IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During 2 Trypanosoma Cruzi Infection

bioRxiv preprint doi: https://doi.org/10.1101/314336; this version posted May 11, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

1 IL-17RA-signaling modulates CD8+ T cell survival and exhaustion during 2 Trypanosoma cruzi infection

3 Jimena Tosello Boari1,2, Cintia L. Araujo Furlan1,2, Facundo Fiocca Vernengo1,2, Constanza 4 Rodriguez1,2, María C. Ramello1,2, María C. Amezcua Vesely1,2, Melisa Gorosito Serrán1,2, 5 Nicolás G. Nuñez3,4, Wilfrid Richer3,4, Eliane Piaggio3,4, Carolina L. Montes1,2, Adriana 6 Gruppi1,2, Eva V. Acosta Rodríguez1,2*.

7 1 Departamento de Bioquímica Clínica. Facultad de Ciencias Químicas, Universidad 8 Nacional de Córdoba, Córdoba, X5000HUA. Argentina.

9 2 Centro de Investigaciones en Bioquímica Clínica e Inmunología. CONICET. Córdoba, 10 X5000HUA. Argentina.

11 3 SiRIC TransImm «Translational Immunotherapy Team», Translational Research 12 Department, Research Center, PSL Research University, INSERM U932, Institut Curie, 13 Paris, 75005. France.

14 4 Centre d’Investigation Clinique Biothérapie CICBT 1428, Institut Curie, Paris, 75005. 15 France.

16 Running title: IL-17RA signaling regulates CD8+ T cell responses to T. cruzi

17 *Corresponding autor:

18 Eva V. Acosta Rodríguez

19 [email protected]

21 Abstract

22 The IL-17 family contributes to host defense against many intracellular pathogens by 23 mechanisms not fully understood. CD8+ T lymphocytes are key elements against 24 intracellular microbes and their survival and appropriate response is orchestrated by several 25 cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen- 26 specific CD8+ T cell immunity. Absence of IL-17RA and IL-17A/F during Trypanosoma 27 cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite- 28 specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite- 29 specific CD8+ T cells while recombinant IL-17 in vitro down-regulated the pro-apoptotic 30 protein BAD and promoted activated CD8+ T cell survival. Phenotypic, functional and 31 trancriptomic profiling showed that T. cruzi-specific CD8+ T cells arising in IL-17RA- 32 deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the 33 magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer 34 experiments established that IL-17RA-signaling is intrinsically required for the proper 35 maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA 36 and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.

39 Introduction

40 IL-17A and IL-17F were initially associated to the pathogenesis of autoinflammatory and 41 autoimmune disorders [1]. Nonetheless, the primary function of these cytokines is likely 42 host protection against microbes. Indeed, IL-17A and/or IL-17F-deficient mice are highly 43 susceptible to a wide array of infections with fungi and extracellular bacteria but also with 44 viruses and parasites [2,3]. We previously demonstrated that cytokines of the IL-17 family 45 play a critical role in host survival by regulating exuberant inflammation and 46 immunopathology during infection with the protozoan Trypanosoma cruzi [4,5]. Additional 47 data from our laboratory suggested that the IL-17 cytokines played additional protective 48 roles against T. cruzi by modulating adaptive immunity. 49 The IL-17 family is constituted by 6 members: IL-17A to IL-17F that have different 50 cellular sources and expression patterns but often show overlapping activities. They signal 51 through a receptor complex composed of at least two identical or different subunits of the 52 IL-17R family (IL-17RA to IL-17RE). IL-17RA is the common signaling subunit used by 53 at least four ligands: IL-17A, IL-17C, IL-17E and IL-17F. IL-17 members regulate 54 inflammation by recruiting and activating neutrophils, NK cells and other cells of the innate 55 immune system and by inducing several pro-inflammatory mediators (i.e. cytokines, 56 chemokines, microbial peptides and metalloproteinases). In addition, these cytokines play 57 important roles during adaptive immune responses including the modulation of germinal 58 center reactions as well as the regulation of Th1 and CD8+ cellular responses [6-9]. 59 Infection with T. cruzi causes Chagas’ disease that is the third most frequent parasitic 60 disease worldwide, endemic in Latin America and increasing globally due to migratory 61 flows. Disease progression, from symptomless to severe cardiac and digestive forms, is 62 linked to parasite heterogeneity and variable host immune response. CD8+ T cell mediated 63 immunity is essential for parasite control throughout all the stages of the infection, although 64 not sufficient for complete parasite elimination [10,11]. CD8+ T cell deficient mice are 65 extremely susceptible to infection [12,13] and strategies that improve specific CD8+ T cell 66 responses result in increased host protection [11,14 ,15]. Considering this and that parasite 67 persistence correlates with disease severity in experimental and human T. cruzi infection 68 [16,17], many efforts are focused at understanding the mechanisms that promote protective 69 anti-parasite CD8+ T cell immunity.

70 The general features of protective CD8+ T cell responses (as defined with model viral and 71 bacterial infections) consist of the generation and expansion of short-lived, highly 72 functional effector populations that operate to clear the microbes. After pathogen control, 73 most of the effector cells die and the few remaining cells differentiate into memory T cells, 74 contributing to long-lived immunological protection [18]. During certain chronic infections, 75 persistently activated CD8+ T cells acquire a state of cell dysfunction characterized by the 76 hierarchical loss of effector functions and proliferation potential accompanied by sustained 77 expression of multiple inhibitory receptors. Severe exhaustion leads to pathogen-specific 78 cells with reduced ability to produce cytokines or to degranulate and prone to deletion. 79 These exhausted CD8+ T cells fail to provide optimal protection contributing to poor 80 pathogen control [19]. Antigen-specific and costimulatory signals are the driving forces of 81 CD8+ T cell responses, but also cytokines (i.e. IL-21, IL-2, IL-12) are required to provide 82 the “third-signal” essential for protective CD8+ T cell immunity [20,21]. 83 Herein, we demonstrate that IL-17RA signaling is required for the maintenance of robust 84 specific CD8+ T cell responses and host resistance to T. cruzi. We show that absence of IL- 85 17RA signaling during this infection alters the transcriptional program of the effector 86 CD8+ T cells affecting their survival, effector function and exhaustion. Our findings are of 87 relevance for a better understanding of the role of IL-17 family on the orchestration of 88 protective immunity against infections. 89

90 Results

91 IL-17RA and IL-17A/IL-17F deficiencies compromise parasite control and reduce the 92 magnitude of the specific CD8+ T cell response during T. cruzi infection.

