/18 stimulation

184 increases PD-1 and TOX expression in T cell subsets and included mock and TCR stimulation conditions

185 as negative and positive controls, respectively. We also included stimulations using rIL-6, rIL-15, or rIL-

186 12 and rIL-18. We chose these additional conditions as IL-6 activates CD8 Tnaïve (as evidenced by CD69

187 upregulation) and to discern individual activating contributions of each cytokine (Supplemental Fig. 4a).

188 Across these conditions, IL-12/15/18- and TCR-mediated stimulations led to the most prominent increase

hi 189 of TOX staining intensity and PD-1 frequency in CD8 Tmem (Fig. 4d). We measured TCF1 expression

190 after mock, IL-12/15/18, and TCR stimulation. A decrease in TCF1 expression accompanied an increase

191 in TOX and PD-1 expression after IL-12/15/18 or TCR stimulation (Supplemental fig. 4b), akin to our

192 mouse stimulation data. We further tested the degree of similarity between human and mouse T cells by

193 measuring PD-1, TCF1, and TOX expression profiles in stimulated human CD8 Tnaïve. Like mouse CD8

194 Tnaïve, only TCR stimulation could lead to appreciable changes in TOX and PD-1 within human CD8 Tnaïve

195 (Fig. 4c, Supplemental fig. 4c). Since IL-6 can activate CD8 Tnaïve, we used this condition to determine

196 if PD-1 and TOX expression could occur in naïve T cells in the absence of a TCR . Despite

197 inducing CD69 expression, we found that IL-6-mediated stimulation failed to increase TOX or PD-1

198 expression in CD8 Tnaïve (Supplemental fig. 4d). Together, these data show that CD8 Tmem differentially

199 expressed TOX, PD-1, and TCF1 at homeostasis and after both IL-12/15/18 and TCR stimulation. We

200 next wanted to better define these changes across different memory T cell subsets.

201

202 Inflammation-induced PD-1 and TOX expression occur in most but not all CD8 Tmem subsets

203 To test if inflammation-induced PD-1 and TOX expression differs across human CD8 Tmem

+ 204 subsets, we used CD45RO and CCR7 staining to further delineate central memory (TCM) (CD45RO bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

+ + – – – 38, 41 205 CCR7 ), TEM (CD45RO CCR7 ), and TEMRA (CD45RO CCR7 ) subsets (Fig. 5a). When we

206 measured TOX, PD-1, and TCF1 expression across these subsets, we noted that a substantial fraction

hi 207 of CD8 TEM events were PD-1 , and both CD8 TEM and TEMRA expressed elevated and lower levels of

208 TOX and TCF1, respectively, at homeostasis (Fig. 5b). While this observation is in with the initial

17 209 report demonstrating TOX heterogeneity in human CD8 Tmem subsets , it remained unknown if these

210 CD8 Tmem subsets are equally capable of further TOX upregulation after stimulation. We observed that

211 TOX, PD-1, and TCF1 expression kinetics in CD8 TCM and TEM largely resembled one another, with both

212 IL-12/15/18 and TCR stimulation increasing the frequency of PD-1hi events and TOX MedFI, but

213 decreasing TCF1 MedFI (Fig. 5c). It is worth noting, that while TCF1 MedFI in CD8 TCM drops profoundly

214 after IL-12/15/18 or TCR stimulation, the loss in frequency of TCF1-expressing cells (as defined by

215 subjective gating) is not as pronounced as what we observed in CD8 TEM (Supplemental fig. 5a). While

216 IL-12/15/18- and TCR-mediated stimulation were both able to significantly increase the frequency of PD-

hi 217 1 events and lower TCF1 MedFI in CD8 TEMRA, the degree of these changes was less pronounced than

218 in CD8 TCM or TEM (Fig. 5c). Moreover, CD8 TEMRA did not significantly upregulate TOX expression after

219 TCR stimulation. This, however, was not due to inability to be stimulated, as CD8 TEMRA readily

220 expressed the activation marker CD69 after cytokine- or TCR-mediated stimulation (Supplemental Fig.

221 5a). Finally, it is worth noting that when stimulated with IL-15 alone, CD8 TCM, unlike CD8 TEM and TEMRA,

222 fail to significantly express PD-1 (Supplemental fig. 5b).

223 We next interrogated T cells with defined TCR specificity, specifically influenza A virus (IAV)-

224 specific CD8 T cells using HLA-A*02 tetramers loaded with the GILGFVFTL peptide (Fig. 6a). We

225 examined this CD8 Tmem population because these cells were reported to not express appreciable levels

17 226 of TOX at homeostasis, likely owing to their TCM phenotype . Within our sample set, IAV-specific CD8 T

227 cells were predominantly TCM in half of the HLA-A*02 PBMC donors (Fig. 6a). Nevertheless, all IAV-

228 specific CD8 T cells were able to substantially upregulate TOX and PD-1 expression after IL-12/15/18

229 stimulation (Fig. 6b), indicating that CD8 Tmem low for TOX and PD-1 at homeostasis, can also contribute

230 to TOX and PD-1 heterogeneity after recent activation. Alongside testing IAV-specific CD8 T cells, we

231 also interrogated the effects of stimulation in mucosal associated invariant T (MAIT) cells. We selected bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

232 this population because 1) MAIT cells are non-conventional T cells, recognizing bacterial metabolites as

233 Ags presented on MHC-related 1 (MR1)42, 2) inflammation is necessary for sustained MAIT cell effector

43, 44 45 234 function and 3) MAIT cells are near-uniformly TEM when defined by CD45RO and CCR7 . We

235 identified MAIT cells using MR1 tetramers loaded with the 5-OP-RU metabolite46, which largely fell into

236 our TEM gate (Fig. 6c). Like IAV-specific CD8 T cells, IL-12/15/18 stimulation led to substantial TOX and

237 PD-1 upregulation in MAIT cells (Fig. 6d). Since inflammation is necessary for sustained MAIT cell

238 effector function, we asked if MAIT cells are differentially capable of responding to other cytokine

239 combinations. Alongside IL-12/15/18, IL-15 alone, or IL-12 and IL-18 in unison could significantly

240 increase both the PD-1hi frequency and TOX MedFI of MAIT cells, but not IAV-specific T cells

241 (Supplemental fig. 6a, b). Together, these data indicate that this cytokine-driven activation program is

242 conserved across conventional and innate-like T cells.

243

244 Cytokine stimulation-induced PD-1 expression is independent of TOX

245 Finally, since PD-1 and TOX upregulation appeared tightly associated following cytokine-driven

246 activation, we next asked if this association is mechanistic in nature. If TOX is necessary for PD-1

247 expression, it would allow to use the surface-expressed PD-1 as a surrogate for the intracellularly

248 expressed TOX. TOX expression appears to drive PD-1 expression in a number of contexts, as

6, 13, 15, 47 249 exhausted Tmem dramatically downregulated PD-1 after TOX deletion or knockdown . Conversely,

250 T cell transduction with TOX-encoding constructs leads to PD-1 upregulation13, 14, 15, 47. While TOX

251 controls PD-1 expression during exhaustion, the role of TOX is less clear in activation. To dissect the

–/– 252 function of TOX in stimulation-mediated PD-1 upregulation, we used wildtype (WT) and Tox P14 Tmem.