93 We previously showed that IL-17RA-signaling and IL-17-secreting B cells are critically 94 required for host survival during T. cruzi infection by, at least in part, regulating innate 95 immunity and inflammation [4,5]. In addition, histological data from these initial studies 96 suggested that infected IL-17RA knockout (KO) mice exhibited increased parasite levels in 97 tissues but similar parasitemia than infected wild-type (WT) counterparts [4]. To confirm 98 this and study possible additional protective mechanisms mediated by IL-17, we evaluated 99 tissue parasitism in infected wild-type (WT) and IL-17RA knockout (KO) mice. As 100 determined by the amounts of parasite DNA quantified in spleen, we observed that T. cruzi 101 infected IL-17RA KO mice controlled tissue parasitism similar to WT mice during the first 102 two weeks of infection (Fig 1A). However, IL-17RA KO mice failed to reduce parasite 103 burden at later stages of infection, as attested by the increased parasitism in the spleen at 22 104 and 130 day post-infection (dpi) (Fig 1A). Furthermore, increased parasite levels were also 105 detected in other organs such as liver and heart (Fig 1B-C).

106 As the aforementioned result suggested a deteriorated adaptive immune response, we 107 focused at studying the role of IL-17RA in the development of parasite-specific CD8+ T 108 cells. To this end, we examined the generation of CD8+ T cells specific for the 109 immunodominant epitope TSKB20 (T. cruzi trans-sialidase amino acids 569-576 – 110 ANYKFTLV–) [22] in infected IL-17RA KO and WT mice. We determined that the 111 frequency of TSKB20-specific CD8+ T cells was reduced in spleen, liver and blood of 112 infected IL-17RA KO mice at 20 dpi (Fig 1D). Kinetics studies showed that the frequency 113 and absolute numbers of spleen TSKB20-specific CD8+ T cells detected in infected IL- 114 17RA KO mice was similar to that infected WT controls up to 14 dpi, but these values 115 dropped dramatically soon after (Fig 1E). Remarkably, total CD8+ T cell numbers at 20 dpi 116 were also reduced in the spleen of infected IL-17RA KO mice (Figure 1F). In agreement 117 with the notion that IL-17A and IL-17F are the major IL-17RA-signaling cytokines able to 118 modulate immune responses, IL-17A/IL-17F double knockout (DKO) mice also showed

119 increased tissue parasitism in spleen and liver (Fig S1A) and reduced frequency of parasite- 120 specific CD8+ T cells in comparison to WT controls (Fig S1B).

121 Considering the kinetics of the TSKB20-specific CD8+ T cell immunity observed in 122 infected IL-17RA KO mice (Fig 1E) as well as our reported results showing that IL-17 123 production peaked around 14 dpi in this experimental infection model [4,5], we speculated 124 that IL-17RA signaling plays a role after priming, during the expansion and maintenance of 125 the parasite-specific CD8+ T cells. To test this, we performed neutralization experiments in 126 which infected WT mice were injected with anti-IL-17A Abs from 13 dpi and during a 127 limited period (Fig 1G). Blockade of IL-17 signaling specifically during the 128 expansion/maintenance phase resulted in a significant decrease in the frequency of 129 TSKB20-specific T cells (Fig 1H). Indeed, mice treated with anti-IL-17 only after the 130 CD8+ T cell response was already established (from 13 to 19 dpi), showed a frequency of 131 parasite-specific T cells comparable to that observed in infected IL-17RA KO mice that 132 lacked IL-17 signaling throughout the infection (Fig 1I).

133

134 Absence of IL-17RA signaling during T. cruzi infection reduces survival of total and 135 specific CD8+ T cells.

136 Considering the conventional kinetics of T cell responses [18], it was conceivable that the 137 abortive CD8+ T cell response observed in infected IL-17RA KO mice could be a 138 consequence of a reduced cell expansion and/or increased contraction. To address the role 139 of IL-17RA in CD8+ T cell proliferation we performed in vivo experiments of BrDU 140 incorporation between days 15 to 20 dpi, time points that directly preceded the dramatic 141 decrease in the numbers of specific CD8+ T cells in infected IL-17RA KO mice. Results 142 depicted in Fig 2A demonstrated that absence of IL-17RA does not compromise 143 proliferation of CD8+ T cells during T. cruzi infection. Complementary studies of ex vivo 144 detection of the Ki-67 antigen led to similar conclusions (Fig S2A). Then, we tested a 145 possible role of IL-17RA signaling in supporting CD8+ T cell survival. In comparison to 146 counterparts from infected WT mice, total and TSKB20-specific CD8+ T cells from 147 infected IL-17RA KO mice showed significantly higher frequencies of apoptotic cells

148 identified by TMRE-based mitochondrial membrane potential assay as early as 10 dpi and 149 also at later time points (20 dpi) (Fig 2B). Similar results were obtained by analyzing 150 Annexin V+/7AAD- cells (Fig S2B). In the same direction, total and parasite-specific 151 CD8+ T cells from infected WT mice treated with anti-IL-17A Abs (Fig 1H) showed 152 slightly higher percentage of apoptotic cells in comparison to counterparts from controls \, 153 reaching levels of apoptosis similar to that determined in CD8+ T cells from infected IL- 154 17RA KO mice (Fig S2C).

155 Given the results described above, we evaluated whether cytokines that signal through IL- 156 17RA were able to directly increase survival of activated CD8+ T cells. CD8+ T cells 157 purified from the spleen of non-infected WT mice were activated during 24 h with plastic- 158 coated anti-CD3 and anti-CD28 Abs in the presence of recombinant cytokines. Addition of 159 IL-17A but not IL-17F, IL-17C and IL-17E lead to a modest but significant reduction in the 160 percentage of apoptotic activated CD8+ T cells (Fig 2C). Of note, the direct anti-apoptotic 161 effect of IL-17 in CD8+ T cells was comparable to that of IL-21, a well-recognized survival 162 factor for this population [23]. Trying to elucidate the mechanisms underlying the pro- 163 survival effects of IL-17A, we evaluated the expression of pro and anti-apoptotic members 164 of the Bcl-2 superfamily that are critically involved in the regulation of T cell death [24]. 165 We determined that IL-17A significantly downregulated the expression of Bad (Fig 2D). 166 Very small or no changes were induced in other members such as Bcl-xL, Bcl-2, Bim and 167 Bax (Fig S2D). Altogether, these results indicate that IL-17A may promote CD8+ T cell 168 survival by down-regulating pro-apoptotic factors of the Bcl-2 family.

169

170 Absence of IL-17RA perturbs the transcriptional program of CD8+ T cells activated during 171 T. cruzi infection.

172 To gain further insights of the overall impact of the absence of IL-17RA signaling, we 173 determined the gene expression profiles of purified CD8+ T cells using Affymetrix 174 microarrays. By normalizing gene expression to the corresponding non-infected samples, 175 we compared the transcriptional changes induced by the infection in CD8+ T cells from IL- 176 17RA KO mice versus counterparts from WT mice in a fold change/fold change plot. We

177 identified 4287 genes that showed a statistically significant difference in expression of over 178 1.2 fold (p<0.05) and were grouped into different sets according to the differential pattern 179 of expression (Fig 3A). Remarkably, 1647 genes (sets pink, orange and red) were expressed 180 at higher level while 2640 genes (sets light green, green and dark) were expressed at lower 181 levels in CD8+ T cells from IL-17RA KO infected mice in comparison to WT counterparts. 182 To further evaluate the biologic characteristics of the transcriptional landscape of CD8+ T 183 cells from infected IL-17RA KO and WT mice, we performed gene-enrichment set analysis 184 (GSEA) in all the genes differentially expressed. After reaching broad results using gene 185 ontology, immunological and curated databases, we decided to focus on specific gene sets 186 (Molecular Signatures DB-MSigDB) related with CD8+ T cell function and differentiation 187 such as GSEA26495 and GSEA41867. This supervised analysis showed that IL-17RA KO 188 CD8+ T cells were significantly enriched in six gene sets within which the most significant 189 enriched CD8+ T cell gene sets in the significance order (size of FWER P values) were 190 PD1hi versus PD1low (p<0.001) and Naïve versus PD1low (p<0.001) (Fig 3B). These results 191 denoted that CD8+ T cells elicited during T. cruzi infection in absence of IL-17RA showed 192 a phenotype with mixed features of naïve and dysfunctional cells.

193 Given the previous results, we then analyzed the expression patterns of genes selected 194 according to categories relevant for CD8+ T cell biology. As observed in the heat maps, 195 gene expression profiles of CD8+ T cells from WT and IL-17RA KO mice were similar 196 before infection but changed significantly after 22 dpi (Fig 3C). Indeed, genes encoding 197 activation and effector cell markers such as the KLRG family molecules, CD11c and CD69 198 showed completely reciprocal pattern of expression in CD8+ T cells from infected IL- 199 17RA KO versus WT mice. Furthermore, CD8+ T cells from infected IL-17RA KO mice 200 showed clearly increased levels of many genes encoding inhibitory and death receptors 201 including Ctla4, Lag3, Tim3, Pd1, Cd160, Tigit, and Fas. We also analyzed how IL-17RA 202 signaling modulated the expression of genes encoding TFs that regulate CD8+ T cell fate. 203 Of note, there was a group of TF-encoding genes that were significantly more up-regulated 204 in CD8+ T cells from infected IL-17RA KO mice. This group included genes such as Irf4 205 and Batf that are critical for the generation of effector CD8+ T cells [25-27]. Finally, we 206 determined that CD8+ T cells from infected WT and IL-17RA mice showed differences in 207 the expression of many genes encoding effector molecules, cytokine receptors and homing

208 and migration molecules that in overall supported the fact that absence of IL-17RA 209 influence CD8+ T cell fate.

210 To broadly analyze the significance of the transcriptional differences between CD8+ T cells 211 from infected IL-17RA KO mice versus WT counterparts, we performed an Ingenuity 212 Pathway Analysis (IPA) on genes marked in red in Figure 3A, which were up-regulated by 213 the infection in the CD8+ T cells from both groups of mice but were significantly expressed 214 at higher levels in CD8+ T cells from IL-17RA KO mice. The top 20 “Diseases and Bio- 215 functions”, “Canonical pathways” and “Up-stream regulators” that were most significantly 216 altered in CD8+ T cells elicited by T. cruzi infection in the absence of IL-17RA are shown 217 in the Fig 3D. Remarkably, “Cell death” and “Apoptosis of Lymphocytes” bio-functions 218 and the “p53 Signaling” canonical pathway emerged as significantly up-regulated. These 219 results support the notion that lack of IL-17RA signaling during T. cruzi infection 220 significantly affected the survival/senescence status and also participated in several 221 biological features of the CD8+ T cells. Finally, to elucidate which molecule/s could 222 govern the observed differences in gene expression, we performed an “upstream regulator 223 analysis”. Many modulators of the inflammatory responses including the cytokines IL-2 224 and IL-6 and the regulators of NFkB as CHUK and IKBKG were significantly up-regulated 225 while MKL-2 and MKL-1 that interacts with transcriptional regulator serum response 226 factor were down-regulated in CD8+ T cells from infected IL-17RA KO mice. 227 Remarkably, IRF4 and at a minor extent other TF such as BATF, STAT1 and AHR 228 emerged as potential upstream regulators of the transcriptional program of the CD8+ T 229 cells induced by T. cruzi infection in absence of IL-17RA signaling.