253 To generate these P14 Tmem, we adoptively transferred wildtype or knockout P14 T cells into C57BL/6J

254 hosts, which we subsequently infected with LCMV Armstrong to form a Tmem population (Supplemental

255 fig. 7a). To determine if TOX deficiency alters stimulation-induced PD-1 upregulation, we cultured

256 MACS-isolated T cells from WT and Tox–/– P14 memory mice (28 days post LCMV Armstrong infection)

257 in the presence of mock, IL-12/15/18, or TCR stimulation (Supplemental fig. 7a). Both WT and Tox–/–

258 P14 Tmem increased PD-1 expression after IL-12/15/18 or TCR stimulation (Fig. 7a, b, c). Together these bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

259 data indicate that TOX alone is not necessary for PD-1 upregulation in cytokine-stimulated CD8 Tmem and

260 suggest other transcription factors are sufficient to drive PD-1 expression in the absence of TOX.

261

262 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

263 Discussion

264 TOX has been foremost studied in TCR-mediated exhaustion of mouse CD8 T cells in context of

265 tumor or chronic infection6, 13, 14. A recent study reported TOX expression in functional circulating human

17, 18 266 CD8 Tmem, suggesting TOX expression does not necessarily dictate dysfunction , which led to the

267 speculation that TOX may have distinct roles across species, specifically mice and humans19.

268 Alternatively, TOX expression heterogeneity in humans may simply reflect the more complex

269 environment that human T cells are exposed to in every day life, that may not be readily appreciable in

270 specific pathogen-free mice, such as routine inflammatory events in barrier tissues. Thus, we asked if

271 pro-inflammatory cues could be sufficient to increase TOX expression and contribute to TOX

272 heterogeneity. While inflammation has been previously shown to enhance TCR-mediated TOX

273 upregulation (in a VEGF-A-dependent manner that necessitates initial TCR signaling)48, our findings are,

274 to the best of our knowledge, the first to demonstrate TOX expression in the absence of agonist TCR

275 signals. Transient IL-12/15/18 and TCR stimulation increased PD-1 and TOX expression in most CD8

276 Tmem. In mouse, dysfunctional P14 Tmem from LCMV Docile infected mice still increased surface PD-1

277 expression after TCR stimulation, while IL-12/15/18 had little to no effect on TOX expression. Similarly,

278 human TEMRA showed limited to no increase in TOX expression following exposure to IL-12/15/18. The

279 underlying mechanisms will require further investigation, but one could speculate that the cytokine

280 stimulation is simply not potent enough to further enhance the already ongoing effector or activation

281 program in these two memory T cell subsets. The notion that TOX, but also PD-1 expression can

282 indicate an ongoing effector or activation program in CD8 T cells is important, since PD-1 and (now also)

283 TOX are used as biomarkers of T cell exhaustion49, 50, 51. Of note, certain features of general activation

284 programs of CD8 Tmem appear to be well conserved and have also been reported as transcriptomic

285 overlap of tissue-resident, recently-activated, and exhausted CD8 T cells52. While infection parameters

286 and inflammatory events are well defined in mouse model studies, most human studies remain agnostic

287 in regard to the infection and activation history of Ag-specific T cells. This in turn makes it difficult to

288 correctly interpret the underlying reason for expression of PD-1 and TOX by human T cells.

289 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

290 Our data emphasize the need for conservative interpretation of TOX in regard to activation and

291 exhaustion and also caution against interpreting TOX expression purely through the lens of recent TCR-

292 mediated activation. TOX expression has been predictive of T cell exhaustion and unfavorable outcome

293 in hepatocellular carcinoma animal models and clinical samples53, in line with the paradigm of TOX-

294 mediated TCR-dependent T cell dysfunction. However, other studies have yielded contradictory data.

295 Meta analyses of TOX expression in breast cancers reported TOX levels paradoxically correlating with

296 increased immune cell function and favorable prognosis54. This is perplexing, as in tumors, TOX

297 expression is associated with T cell dysfunction6, 13, 14. This discrepancy could in part be explained by

298 TOX upregulation during activation, akin to what we observed during T cell activation in TCR-dependent

299 and -independent stimulations. Thus, our data stress that all possible activation pathways of TOX and

300 PD-1 induction must be considered before interpreting TOX as a biomarker of T cell dysfunction. A well

301 done human study that interrogated TOX heterogeneity found elevated TOX in CMV- and EBV-specific

302 CD8 Tmem and hypothesized recent viral reactivation provided cognate Ag to facilitate TCR-mediated

303 upregulation of TOX. This is certainly a plausible explanation, but our data highlight the need to also

304 consider recent exposure to inflammation as a critical parameter affecting TOX expression. Conventional

305 CD8 TEM and TEMRA (the predominant phenotype of CMV- and EBV-specific CD8 T cells) express

306 elevated levels of TOX basally, TCM (including IAV-specific CD8 T cells) and innate-like MAIT cells can,

307 too, upregulate TOX expression following inflammation-mediated activation. Importantly, our data

308 highlight that this mechanism of TOX expression is conserved across species, conventional CD8 Tmem

309 subsets, and innate-like MAIT cells.

310 Since pro-inflammatory cytokines can concurrently induce TOX and PD-1 expression, these

311 signals may drive TOX heterogeneity in other contexts. P14 tissue-resident memory T cells showed

312 increased Tox expression at homeostasis, which has been observed 90 days post priming with LCMV

313 Armstrong20. Since the acute infection is cleared well before this timepoint, it is unlikely that continued

314 TCR signaling by cognate Ag drives this phenotype, despite elevated transcripts encoding mediators of

315 TCR signaling20. IL-15, however, is likely present within the tissue microenvironment. IL-15 has been

25, 26 316 implicated in TRM maintenance , and transcriptional profiles indicative of IL-15/STAT5 signaling can be bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

23, 24 317 detected in human TRM . Thus, IL-15 in tissue microenvironments may also contribute to TOX

318 heterogeneity. Future work will be necessary to dissect the role of these inflammatory cues versus other

21 319 signals that can shape TRM phenotype, such as co-stimulation and tonic TCR signaling .

320 Previous studies have demonstrated that TOX ablation or knockdown leads to PD-1

321 downregulation in models of exhaustion6, 13, 15, 47, and conversely, introduction of TOX-expressing

322 constructs enhances PD-1 expression15, 47. Similarly, our data showed a close correlation in regards to

323 TOX and PD-1 expression levels, but we found that PD-1 expression could be induced in stimulated Tox–

/– –/– 324 P14 Tmem. Of note, these Tox P14 Tmem lack exon 5, which abrogates the ability to function as a

325 transcription factor, but the truncated protein is still expressed and detected by the TOX antibody. Alfei et

–/– 326 al. previously showed that the early wave of effector cells formed from Tox Tnaïve expressed significant

327 levels of PD-1 independently of functional TOX. However, TOX was required for the expression of high

328 levels of PD-1 at later stages, once the initial population of exhausted effector T cells had been replaced

329 by a proliferation competent TCF1 progenitor population31. Together, these data suggest that long-term

330 expression of PD-1 requires TOX, but activation-induced expression of PD-1 is TOX-independent. In the

331 absence of TOX, PD-1 expression could be driven TOX2, which can induce PD-1 expression in CD8 T

332 cells15, 47; however, it remains unclear if TOX2 is also upregulated by transient TCR- or cytokine-

333 mediated stimulation. Similarly, how different activating signals integrate to regulate TOX expression also

334 requires further studies: while inflammatory cues increase TOX expression in memory T cells, increased

335 IL-12 signaling during the priming of Tnaïve has been shown to limit subsequent TOX expression at steady

336 state55, 56.