230 231 Absence of IL-17RA signaling during T. cruzi infection results in CD8+ T cell exhaustion. 232 Given microarray data and considering that the increased T cell deletion could be 233 consequence of a process of cell exhaustion, we analyzed the expression of inhibitory and 234 death receptors in CD8+ T cells from infected WT and IL-17RA KO mice. Compared to 235 CD8+ T cells from non-infected mice, total and TSKB20-specific CD8+ T cells from 236 infected WT mice presented a slight upregulation of PD-1 and TIGIT as well as similar 237 frequency of CTLA-4+ cells and reduced frequency of BTLA+ cells (Fig 4A). Noteworthy,

238 total and, particularly, TSKB20-specific CD8+ T cells from infected IL-17RA KO mice 239 showed significantly higher expression of PD-1 and TIGIT together with increased 240 frequency of CTLA-4+ and BTLA+ cells. Furthermore, these cells presented an apoptosis- 241 prone phenotype characterized by the expression of high levels of CD95/Fas and 242 CD120b/TNF-R2 (Figure 4B). Total and TSKB20-specific CD8+ T cells from infected IL- 243 17A/IL-17F DKO mice showed a profile of expression of inhibitory (PD-1) and death 244 receptors (Fas/CD95) that was comparable to that of counterparts from infected IL-17RA 245 KO mice (Fig S1C and D). 246 To address whether the phenotypic features of CD8+ T cells elicited in absence of IL-17RA 247 correlated with altered functionality, we compared the effector function of CD8+ T cells 248 from infected WT and IL-17RA KO mice. To this end, we analyzed the mobilization of 249 CD107a and secretion of effector cytokines upon specific and polyclonal activation in vitro. 250 Stimulation of splenocytes from infected IL-17RA KO mice with the TSKB20 peptide 251 resulted in significant reduced frequency of CD8+ T cells showing polyfunctional 252 characteristics (surface CD107a expression and IFNγ and/or TNF production) in 253 comparison to WT controls (Fig 4C, left panel and Fig S3). Although a poor response was 254 anticipated due to the low frequency of TSKB20-specific CD8+ T cells in infected IL- 255 17RA KO mice, the antigen-specific effector response of CD8+ T cells was barely different 256 from background. Stronger polyclonal stimulation with PMA and Ionomycin increased the 257 percentage of CD8+ T cells within splenocytes from infected IL-17RA KO mice showing a 258 polyfunctional response; however, the magnitude of this effector response was significantly 259 lower than that of WT counterparts (Fig 4C, right panel and Fig S3). 260 To determine if the higher parasite burden in infected IL-17RA KO mice may underlie the 261 upregulation of the inhibitory receptors observed in their CD8+ T cells, we evaluated the 262 phenotype of CD8+ T cells from WT mice infected with increasing parasite doses. 263 Remarkably, increasing loads of T. cruzi did not diminish the frequency of parasite-specific 264 CD8+ T cells nor promoted the upregulation of the inhibitory and death receptors evaluated 265 (Fig S4). 266

267

268 Checkpoint blockade partially restores parasite-specific CD8+ T cell immunity and 269 enhances parasite control in infected IL-17RA KO mice.

270 CD8+ T cell exhaustion has been associated with poor microbial control during many 271 infections as well as with tumor progression in cancer. In these settings, checkpoint 272 blockade has shown success to restore the magnitude and functionality of effector T cells 273 and to enhance the control of the insult [28]. Furthermore, PD-L1 blockade has been 274 effective to prevent T cell apoptosis in murine and human sepsis [29,30]. Our results 275 showing that lack of IL-17RA signaling during T. cruzi infection lead to poor CD8+ T cell 276 effector function and parasite persistence, prompted us to evaluate whether checkpoint 277 blockade could restore a resistant phenotype in infected IL-17RA KO mice. As PD-1 is 278 highly expressed on parasite-specific CD8+ T cells from infected IL-17RA KO mice, we 279 targeted this checkpoint receptor by injecting blocking anti PD-L1 Abs at 15 and 18 dpi, 280 before the contraction of parasite-specific CD8+ T cell response (Fig 5A). We determined 281 that PD-L1 blockade in infected IL-17RA KO significantly augmented the magnitude of the 282 TSKB20-specific CD8+ T cell response by 21dpi, particularly in spleen and liver (Fig 5B). 283 Similar response to treatment was observed in infected WT mice. Remarkably, the 284 enhanced T. cruzi-specific CD8+ T cell immunity correlated with a clear reduction in the 285 levels of tissue parasitism (Figure 5C). In addition, inhibition of PD-L1 also lead to a 286 milder infection as highlighted by the significantly reduced levels of several damage 287 biomarkers such as the transaminase aspartate aminotransferase (AST), and creatinin kinase 288 total (CK) and myocardial band (CK-MB) (Fig 5D). The reduction in the severity of the 289 infection (parasite persistence and tissue damage) upon PD-L1 checkpoint blockade was 290 more impressive in IL-17RA KO mice as they show an extremely high susceptibility to T. 291 cruzi infection in absence of treatment.