337 Overall, our data suggest that the mechanisms that regulate TOX expression, both at

338 homeostasis and after transient TCR or cytokine stimulation, are remarkably similar and quite possibly

339 highly conserved between humans and mice. Our data further highlight the need to consider TOX and

340 PD-1 expression as prominent indicators of ongoing activation and effector programs in Tmem instead of

341 exclusive biomarkers of exhaustion.

342 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

343 Materials and methods

344

345 Mice

346 Mouse protocols and experimentation conducted at the Fred Hutchinson Cancer Research

347 Center were approved by and in compliance with the ethical regulations of the Fred Hutchinson Cancer

348 Research Center’s Institutional Animal Care and Use Committee. Experiments performed at the

349 Technical University of Munich were in compliance with institutional and governmental regulations in

350 Germany and approved by the veterinarian authorities of the Regierung von Oberbayern in Germany. All

351 animals were maintained in specific pathogen-free facilities and infected in modified pathogen-free

352 facilities. Experimental groups were non-blinded, animals were randomly assigned to experimental

353 groups, and no specific method was used to calculate sample sizes.

354

355 We purchased 6-week-old female C67BL/6J mice from the Jackson Laboratory; Tox–/– P14 mice

356 (P14 Toxtm1c(KOMP)Wtsi;MxCre;Rosa26-STOP-eYFP) were generated as previously described 6. Both WT

357 and Tox–/– P14 mice, OT-I mice, and gBT-I mice were maintained on CD45.1 congenic backgrounds. We

358 euthanized mice in accordance with institutional protocols and subsequently collected spleens and lymph

359 nodes (LNs) for experimentation.

360

361 Development of memory mice

362 We prepared a single-cell suspension of LN cells that were harvested from female OT-I, P14, or

363 gBT-I mice by mechanically passing LN tissue through a 70-100µm strainer. To enrich transgenic T cells,

364 we used MACS with a CD8 negative selection kit (Miltenyi Biotec).

365

366 For OT-I memory mice, we adoptively transferred 1×104 OT-I T cells in sterile 1× PBS i.v. per

367 C57BL/6J recipient, and subsequently infected recipients i.v. with 1-2 ×107 PFU OVA-expressing

368 vesicular stomatitis virus (VSV-OVA) or 4 ×103 CFU OVA-expressing Listeria monocytogenes (LM-

369 OVA). For gBT-I memory mice, we adoptively transferred 5 ×104 gBT-I T cells i.v. and subsequently bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

370 infected recipient mice i.v. with or 4 ×103 CFU herpes simplex virus 2 (HSV2) glycoprotein B (gB)-

371 expressing L. monocytogenes (LM-gB). We allowed ≥ 60 days to pass after initial VSV or LM infections

372 before assaying tissues.

373

374 For P14 memory mice, we adoptively transferred 2 ×103 WT P14 T cells i.v. and subsequently

375 infected recipient mice i.v. with 2 ×105 PFU LCMV Armstrong clone (LCMV Arm.) or 2 ×106 PFU

376 LCMV Docile clone (LCMV Doc.). For Tox–/– P14 memory mice, we adoptively transferred 2 ×103 Tox–/–

377 P14 memory mice and subsequently infected with 2 ×105 PFU LCMV Arm.; we allowed 28 days to pass

378 after initial LCMV infection before assaying tissues.

379

380 Human PBMC and study approval

381 Twenty-three healthy, HIV-uninfected adults were recruited by the Seattle HIV Vaccine Trials Unit

382 (Seattle, Washington, USA) as part of the study “Establishing Immunologic Assays for Determining HIV-1

383 Prevention and Control.” These samples are also known as the Seattle Area Control (SAC) Cohort. All

384 participants were provided and signed informed consent, and the Fred Hutchinson Cancer Research

385 Center Institutional Review Board approved the study protocol.

386

387 T cell isolation and in vitro stimulation

388 We harvested spleen and LN from memory mice and mechanically prepared single-cell

389 suspensions. We thawed ~4 ×107 cryopreserved PBMC in human RP10 media (RPMI1640

390 supplemented with 10% FBS, 2mM L-glutamine, 100 U/mL penicillin-streptomycin). To enrich bulk T cells

391 from single cell suspensions, we respectively used mouse- and human-specific T cell negative isolation

392 MACS (STEMCELL Technologies, Canada). We plated 0.5–1 ×106 T cells per well in 96-well V-bottom

393 tissue culture plates. We cultured cells in human RP10 or mouse RP10 media (RPMI 1640

394 supplemented with 10% FBS, 2mM L-glutamine, 100 U/mL penicillin-streptomycin, 1mM sodium

395 pyruvate, 0.05mM β-mercaptoethanol, and 1mM HEPES). To stimulate cells, we cultured mouse T cells

396 in mouse RP10 with rIL-12, rIL-15, and rIL-18 (BioLegend) (each at 100ng/mL), with Dynabeads mouse bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

397 T-Activator (Thermo Fisher) anti-CD3/CD28 beads (at a 1:1 bead:cell ratio), or with media alone. For

398 human T cell stimulations, we used human RP10 media with combinations of rIL-6 (BioLegend), rIL-12,

399 rIL-15, and/or rIL-18 (Peprotech) (each at 100ng/mL), with Dynabeads human T-Activator (Thermo

400 Fisher) anti-CD3/CD28 beads (at a 1:1 bead:cell ratio), or with RP10 alone. We cultured cells at 37°C,

401 5% CO2, sampling cells at 0, 24, and 48 hours for flow staining. For intracellular cytokine staining (ICS),

402 we added GolgiPlug (BD Biosciences) at a 1:1,000 dilution 8 hours prior to cell harvest.

403

404 Flow cytometric analysis

405 We conducted all flow staining for mouse and human T cells on ice and at room temperature,

406 respectively. All mouse and human flow panel reagent information, stain conditions, and gating are

407 included in (Supplemental Fig. 8-11, Supplemental tables 1-6). We conducted LIVE/DEAD fixable

408 aqua or blue viability dye (AViD or BViD, repectively) or Zombie Near-IR viability dye (NIRViD) staining in

409 1× PBS. For surface staining, we utilized FACSWash (1× PBS supplemented with 2% FBS and 0.2%

410 sodium azide) as the stain diluent. For all TOX staining panels, we fixed cells with the FOXP3

411 fixation/permeabilization buffer kit (Thermo Fisher) and conducted intranuclear stains using the FOXP3

412 permeabilization buffer (Thermo Fisher) as diluent. To minimize day-to-day variation for TOX staining, we

413 conducted all intracellular stains within a batch (0, 24, and 48-hour samples) at the same time. We

414 resuspended cells in FACSWash and acquired events on a FACSSymphony, which we analyzed using

415 FlowJo v10 (BD Biosciences). We conducted statistical testing using Prism v8 (GraphPad).

416

417 Acknowledgements:

418 We thank Andrea Schietinger for helpful discussions and critical review of the manuscript. We

419 also thank the Prlic lab, especially Jami Erickson, Florian Mair, Marie Frutoso, and Veronica Davé for

420 critical review of the manuscript. This work was supported by National Institutes of Health grant R01

421 AI123323 (to M.P.), National Cancer Institute Grant F99 CA245735 (to N.J.M). N.J.M. is a Leslie and

422 Pete Higgins Achievement Rewards for College Scientists Fellow and Dr. Nancy Herrigel-Babienko bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

423 Memorial Scholar. D.Z. and J.B were supported by a European Research Council consolidator grant

424 (ToCCaTa) and by the German Research Foundation (SFB1054 and SFB1371).

425 426 References

427 1. Zajac, A.J. et al. Viral immune evasion due to persistence of activated T cells without 428 effector function. J Exp Med 188, 2205-2213 (1998). 429 430 2. Pauken, K.E. & Wherry, E.J. Overcoming T cell exhaustion in infection and cancer. 431 Trends Immunol 36, 265-276 (2015). 432 433 3. Schietinger, A. & Greenberg, P.D. Tolerance and exhaustion: defining mechanisms of T 434 cell dysfunction. Trends Immunol 35, 51-60 (2014). 435 436 4. Goepfert, P.A. et al. A significant number of human immunodeficiency virus epitope- 437 specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma 438 interferon. J Virol 74, 10249-10255 (2000). 439 440 5. Zehn, D., Utzschneider, D.T. & Thimme, ="tag">R. Immune-surveillance through exhausted 441 effector T-cells. Curr Opin Virol 16, 49-54 (2016). 442 443 6. Alfei, F. et al. TOX reinforces the phenotype and longevity of exhausted T cells in chronic 444 viral infection. Nature 571, 265-269 (2019). 445 446 7. Lee, P.P. et al. Characterization of circulating T cells specific for tumor-associated 447 antigens in melanoma patients. Nat Med 5, 677-685 (1999). 448 449 8. Utzschneider, D.T. et al. T cells maintain an exhausted phenotype after antigen 450 withdrawal and population reexpansion. Nat Immunol 14, 603-610 (2013). 451 452 9. Speiser, D.E. et al. T cell differentiation in chronic infection and cancer: functional 453 adaptation or exhaustion? Nat Rev Immunol 14, 768-774 (2014). 454 455 10. Barber, D.L. et al. Restoring function in exhausted CD8 T cells during chronic viral 456 infection. Nature 439, 682-687 (2006). 457 458 11. Schietinger, A. et al. Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven 459 Differentiation Program Initiated Early during Tumorigenesis. Immunity 45, 389-401 460 (2016). 461 462 12. Blackburn, S.D., Shin, H., Freeman, G.J. & Wherry, E.J. Selective expansion of a subset 463 of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci U S A 105, 464 15016-15021 (2008). 465 466 13. Scott, A.C. et al. TOX is a critical regulator of tumour-specific T cell differentiation. Nature 467 571, 270-274 (2019). 468 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

469 14. Khan, O. et al. TOX transcriptionally and epigenetically programs CD8(+) T cell 470 exhaustion. Nature 571, 211-218 (2019). 471 472 15. Seo, H. et al. TOX and TOX2 transcription factors cooperate with NR4A transcription 473 factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci U S A 116, 12410-12415 474 (2019). 475 476 16. Yao, C. et al. Single-cell RNA-seq reveals TOX as a key regulator of CD8(+) T cell 477 persistence in chronic infection. Nat Immunol 20, 890-901 (2019). 478 479 17. Sekine, T. et al. TOX is expressed by exhausted and polyfunctional human effector 480 memory CD8(+) T cells. Sci Immunol 5 (2020). 481 482 18. Galletti, G. et al. Two subsets of stem-like CD8(+) memory T cell progenitors with distinct 483 fate commitments in humans. Nat Immunol 21, 1552-1562 (2020). 484 485 19. Utzschneider, D.T. & Kallies, A. Human effector T cells express TOX-Not so "TOX"ic 486 after all. Sci Immunol 5 (2020). 487 488 20. Kurd, N.S. et al. Early precursors and molecular determinants of tissue-resident memory 489 CD8(+) T lymphocytes revealed by single-cell RNA sequencing. Sci Immunol 5 (2020). 490 491 21. Wang, Z. et al. PD-1(hi) CD8(+) resident memory T cells balance immunity and fibrotic 492 sequelae. Sci Immunol 4 (2019). 493 494 22. Kinter, A.L. et al. The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce 495 the expression of programmed death-1 and its ligands. J Immunol 181, 6738-6746 496 (2008). 497 498 23. Hombrink, P. et al. Erratum: Programs for the persistence, vigilance and control of human 499 CD8(+) lung-resident memory T cells. Nat Immunol 18, 246 (2017). 500 501 24. Hombrink, P. et al. Programs for the persistence, vigilance and control of human CD8(+) 502 lung-resident memory T cells. Nat Immunol 17, 1467-1478 (2016). 503 504 25. Mackay, L.K. et al. The developmental pathway for CD103(+)CD8+ tissue-resident 505 memory T cells of skin. Nat Immunol 14, 1294-1301 (2013). 506 507 26. Mackay, L.K. et al. T-box Transcription Factors Combine with the Cytokines TGF-beta 508 and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43, 1101-1111 509 (2015). 510 511 27. Freeman, B.E., Hammarlund, E., Raue, H.P. & Slifka, M.K. Regulation of innate CD8+ T- 512 cell activation mediated by cytokines. Proc Natl Acad Sci U S A 109, 9971-9976 (2012). 513 514 28. Liu, K., Catalfamo, M., Li, Y., Henkart, P.A. & Weng, N.P. IL-15 mimics T cell receptor 515 crosslinking in the induction of cellular proliferation, expression, and cytotoxicity in 516 CD8+ memory T cells. Proc Natl Acad Sci U S A 99, 6192-6197 (2002). 517 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

518 29. Smeltz, R.B. Profound enhancement of the IL-12/IL-18 pathway of IFN-gamma secretion 519 in human CD8+ memory T cell subsets via IL-15. J Immunol 178, 4786-4792 (2007). 520 521 30. Martin, M.D. et al. Bystander responses impact accurate detection of murine and human 522 antigen-specific CD8 T cells. J Clin Invest 129, 3894-3908 (2019). 523 524 31. Utzschneider, D.T. et al. T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain 525 the Immune Response to Chronic Viral Infections. Immunity 45, 415-427 (2016). 526 527 32. Hudson, W.H. et al. Proliferating Transitory T Cells with an Effector-like Transcriptional 528 Signature Emerge from PD-1(+) Stem-like CD8(+) T Cells during Chronic Infection. 529 Immunity 51, 1043-1058 e1044 (2019). 530 531 33. Yu, S. et al. The TCF-1 and LEF-1 transcription factors have cooperative and opposing 532 roles in T cell development and malignancy. Immunity 37, 813-826 (2012). 533 534 34. Zhou, X. et al. Differentiation and persistence of memory CD8(+) T cells depend on T cell 535 factor 1. Immunity 33, 229-240 (2010). 536 537 35. Maurice, N.J., Taber, A.K. & Prlic, M. The Ugly Duckling Turned to Swan: A Change in 538 Perception of Bystander-Activated Memory CD8 T Cells. J Immunol 206, 455-462 (2021). 539 540 36. Li, W., Kashiwamura, S., Ueda, H., Sekiyama, A. & Okamura, H. Protection of CD8+ T 541 cells from activation-induced cell death by IL-18. J Leukoc Biol 82, 142-151 (2007). 542 543 37. Valenzuela, J., Schmidt, C. & Mescher, M. The roles of IL-12 in providing a third signal 544 for clonal expansion of naive CD8 T cells. J Immunol 169, 6842-6849 (2002). 545 546 38. Jameson, S.C. & Masopust, D. Understanding Subset Diversity in T Cell Memory. 547 Immunity 48, 214-226 (2018). 548 549 39. Duraiswamy, J. et al. Phenotype, function, and profiles of programmed 550 death-1(hi) CD8 T cells in healthy human adults. J Immunol 186, 4200-4212 (2011). 551 552 40. Beura, L.K. et al. Normalizing the environment recapitulates adult human immune traits in 553 laboratory mice. Nature 532, 512-516 (2016). 554 555 41. Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T 556 lymphocytes with distinct homing potentials and effector functions. Nature 401, 708-712 557 (1999). 558 559 42. Kjer-Nielsen, L. et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 560 491, 717-723 (2012). 561 562 43. Berkson, J.D. & Prlic, M. The MAIT conundrum - how human MAIT cells distinguish 563 bacterial colonization from infection in mucosal barrier tissues. Immunol Lett 192, 7-11 564 (2017). 565 566 44. Slichter, C.K. et al. Distinct activation thresholds of human conventional and innate-like 567 memory T cells. JCI Insight 1 (2016). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