292

293 Intrinsic IL-17RA signaling modulates the maintenance and phenotype of CD8+ T cells 294 activated during T. cruzi infection.

295 To understand whether IL-17RA played direct or indirect roles in the modulation of CD8+ 296 T cell responses to T. cruzi, we first quantified in serum the concentration of IL-21, known

297 to modulate CD8+ T cell immunity. Of note, we determined that in comparison to WT 298 counterparts, infected IL-17RA KO mice exhibited increased concentration of IL-21 (Fig 299 6A). This result pointed out that high amounts of IL-21 are not able to compensate the lack 300 of IL-17RA signaling for the induction of robust CD8+ T cell responses during this 301 infection.

302 We next evaluated the possibility of a direct effect of IL-17RA signaling in the modulation 303 of CD8+ T cell responses based on the fact that CD8+ T cells not only express IL-17RA 304 but also upregulate it in response to cytokines that boost CD8+ T cell responses, such as IL- 305 21 [23,31]. The IL-17RA subunit interacts with IL-17RC to form the functional receptor for 306 both IL-17A and IL-17F (reviewed in [6]), but also with IL-17RD that differentially 307 regulate IL-17A-inducing signaling pathways [32,33]. Therefore, we evaluated by 308 quantitative PCR the amounts of mRNA encoding IL-17RA, IL-17RC and IL-17RD. In 309 agreement with published data, significant amounts of mRNA encoding IL-17RA were 310 detected in spleen CD8+ T cells (Fig 6B). These cells also presented the Il17rc and Il17rd 311 transcripts but at remarkably lower levels in comparison to Il17ra. Studies by flow 312 cytometry confirmed expression of IL-17RA on CD8+ T cells although at different levels 313 according to the CD8+ T cell subset, being maximal in memory cells, and lower in the 314 other subsets. T. cruzi infection upregulated IL-17RA expression in naïve and memory 315 CD8+ T cell subsets (Fig 6C).

316 To directly address the impact of intrinsic IL-17RA signaling in the development of CD8+ 317 T cell responses during T. cruzi infection, we performed a series of adoptive transfer 318 experiments. First, we transferred equal number of congenically marked IL-17RA deficient 319 (CD45.2+) and WT (CD45.1+) CD8+ T cells into F1 (CD45.1+/CD45.2+) WT hosts. 320 Recipient mice were infected and at 20 dpi we examined the presence of injected cells in 321 the total and TSKB20-specific CD8+ T cell pools. As expected, no CD45.2+ KO or 322 CD45.1+ WT CD8+ T cells were detected in infected F1 (CD45.1+/CD45.2+) WT hosts 323 that were non-injected (control mice). When analyzing the competitive adoptive transfer 324 experiment, we observed that within the population of injected cells, IL-17RA KO CD8+ T 325 cells were outcompeted by their IL-17RA-sufficient WT counterparts in total and parasite- 326 specific CD8+ T cell subsets all the organs examined including blood and spleen (Fig 6D).

327 To overcome the possible limitations derived from the presence of endogenous WT CD8+ 328 T cells in lymphoreplete hosts, we repeated this competitive experiment in CD8α-/- mice. 329 At 17 dpi, the frequency of IL-17RA KO cells doubled that of WT cells within the total 330 CD8+ T cells (Fig. 6E), likely as result of an increased homeostatic proliferation of 331 polyclonal IL-17RA KO CD8+ T cells that exhibit increased proliferative capacity (Fig 332 2A). In contrast, WT cells significantly outnumbered IL-17RA KO cells within the 333 TSKB20-specific CD8+ T cell pool, indicating that intrinsic expression of IL-17RA 334 provides a maintenance advantage particularly for parasite-specific CD8+ T cells. Indeed, 335 the ratio between the percentage of WT CD8+ T cells within TSKB20-specific gate and the 336 polyclonal total CD8+ T cells is significantly higher when compared to the same ratio of 337 IL-17RA KO CD8+ T cells (Fig 6F).

338 Phenotypic evaluation determined IL-17RA KO CD8+ T cells exhibited a higher 339 expression of the inhibitory receptor PD-1 (Fig 6G). Upon antigen specific stimulation with 340 TSKB20, WT CD8+ T cells exhibited a significantly higher polyfunctional effector 341 response in comparison to IL-17RA KO CD8+ T cell from the same host (Fig 6H, left 342 panel and Fig S5). An identical result was observed after polyclonal stimulation (Fig 6H, 343 right panel and Fig S5).

344 Discussion 345 During the last decades, many reports have delineated the immunological mechanisms 346 underlying IL-17-mediated roles in host protection against infection as well as in 347 inflammatory diseases [34,35]. In this paper, we uncover a novel mechanism by which IL- 348 17RA signaling regulates CD8+ T cell responses and promotes protection against a 349 protozoan infection. Our findings provide new elements to elucidate the cytokines and 350 pathways leading to robust CD8+ T cell responses against infections with intracellular 351 microbes and consequently, may have important implications for vaccine and therapy 352 design.

353 The IL-17 family has a well-established importance in host defense against extracellular 354 bacterial and fungal pathogens and numerous recent studies indicate that it also potentiates 355 immunity to intracellular bacteria, viruses and parasites [3]. So far, the IL-17-dependent 356 protective mechanisms were described to depend on the activation of non-immune and 357 innate immune cells to sustain inflammation [8] and, only indirectly, promote adaptive 358 cellular immune responses [36,37]. In this regard, there are antecedents about the role of 359 IL-17 cytokines in promoting specific CD8+ T cell responses that favored host resistance to 360 Listeria infection and melanoma [38,39]. In these settings, the IL-17-mediated induction of 361 protective CD8+ T cells depended on an enhanced recruitment of cross-presenting dendritic 362 cells but a direct role on CD8+ T cells was not ruled out. Very recently, IL-17 was shown 363 to directly potentiate CD8+ T cell cytotoxicity against West Nile Virus infection [9]. In the 364 same direction, we demonstrate that mice deficient in IL-17RA showed an abortive CD8+ 365 T cell response during T. cruzi infection characterized by an early reduction in the number 366 of parasite-specific CD8+ T cells. Concomitantly, infected IL-17RA KO mice showed poor 367 control of the parasite in target tissues such as spleen, liver and heart.