568 569 45. Voillet, V. et al. Human MAIT cells exit peripheral tissues and recirculate via lymph in 570 steady state conditions. JCI Insight 3 (2018). 571 572 46. Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor 573 heterogeneity in mucosal-associated invariant T cells. J Exp Med 210, 2305-2320 (2013). 574 575 47. Correction for Seo et al., TOX and TOX2 transcription factors cooperate with NR4A 576 transcription factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci U S A 116, 577 19761 (2019). 578 579 48. Kim, C.G. et al. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant 580 microsatellite stable colorectal cancers. Sci Immunol 4 (2019). 581 582 49. Blank, C.U. et al. Defining 'T cell exhaustion'. Nat Rev Immunol 19, 665-674 (2019). 583 584 50. Heim, K. et al. TOX defines the degree of CD8+ T cell dysfunction in distinct phases of 585 chronic HBV infection. Gut (2020). 586 587 51. Guo, L. et al. TOX correlates with prognosis, immune infiltration, and T cells exhaustion 588 in lung adenocarcinoma. Cancer Med 9, 6694-6709 (2020). 589 590 52. Kumar, B.V. et al. Human Tissue-Resident Memory T Cells Are Defined by Core 591 Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites. Cell Rep 20, 592 2921-2934 (2017). 593 594 53. Wang, X. et al. TOX promotes the exhaustion of antitumor CD8(+) T cells by preventing 595 PD1 degradation in hepatocellular carcinoma. J Hepatol 71, 731-741 (2019). 596 597 54. Arora, M., Kumari, S., Singh, J., Chopra, A. & Chauhan, S.S. Expression pattern, 598 regulation, and clinical significance of TOX in breast cancer. Cancer Immunol 599 Immunother (2020). 600 601 55. Tucker, C.G. et al. Adoptive T Cell Therapy with IL-12-Preconditioned Low-Avidity T Cells 602 Prevents Exhaustion and Results in Enhanced T Cell Activation, Enhanced Tumor 603 Clearance, and Decreased Risk for Autoimmunity. J Immunol 205, 1449-1460 (2020). 604 605 56. Page, N. et al. Expression of the DNA-Binding Factor TOX Promotes the 606 Encephalitogenic Potential of Microbe-Induced Autoreactive CD8(+) T Cells. Immunity 607 48, 937-950 e938 (2018). 608 609 610 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

611 Figure Legends

612

613 Figure 1. Cytokine stimulation induces TOX expression in murine CD8 Tmem

614 a Schematic of OT-I memory mouse generation (top) and subsequent stimulation assays (bottom). OT-I

615 Tnaïve were transfered and expanded with VSV-OVA, then aged to stable memory contraction; after, T

616 cells were enriched from VSV-OVA expanded OT-I memory animals and stimulated with media alone

617 (mock), IL-12, -15, and -18 in combination (IL-12/15/18) (each at 100ng/mL), or anti-CD3/CD28

618 microbeads (TCR) at a ~1:1 bead:cell ratio. b-c expression of b PD-1, c TOX, and d TCF1 within

619 stimulated OT-I Tmem throughout experiment time course. TOX MedFI fold change in c was calculated

620 against average TOX MedFI from mock stimulations in a subset-specific, batch-specific, and timepoint-

621 specific manner. In b and c, bar chart symbols represent one animal at a unique timepoint/condition and

622 are connected by animal identity, with bar indicating mean; the indicated statistical significances were

623 calculated using Wilcoxon matched-pairs signed rank tests. In b-d, symbols in line plots comparing

624 stimulation conditions represent the mean across all animals for a specific timepoint/condition ± SD; the

625 indicated statistical significances were calculated using Mann-Whitney tests. Figures in b and c depict

626 results from n = 14 mice across 7 experiments. Figures in d depict results from n = 9 mice across 2

627 experiments. All representative flow plots are sourced from the same animal.

628

629 Figure 2. TOX and PD-1 expression occur in functional CD8 T cells

630 a-c Intracellular cytokine staining (ICS) in tandem with TOX interrogation. a Experiment schematic, in

631 which bulk T cells from VSV-OVA OT-I memory mice were stimulated (mock, black; IL-12/15/18, blue;

632 TCR, red). Cells were treated with GolgiPlug 18h into stimulation and harvested for flow staining and

+ – 633 analysis at 24h. b, c Expression of b TOX and c PD-1 in IFNγ and IFNγ OT-I Tmem. Representative

634 plots depict cells from the same animal across different stimulation conditions. Symbols in b and c

635 represent a T cell population within a unique animal with symbols connected by animal identity (n = 6

636 across 2 experiments). Bars represent mean and indicated statistical significances were calculated by

637 Wilcoxon matched-pairs signed rank test. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

638

639 Figure 3 Cytokine-mediated TOX induction is limited in exhausted T cells

640 a-b Changes in TOX expression within LM-expanded TCR transgenic Tmem: OT-I, specific for OVA Ag

641 and gBT-I, specific for gB Ag. MACS-enriched T cells from LM-expanded OT-I or gBT-I memory mice

642 were stimulated with media alone (mock), recombinant IL-12, -15, and -18 in combination (IL-12/15/18)

643 (each at 100ng/mL), or anti-CD3/CD28 microbeads (TCR) at a ~1:1 cell:bead ratio. a, b Representative

644 TOX expression and TOX MedFI fold change during stimulation in LM-primed a OT-I and b gBT-I Tmem.

645 c-d Changes in TOX expression within LCMV-specific TCR transgenic P14 T cells expanded by acute

646 (Armstrong, Arm.) or chronic (Docile, Doc.) LCMV infection. c, d Representative TOX expression and

647 TOX MedFI fold change during stimulation in P14 T cells primed by c LCMV Armstrong and d LCMV

648 Docile. TOX MedFI fold change in a-d was calculated against average TOX MedFI within mock

649 stimulation in a batch-specific, timepoint-specific manner. We calculated indicated statistical

650 significances in a-d using Wilcoxon matched-pairs signed rank tests. Each symbol in a-d represents a

651 sample at a unique timepoint/condition, with bars delineating mean, which are connected by donor in a-d

652 (n = 4 LM-OVA expanded OT-I memory mice across 2 experiments; n = 10 LM-gB expanded gBT-I

653 memory mice across 2 experiments; n = 17 LCMV Armstrong-expanded P14 memory mice across 4

654 experiments; n = 8 LCMV Docile-expanded P14 memory mice across 2 experiments). Mouse identities

655 are consistent between representative flow plots within the same generation/adoptive transfer condition.