368 By using different experimental approaches that included a detailed phenotypic, functional 369 and transcriptional profiling of CD8+ T cell arising during T. cruzi infection in IL-17RA 370 KO mice as well as in vitro and in vivo assays, we demonstrated that CD8+ T cell-intrinsic 371 IL-17RA signaling, particularly during the expansion phase, is required to sustain CD8+ T 372 cell survival and therefore, a robust pathogen-specific CD8+ T cell immunity. Still, it 373 remains possible that the cross-talk of IL-17 with other cytokines that regulate CD8+ T cell

374 fate such as IL-21 may be indirectly involved in the effects that we observe. In this regard, 375 Hoft and colleagues described that TCR-transgenic CD4+ T cells specific for an 376 immunodominant peptide of T. cruzi and polarized in vitro into a Th17 cells potentiated 377 CD8+ T cell immunity in a mechanism dependent on IL-21 but independent on IL-17 [40]. 378 Whether these in vitro generated Th17 cells are equivalent those differentiated in vivo 379 within the environment of the natural T. cruzi infection remains to be established. Indeed, 380 we determined that infected IL-17RA KO mice presented higher concentration of IL-21 in 381 serum than infected WT controls, ruling out deficient IL-21 production as the cause of the 382 altered CD8+ T cell response observed in absence of IL-17RA signaling.

383 CD8+ T cells from infected IL-17RA KO mice showed increased apoptosis and a 384 transcriptional landscape associated to bio-functions and canonical path of cell death and 385 senescence. In addition, these cells exhibited high expression of multiple inhibitory and 386 death receptors that correlated with reduced ex vivo effector responses and a gene signature 387 that positively correlated with those defined for dysfunctional PD-1high CD8+ T cells 388 [41,42]. Altogether these features partially resembled those of dysfunctional CD8+ T cells 389 described in many chronic infections and cancer and characterized by the progressive loss 390 of T cell function, sustained expression of inhibitory receptors and high susceptibility to 391 deletion [43]. Interestingly, we found that the functional CD8+ T cell response observed in 392 T. cruzi-infected WT mice (this manuscript and [44]) turned into a poorly-functional 393 response in the absence of IL-17RA signaling. In this regard, although it is conceivable that 394 the increased tissue parasitism in IL-17RA KO mice favored CD8+ T cell exhaustion, 395 infection of WT mice with increasing parasite loads does not induce expression of 396 inhibitory receptors or deletion of parasite-specific CD8+ T cells nor results in lower CD8+ 397 T cell cytotoxic effector function [45]. As cytokines play essential roles in the regulation of 398 exhaustion [20], we performed adoptive transfer experiments that supported the notion that 399 IL-17 signaling directly influenced CD8+ T cell survival and function. In this line, we 400 demonstrated that anti-PD-L1 Abs during the CD8+ T cell expansion phase reinvigorated 401 parasite-specific CD8+ T cell responses in infected IL-17RA KO mice, restoring a robust 402 parasite control and reducing pathology to levels comparable to infected WT controls. 403 Remarkably, we observed that a short regimen of PD-L1 neutralization increased the 404 frequency of parasite-specific CD8+ T cells by two-fold in both treated groups, but the

405 magnitude of the response in infected IL-17RA KO mice was still lower than in WT 406 counterparts. In this regard, it is likely that, given the high levels of several inhibitory 407 receptors in CD8+ T cells elicited by T. cruzi infection in absence of IL-17RA, the 408 combination of different anti checkpoint Abs may have stronger effects in these settings, as 409 reported in cancer [46].

410 So far, the role of checkpoint inhibitors and, particularly the PD-1/PD-L1 axis has been 411 scarcely evaluated during T. cruzi infection. One report demonstrated that genetic 412 disruption of this inhibitory pathway increased the effector immune response and 413 consequently, favored parasite control but also promoted cardiac pathology and 414 compromised host resistant to the infection [47]. In this context, our data provide evidences 415 that a PD-L1 checkpoint blockade temporarily limited to the CD8+ T cell expansion phase 416 not only reverses the increased susceptibility to T. cruzi absence of IL-17RA signaling but 417 also boosts protective CD8+ T cell immunity during the natural infection. These findings 418 are relevant to a growing area of research aimed at investigating the therapeutic potential of 419 targeting immune checkpoint pathways during chronic infections [48]. In the last years, 420 several reports have shown that immune cell exhaustion or dysfunction is a common 421 finding in human Chagas´ disease, particularly in patients with the most severe conditions 422 [49-52]. There is certain consensus that even the very low parasite load present in tissues 423 may drive, in the long term of the human infection (two decades), the expression of 424 inhibitory receptors and cell dysfunction in T cells, likely resulting in an immune 425 modulatory mechanism and reduced tissue damage in the late stage of infection. 426 Interestingly, many reports have described that patients with moderate and severe chronic 427 chagasic cardiomyopathy present reduced production of IL-17, supporting the notion that 428 this cytokine is protective against cardiac damage [53,54]. Altogether, these and our results 429 support the hypothesis that high production of IL-17 in certain patients may prevent, in the 430 context of persistent parasite levels, the induction of T cell dysfunction and the associated 431 immune dysbalance that cause chronic myocarditis. This hypotheses deserves further 432 evaluation given the relevance for the understanding of the immunopathology during 433 human Chagas´ disease,