656

657 Figure 4. Inflammatory cytokines are potent inducers of TOX and PD-1 in human Tmem

658 a Basal expression of TOX and PD-1 in CD8 Tmem and Tnaïve. b TOX MedFI across PD-1 low, medium,

659 and high expressing CD8 Tmem. c Schematic detailing T cell isolation from cryopreserved PBMCs and

660 subsequent stimulation with recombinant IL-6, IL-15, IL-12 and -18, IL-12 and -15 and -18 (all at

661 100ng/mL, each), or anti-CD3/CD28 microbeads (TCR, 1:1 bead to cell ratio) and subsequent flow

hi 662 interrogation. d TOX expression (MedFI) and PD-1 frequency in CD8 Tmem throughout stimulation time

663 course. e, f Comparison of TOX MedFI and PD-1hi frequency in mock-, IL-12/15/18-, and TCR-stimulated

664 e CD8 Tmem and f CD8 Tnaïve. In a, b, d, e, f we calculated indicated statistical significances by a, d bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

665 Wilcoxon matched-pairs signed rank tests, b Friedman test with Dunn’s multiple comparisons tests, or e,

666 f Mann-Whitney tests. In a, d each symbol represents a unique timepoint/treatment connected by donor

667 with bars indicating mean a (n = 23 across four experiments) d (n = 11 across two experiments). In e, f

668 each symbol represents the mean ± SD of the stimulation condition from n = 23 donors across four

669 experiments. Representative plots from a, d, f are sourced from the same donor.

670

671 Figure 5. TOX and PD-1 upregulation are largely independent of Tmem subset

672 a-c Basal and stimulation-induced TOX and PD-1 expression in CD8 memory subsets. a Representative

673 gating of CD8 T cells into naïve (Tnaïve, grey), central memory (TCM, orange), effector memory (TEM,

674 purple), and effector memory CD45RA-expressing (TEMRA, green) subsets. b basal expression levels

675 (MedFI) of TOX and TCF1 and frequency of PD-1hi cells across CD8 T cell memory subsets. c TOX

676 MedFI, PD-1hi frequency, and TCF1 MedFI after mock (black), IL-12/15/18 (each at 100ng/mL, blue), or

677 TCR (1:1 bead to cell ratio, red) stimulation in CD8 TCM (left column), CD8 TEM (center column), and CD8

678 TEMRA (right column). Symbols in b and c represent unique samples (by timepoint/condition/subset) and

679 are connected by donor identity, with bars representing mean. We determined statistical significances in

680 b and c respectively using Friedman tests and Wilcoxon matched-pairs signed rank tests. b and c depict

681 n = 23 donors across four experiments, except for TCF1 plots, which depict n = 12 donors across two

682 experiments.

683

684 Figure 6. Stimulation induces TOX and PD-1 expression in conventional and innate-like T cells

685 a-b TOX and PD-1 induction in influenza A virus (IAV)-specific CD8 T cells. a Gating and memory

686 phenotyping of IAV-specific CD8 T cells. b Induction of TOX and PD-1 in IAV- specific CD8 T cells by

687 mock (black) or IL-12/15/18 (each at 100ng/mL, blue) stimulation. c-d TOX and PD-1 induction in

688 mucosal associated invariant T (MAIT) cells. c Gating and memory phenotyping of MAIT cells. d

689 Induction of TOX and PD-1 in MAIT cells by mock (black) or IL-12/15/18 (each at 100ng/mL, blue)

690 stimulation. Representative plots in a-d are sourced from the same donor. Symbols in a-d represent

691 unique samples (by timepoint/condition/subset) and are connected by donor identity, with bars bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

692 representing mean. We determined statistical significances in b and d using Wilcoxon matched-pairs

693 signed rank tests. a and b depict n = 8 donors across two experiments; c and d depict n = 23 donors

694 across four experiments.

695

696 Figure 7. TOX deficiency does not abrogate stimulation-induced PD-1 expression

–/– 697 a-c Stimulation-induced PD-1 expression in WT and Tox P14 Tmem. T cells were stimulated with media

698 alone (mock), recombinant IL-12, -15, and -18 in combination (IL-12/15/18 or ILs) (each at 100ng/mL), or

699 with anti-CD3/CD28 microbeads at an ~1:1 cell:bead ratio (TCR). a, b PD-1 MedFI and expression

–/– 700 frequencies in a WT or b Tox P14 Tmem over stimulation time course. c Comparison of PD-1 MedFI and

–/– 701 expression frequencies between IL-12/15/18 (left) or TCR (right) stimulated WT and Tox P14 Tmem. All

702 indicated statistical significances were calculated using Mann-Whitney tests. Symbols in a and b

703 represent the mean ± SD from all animals at a specific time/condition; and symbols in c represent

–/– 704 stimulated P14 Tmem populations within a single animal (n = 9 WT P14 recipients and n = 10 Tox P14

705 recipient across 2 experiments). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license. a b OT-I T 1×104 CD8 mem p = 0.0001 1×106 PFU OT-I T 24h p = 0.0001 naïve VSV-OVA 10000 p = 0.0001 100 (CD45.1) 8000 80 p = 0.0001 i.v. 6000 60 4000 40 IL-12/15/18 48h 2000 %PD- 1 20 >60d Mock PD-1 MedF I 0 0 TCR 0 24 48 0 24 48 C57BL/6J VSV-OVA Time (hours) Time (hours)

(CD45.2) OT-I memory 4 5 0 10 10 IL-12/15/18 stim IL-12/15/18 stim mouse PD-1 (BV605)

20000 IL-12/15/18 24h 48h Harvest Harvest Harvest 15000 Mock Mock v ILs Mock v ILs Spleen (t = 0h) (t = 24h) (t = 48h) 10000 TCR p = 0.1371 p < 0.0001 and LN T cells 5000 Mock v TCR Mock v TCR p < 0.0001 p < 0.0001 Flow PD-1 MedF I 0 0 24 48 analysis MACS Time (hours) VSV-OVA +Media “mock” 100 24h 48h OT-I memory or 80 Mock v ILs Mock v ILs mouse +rIL-12, rIL-15, rIL-18 60 (100ng/mL each) “ ” p = 0.0690 p = 0.0001 IL-12/15/18 40 Mock v TCR Mock v TCR or %PD- 1 20 p < 0.0001 p < 0.0001 +anti-CD3/CD28 beads 0 (1:1 bead:cell) “TCR” 0 24 48 Time (hours) c d OT-I Tmem IL-12/15/1824h24h Mock 6000 TCR 24h 48h 4000 Mock v ILs Mock v ILs OT-I T p = 0.0274 p = 0.0021 mem 48h48h 2000 Mock v TCR Mock v TCR IL-12/15/18 24h OX MedF I Mock 100 T p = 0.0030 p < 0.0001 24h 48h 0 TCR 80 0 24 48 Mock v ILs Mock v ILs 60 Time (hours) p = 0.0002 p < 0.0001 4 40

%TCF1 Mock v TCR Mock v TCR 0 10 48h 20 p = 0.1297 p = 0.0012 TOX (APC) 0 0 24 48 Time (hours) p = 0.0001 4 5 0 10 10 p = 0.0001 5 5 p = 0.0001 TCF1 (PE) 4 p = 0.0001 4 3 3 2 2 OX MedF I 1 OX MedF I 1 T T Fold chang e 0 Fold chang e 0 0 24 48 0 24 48 Time (hours) Time (hours) IL-12/15/18 stim TCR stim bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

a +GolgiPlug 1:1000 Harvest Spleen (t = 0h) (t = 18h) (t = 24h) and LN T cells Flow analysis MACS VSV-OVA +Media “ ” OT-I memory mock or mouse +rIL-12, rIL-15, rIL-18 (100ng/mL each) “IL-12/15/18” or +anti-CD3/CD28 beads (1:1 bead:cell) “TCR” b OT-I Tmem IL-12/15/18 TCR p = 0.0312 1000 Mock Mock p = 0.0312 800 3 10 600 400