434 From the IPA analysis of the transcriptome of CD8+ T cells arising during T. cruzi 435 infection in the absence of IL-17RA, it emerged as potential upstream regulators IRF4 and, 436 at a minor extent, BATF, two TFs reported to be individually required for survival and 437 differentiation of early effector CD8+ T cells [25-27,55-57]. IRF4 promotes CD8+ T cell 438 expansion and differentiation [25], but it also induces the expression of molecules 439 associated with an exhausted status such as Blimp-1, CTLA-4 and IL-10 [58-60]. 440 Furthermore, BATF expression may be triggered by PD-1 ligation and associated with 441 CD8+ T cell exhaustion during chronic viral infection in humans and mice [61]. Very 442 recently, IRF4 and BATF have been shown to form a TCR-responsive transcriptional 443 circuit that establishes and sustains T cell exhaustion during a chronic viral infection [62]. 444 Therefore, it is conceivable that sustained high levels of both TFs could result in the 445 induction of a cell death and exhaustion program as that observed in CD8+ T cells from 446 infected IL-17RA KO mice. How the triangle formed by of loss of IL-17RA signaling, 447 increased expression of BATF and IRF-4 and CD8+ T cell exhaustion is actually linked 448 deserves further studies using genetic dissection approaches.

449 In conclusion, our results provide evidences for a novel IL-17RA-mediated mechanism that 450 potentiates immunity to an intracellular pathogen like T. cruzi by improving adaptive CD8+ 451 T cell responses. This, together with the report showing that IL-17 controls functional 452 competence of NK cells during a fungal infection [63], suggests that cytokines of the IL-17 453 family would be important for both the innate and the adaptive arms of the cytotoxic 454 response against pathogens. Although, it remains to be determined whether this mechanism 455 is also operative in other infections where IL-17 cytokines have been shown to increase 456 host resistance, these findings have two fundamental repercussions in the field of IL-17- 457 mediated immune responses. The first is that the prolonged targeting of the IL-17 pathways 458 as part of an anti-inflammatory treatment during autoimmune diseases or cancer could 459 potentially lead to severe undesired effects due to defective cytotoxic responses. The 460 second is the notion that promoting the production of IL-17 cytokines during infection or 461 vaccination may help to elicit stronger cytotoxic responses to fight against microbes, and 462 eventually, tumors.

463 Material and Methods

464 Mice.

465 Mice used for experiments were sex- and age-matched (6 to 10 week-old). C57BL/6 mice 466 were obtained from School of Veterinary, La Plata National University (La Plata, 467 Argentina). IL-17RA KO mice were provided by Amgen Inc. (Master Agreement N° 468 200716544-002). IL-17A/IL-17F double KO mice [64] were kindly provided by Dr Immo 469 Prinz. CD45.1 C57BL/6 mice (B6.SJL-Ptprca Pepcb/Boy) and CD8α KO mice (B6.129S2- 470 Cd8atm1Mak/J) were obtained from The Jackson Laboratories (USA). CD45.1 x CD45.2 471 F1 mice bred in our animal facility. All animals were housed in the Facultad de Ciencias 472 Químicas, Universidad Nacional de Córdoba. 473 474 Animal Welfare 475 All animal experiments were approved by and conducted in accordance with guidelines of 476 the Institutional Animal Care and Use Committee (IACUC) Facultad de Ciencias 477 Químicas, Universidad Nacional de Córdoba (Approval Number 981/15) (OLAW 478 Assurance number A5802-01). The IACUC adhered to the guidelines from “Guide to the 479 care and use of experimental animals” (Canadian Council on Animal Care, 1993) and 480 “Institutional Animal Care and Use Committee Guidebook” (ARENA/OLAW IACUC 481 Guidebook, National Institutes of Health, 2002). 482 483 Parasites and experimental infection. 484 Bloodstream trypomastigotes of the Tulahuén strain of T. cruzi were obtained from 485 BALB/c mice infected 10 days earlier. For experimental infection mice were inoculated 486 intraperitoneally with 0.2 ml PBS containing 5×103 trypomastigotes (usual dose) or doses 487 of 500 and 5×104 trypomastigotes when indicated. For infection of transferred CD8α 488 knockout mice, parasite dose was 1000 trypomastigotes. 489

490 Quantification of parasite DNA in tissues.

491 Genomic DNA was purified from 50 µg of tissue (spleen, liver and heart) using TRIzol 492 Reagent (Life Technologies) following manufacturer´s instructions. Satellite DNA from T. 493 cruzi (GenBank AY520036) was quantified by real time PCR using specific Custom 494 Taqman Gene Expression Assay (Applied Biosystem) using the primer and probe 495 sequences described by Piron et al [65]. A sample was considered positive for the T. cruzi 496 target when CT<45. Abundance of satellite DNA from T. cruzi was normalized to GAPDH 497 abundance (Taqman Rodent GAPDH Control Reagent, Applied Biosystem) and expressed 498 as arbitrary units. 499

500 Cells and culture.

501 CD8+ T cells were isolated from the spleen by magnetic negative selection or cell sorting 502 from pools of at least 3-5 mice. For magnetic cell purification the EasySep™ Mouse CD8+ 503 T Cell Isolation Kit (Stemcell Technologies) were used according to the manufacturer’s 504 protocol. For cell sorting, spleen cell suspensions were surface stained and CD3+CD8+ T 505 cells were sorted with a FACSAria II (BD Biosciences). During in vitro studies, CD8+ T 506 cells (2 x 105) purified by magnetic selection were stimulated in 96-well plates coated with 507 anti-CD3/anti-CD28 Abs (eBioscience, 2 μg/ml and 1 μg/ml respectively) and incubated 508 during 24 h in the presence of 100 ng/ml of recombinant IL-17A, IL-17F, IL-17C and IL- 509 17E (ImmunoTools, GmbH) and/or 50ng/mL of IL-21 and TNF (ImmunoTools, GmbH).