0 OX MedF I 200 TOX (APC) T 0 IFNγ– IFNγ– IFNγ+ IFNγ– IFNγ+ 3 4 5 3 4 5 0 10 10 10 0 10 10 10 IFNγ (BUV737) Mock IL-12/15/18 TCR c OT-I Tmem p = 0.0312 5 10 IL-12/15/18 TCR 100 Mock Mock p = 0.0312 4 10 80 60 3 10 40 %PD- 1 20 PD-1 (BV605) 0 0 IFNγ– IFNγ– IFNγ+ IFNγ– IFNγ+ 3 4 5 3 4 5 0 10 10 10 0 10 10 10 IFNγ (BUV737) Mock IL-12/15/18 TCR bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

a b p = 0.0020

5 p = 0.0286 5 p = 0.0020 OT-I Tmem 4 p = 0.0286 gBT-I Tmem 4 24h 3 IL-12/15/18 24h 3 2 2

Mock ) ) 1 1 TCR mem mem 0 0 p = 0.0020 IL-12/15/18 48h p = 0.0286 48h p = 0.0020 5 5 Mock p = 0.0286 (of OT-I T TCR 4 (of gBT-I T 4 3 3 LM-gB expanded OT-I memory mice gBT-I memory mice LM-OVA expanded

2 Fold change TOX MedFI 2 4 Fold change TOX MedFI 4 0 10 1 0 10 1 TOX (APC) 0 TOX (APC) 0 0 24 48 0 24 48 Time (hours) Time (hours) IL-12/15/18 or TCR stim IL-12/15/18 or TCR stim

c d exhausted 5 p < 0.0001 5 p = 0.1094 P14 Tmem 4 p < 0.0001 P14 Tmem 4 24h 3 24h 3 p = 0.6406 2 2 ) 1 ) 1 mem 0 mem 0

IL-12/15/18 48h p < 0.0001 IL-12/15/18 48h Mock 5 Mock 5 p = 0.0781 (of P14 T 4 p < 0.0001 (of P14 T 4 TCR LCMV Docile TCR p = 0.0078 3 3 LCMV Armstrong P14 memory mice P14 memory mice 2

Fold change TOX MedFI 2 3 4 3 4 Fold change TOX MedFI 0 10 10 1 0 10 10 1 TOX (e660) 0 TOX (e660) 0 0 24 48 0 24 48 Time (hours) Time (hours) IL-12/15/18 or TCR stim IL-12/15/18 or TCR stim bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

a b CD8 T Mid mem Lo Hi CD8 T CD8 T cells naïve

5 10 e 3 3 4 3 4 5 3 4 5 CD8 -10 0 10 10 0 10 10 10 0 10 10 10

4 ) 10 TOX (APC) PD-1 (BB700) PD-1 (BB700) Tmem 1500 24h 48h me m 3 Mock v ILs Mock v ILs 10 p < 0.0001 1000 CD8 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.0012 0 500 Mock v TCR Mock v TCR Tnaïve OX MedF I

CD45RO (BB515) 800 1500 p < 0.0001 1500 p = 0.0059 p < 0.0001 p < 0.0001 T 3 4 5

0 10 10 10 600 T (of CD8 0 1000 1000 CCR7 (PE-Cy7) 400 0 24 48 500 200 500 Time (hours) OX MedF I OX MedF I PD-1 MedF I T 0 0 T 0 lo hi mi d me m me m naïv e naïv e PD- 1 PD- 1 ) PD- 1 100 T CD8 T CD8 T CD8 IL-12/15/18 T CD8 CD8 Tmem 24h 48h hi me m 80 TCR Mock v ILs Mock v ILs 60 Mock p = 0.0004 p < 0.0001 40 Mock v TCR Mock v TCR %PD- 1 20 p < 0.0001 p < 0.0001 c Harvest Harvest Harvest T (of CD8 0 (t = 0h) (t = 24h) (t = 48h) 0 24 48 Bulk cells T cells Time (hours) Flow MACS analysis Human +Media “mock” or PBMC +rIL-6 (100ng/mL each) “IL-6” or +rIL-15 (100ng/mL each) “IL-15” or +rIL-12, rIL-18 (100ng/mL each) “IL-12/18” or +rIL-12, rIL-15, rIL-18 (100ng/mL each) “IL-12/15/18” or +anti-CD3/CD28 beads (1:1 bead:cell) “TCR” f Gated on CD8 Tnaïve Mock IL-12/15/18 TCR 24h 24h d Gated on CD8 T Gated on CD8 T mem mem 48h 48h 24h 48h 24h 48h Stimulation TCR

3 3 4 3 4 5 IL-12/15/18 -10 0 10 10 0 10 10 10 TOX (APC) PD-1 (BB700) IL-12/18 IL-15 IL-6 ) Mock 1500 IL-12/15/18 24h 48h 3 3 4 3 3 4 3 4 5 3 4 5 -10 0 10 10 -10 0 10 10 0 10 10 10 0 10 10 10 na ïv e 1000 TCR Mock v ILs Mock v ILs TOX (APC) PD-1 (BB700) Mock p < 0.0001 p < 0.0001 500 Mock v TCR Mock v TCR OX MedF I

T p < 0.0001 p < 0.0001

T (of CD8 0 Mock IL-6 IL-15 IL-12/18 IL-12/15/18 TCR 0 24 48 p = 0.0068 Time (hours) p > 0.9999 p = 0.0254 p = 0.0010 p = 0.1192 p = 0.0010 1500 p = 0.0674 p = 0.0010 p = 0.0029 ) p > 0.9999 p = 0.5771 p = 0.0527

me m 1000 ) 100 24h 48h hi 500 na ïv e 80 OX MedF I Mock v ILs Mock v ILs T 60 T (of CD8 p = 0.0140 p < 0.0001 40 0 Mock v TCR Mock v TCR %PD- 1 20 p = 0.0010 p < 0.0001 p < 0.0001

T (of CD8 0 100 p = 0.0010 ) 0 24 48 80 hi me m p = 0.0010 Time (hours) 60 p = 0.0010 p = 0.0010 p = 0.0537 p = 0.7646 p = 0.2754 40 p = 0.0010 p = 0.0010 p = 0.4648 p = 0.0273 %PD- 1 20 T (of CD8 0 0 24 48 0 24 48 0 24 48 0 24 48 0 24 48 0 24 48 Time (hours) bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

a CD8 T cells b 5 1500 40 6000 10 Tnaïve v TCM p = 0.0368 Tnaïve v TCM p = 0.0002 Tnaïve v TCM p = 0.3467 T v T p < 0.0001 30 T v T p < 0.0001 T v T p < 0.0001 4 naïve EM hi naïve EM naïve EM 10 1000 4000 T v T p < 0.0001 Tnaïve v TEMRA p = 0.0006 T v T p < 0.0001 naïve EMRA 20 naïve EMRA 3 10 TCM v TEM p = 0.0024 TCM v TEM p = 0.0015 TCM v TEM p = 0.0685

500 %PD- 1 2000 OX MedF I T v T p = 0.0004 T v T p > 0.9999 T v T p = 0.0094 T CM EMRA 10 CM EMRA CM EMRA TCF1 MedF I 0 TEM v TEMRA p > 0.9999 TEM v TEMRA p = 0.0006 TEM v TEMRA p > 0.9999 CD45RO (BB515) 0 0 0 3 4 5 0 10 10 10 EM EM EM CM CM CM naïv e naïv e naïv e EMR A EMR A CCR7 (PE-Cy7) EMR A T CD8 T CD8 T T T CD8 T CD8 T CD8 EM CM T CD8 T CD8 T CD8 T CD8 T CD8 T CD8 T CD8 TEMRA Tnaïve c CD8 TCM CD8 TEM CD8 TEMRA Mock IL-12/15/18 TCR Mock IL-12/15/18 TCR Mock IL-12/15/18 TCR

p < 0.0001 p < 0.0001 p < 0.0001 p = 0.0003 p = 0.0005 ) p = 0.0099 p = 0.3806 ) p < 0.0001 p < 0.0001 ) p = 0.0023 2000 p = 0.0009 2000 p < 0.0001 p < 0.0001 2000 p < 0.0001 EM CM p = 0.0698 p = 0.0026

p = 0.6010 EMR A 1500 p = 0.4318 1500 1500 1000 1000 1000 500 500 500 OX MedF I OX MedF I OX MedF I T T T T (of CD8 T (of CD8 0 0 0 T (of CD8

p < 0.0001 p < 0.0001

p < 0.0001 ) ) p < 0.0001 ) p < 0.0001 p < 0.0001 100 100 p = 0.0001 100 EM CM hi 80 p < 0.0001 p < 0.0001 hi 80 p = 0.0010 hi 80 p = 0.0016 p < 0.0001 p < 0.0001 EMR A 60 p = 0.0005 p = 0.2112 60 p < 0.0001 60 p = 0.0029 p < 0.0001 40 40 40 %PD- 1 20 %PD- 1 20 %PD- 1 20 T (of CD8 T (of CD8 0 0 0 T (of CD8

p = 0.8501 p = 0.0005 p = 0.0005

p = 0.5186 p = 0.0005 p = 0.0005 p = 0.0049 p = 0.0005 p = 0.0005 ) p = 0.0049 )

) p = 0.0005 p = 0.0005 5000 5000 5000 p = 0.1099 EM CM p = 0.0005 p = 0.0024 4000 4000 p = 0.0005 4000 p = 0.0005 p = 0.0005 EMR A 3000 3000 3000 2000 2000 2000 1000 1000 1000 TCF1 MedF I TCF1 MedF I TCF1 MedF I T (of CD8 T (of CD8 0 0 0 0 24 48 0 24 48 0 24 48 0 24 48 0 24 48 0 24 48 T (of CD8 0 24 48 0 24 48 0 24 48 Time (hours) Time (hours) Time (hours) (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade b a bioRxiv preprint 48h 24h -10 -10 IAV tetramer (PE) 10 10 10 10 0 2 2 3 4 5 3 IAV-specific CD8 T cells TOX (APC) 0 CCR7 (PE-Cy7) 0 CD8 Tcells 10 3 IAV-specific CD8 Tcells Gated on 10 3 10 4 10 4 48h 24h PD-1 (BB700) 0 10 IL-12/15/18 5 doi: 10 3 IAV-specific CD8 Tcells https://doi.org/10.1101/2021.03.15.435527 Mock 10

4 CD45RO (BB515) 10 10 10 0 3 4 5 10 5 CCR7 (PE-Cy7) 0

TOX MedFI hi 10 3 %PD-1 (of IAV-specific CD8 Tcells (of IAV-specific

CD8 T ) 10

1000 me1500 m 2000 4 CD8 Tmem) 500 20 40 60 80 0 0 10 5 p p T T Time Time =0.1953 =0.6875 448 24 0 448 24 0 EM EMRA p p =0.4609 =0.9453 Mock available undera ( ( T hours) hours) T CM naïve

% of IAV-specific

1000 1500 2000 CD8 T cells 100 500 20 40 60 80 20 40 60 80 0 0 0 p p Time Time =0.0156 =0.0156 IL-12/15/18 448 24 0 448 24 0 Tnaïve p p =0.0078 =0.0078 CC-BY-ND 4.0Internationallicense ( ( TCM hours) hours) T EM ; this versionpostedMarch16,2021. TEMRA d c

MR1-10 tetramer (BV421) 48h 24h -10 10 10 10 0 3 3 4 5 3 TOX (APC) -10 Gated onMAIT cells 0 3 CD8 (BUV395) Bulk Tcells MAIT cells 0 10 3 10 3 10 4 10 4 48h 24h PD-1 (BB700) 0 10 IL-12/15/18 5 10 3 Mock 10 MAIT cells 4 CD45RO (BB515) 10 10 10 0 . 3 4 5 10 5 CCR7 (PE-Cy7) 0 The copyrightholderforthispreprint

TOX MedFI 10

hi 3 %PD-1 (of MAIT) CD8 Tcells (of MAIT) 1000 2000 3000 100 10 20 40 60 80 4 0 0 10 5 p p Time Time =0.7809 <0.0001 448 24 0 448 24 0 T T p p EM =0.2002 =0.0327 EMRA Mock ( ( hours) hours) T T CM naïve

1000 2000 3000 % of MAIT 100 100 20 40 60 80 20 40 60 80 0 0 0 p p <0.0001 Time Time <0.0001 IL-12/15/18 448 24 0 448 24 0 Tnaïve p p <0.0001 <0.0001 T ( ( CM hours) hours)

TEM

TEMRA bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435527; this version posted March 16, 2021. 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-ND 4.0 International license.

25000 30000 a 24h 48h c p = 0.3154 20000 Mock v ILs Mock v ILs 20000 IL-12/15/18 24h 15000 p < 0.0001 p < 0.0001 p = 0.4967 Mock 10000 Mock v TCR Mock v TCR 10000 mem TCR 5000 p < 0.0001 p < 0.0001 PD-1 MedF I PD-1 MedF I 0 0 0 24 48 p = 0.0052 48h Time (hours) 100 p = 0.4123 80 100 24h 48h 60 WT P14 T 80 Mock v ILs Mock v ILs 60 40 p < 0.0001 p < 0.0001 %PD- 1 20 4 5 40 0 10 10 Mock v TCR Mock v TCR %PD- 1 0 PD-1 (BV605) 20 p < 0.0001 p < 0.0001 WT Tox–/– WT Tox–/– 0 P14 P14 P14 P14 0 24 48 24h 48h Time (hours) IL-12/15/18

b p = 0.2110 25000 24h 48h 30000 p = 0.0329 20000 Mock v ILs Mock v ILs 20000 IL-12/15/18 24h 15000 p < 0.0001 p < 0.0001 Mock 10000 mem Mock v TCR Mock v TCR 10000 TCR 5000 p < 0.0001 p < 0.0001 PD-1 MedF I PD-1 MedF I 0 0 0 24 48 p = 0.0750 p = 0.6163

P14 T 48h Time (hours) 100

–/– 80 100 24h 48h 60 80 Mock v ILs Mock v ILs Tox 60 40 p < 0.0001 p < 0.0001 %PD- 1 20 4 5 40 0 10 10 Mock v TCR Mock v TCR %PD- 1 0 PD-1 (BV605) 20 p < 0.0001 p < 0.0001 WT Tox–/– WT Tox–/– 0 P14 P14 P14 P14 0 24 48 24h 48h Time (hours) TCR