510

511 Antibodies and Flow cytometry.

512 Cell suspensions were washed in PBS and incubated with LIVE/DEAD Fixable Cell Dead 513 Stain (eBioscience) during 15 min at RT. Next, cells were washed and incubated with 514 fluorochrome labeled-Abs for 20 min at 4°C (see below for details). To detect T. cruzi 515 specific CD8+ T cells, H-2K(b) T. cruzi trans-sialidase amino acids 567-574 ANYKFTLV 516 (TSKB20) APC-Labeled Tetramer (NIH Tetramer Core Facility) were incubated 20 min at 517 4°C before further surface staining with additional Abs. After further surface staining, cells 518 were washed and acquired in a FACSCanto II (BD Biosciences). Blood was directly

519 incubated with the indicated antibodies and erythrocytes were lysed with a 0.87% NH4Cl

520 buffer previously to acquisition. For Ki-67 intranuclear staining, cells were first stained on 521 surface, washed and then fixed, permeabilized and stained with Foxp3/Transcription Factor 522 Staining Buffers (eBioscience) following eBioscience One-step protocol: intracellular 523 (nuclear) proteins.

524 Flow cytometry and/or cell sorting was performed with a combination of the following Abs 525 (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or 526 Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), Bcl-2 (10C4), IgG 527 (polyclonal goat) and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), 528 CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b 529 (TR75-89), Bim (rabbit. C34C5), rabbit IgG control isotype (DA1E); PECy7-labeled anti- 530 mouse: CD8 (53-6.7), PD-1 (RMP1-30), FAS (Jo2), PerCPCy5.5-labeled anti-mouse: CD3 531 (145-2C11), CD45.2 (104); PerCP-eFluor 710 labeled anti-mouse: TIGIT (GIGD7); APC- 532 Cy7/AF780-labeled anti-mouse: CD45.1 (A20); Biotin-labeled anti-mouse: BTLA (8F4) 533 and PE-labeled Streptavidin; unlabeled anti-mouse: Bcl-xL (rabbit, 54H7), Bcl-2-associated 534 death promoter (BAD) (rabbit, C-20) , Bax (rabbit, P-19).

535

536 Quantification of IL-21.

537 The concentration of IL-21 in the plasma of in plasma of infected WT and IL-17RA KO 538 mice was assessed by ELISA using paired specific Abs (eBiosciences) according to 539 standard protocols.

540

541 Evaluation of Proliferation and Apoptosis.

542 T. cruzi-infected WT and IL-17RA KO mice were given BrdU (1 mg/ml, Sigma- 543 Aldrich)/1% sucrose in the drinking water (carefully protected from light) ad libitum. After 544 5 days, mice were sacrificed and spleen mononuclear cells were collected and stained with 545 surface antibodies. Incorporated BrdU was detected with a BrdU Flow kit according to the 546 manufacturer’s specifications (BD Biosciences).

547 Apoptosis was determined by Annexin V and 7AAD staining according to the 548 manufacturer’s specifications (BD Biosciences). Mitochondrial depolarization (ψ) was 549 measured by FACS using 50 nM TMRE (Invitrogen) as described [66].

550

551 Determination of CD8+ T cell effector function in vitro.

552 Spleen cell suspensions were cultured during 5 h with medium, 5 µg/ml TSKB20 553 (ANYKFTLV) peptide (Genscript Inc.) or 50 nM PMA plus 0.5 µg/ml ionomycin (Sigma- 554 Aldrich) in the presence of Monensin (eBioscience) and a PE-labeled anti-CD107a mAb 555 (eBioscience, eBio1D4B). After culture, the cells were surface stained, fixed and 556 permeabilized with BD Cytofix/Cytoperm and Perm/Wash (BD Biosciences) according 557 manufacturer’s instruction. Cells were incubated with FITC-labeled antibody to IFNγ 558 (eBioscience, XMG1.2) and PerCP/APC-labeled antibody to TNF (Biolegend, MP6-XT22) 559 and acquired on FACSCanto II (BD Biosciences).

560

561 Adoptive cell transfer.

562 Competitive adoptive transfer experiments were performed by i.v. injection of 563 CD45.1/CD45.2 F1 recipient WT mice or CD8α knockout mice with a mixture 1:1 of 564 CD8+ T cells purified from spleen of CD45.1+ WT mice and CD45.2+ IL-17RA KO mice. 565 Total 5x106 cells and 15x106 cells were injected in WT and CD8α knockout recipients, 566 respectively. Recipient mice were immediately infected and frequency of the injected 567 CD45.1+ WT and CD45.2+ IL-17RA KO cells within the total T cell population were 568 determined in blood, spleen and liver at different days pi.

569

570 Treatment with neutralizing anti- IL-17A and PD-L1 Abs

571 To block the PD-1/PD-L1 pathway, infected WT and IL-17RA KO mice were injected i.p. 572 with 200 μg rat anti-mouse PD-L1 antibody (10F.9G2, BioXcell) or total rat IgG isotype

573 control (Jackson Research) every 3 days from 15 dpi until sacrifice at day 21 dpi. Infection 574 matched WT mice were assayed in parallel as controls.

575 For the in vivo neutralization of IL-17A, infected WT mice were injected i.p. with 200 μg 576 rat anti-mouse IL-17 antibody (17F3, BioXcell) or rat total IgG1 isotype control (MOPC- 577 21, BioXcell) every 3 days from day 13 dpi until sacrifice at 21 dpi. Infection-matched IL- 578 17RA KO mice were assayed in parallel as controls.

579

580 Real-Time quantitative PCR and DNA microarray.

581 RNA from total spleen tissue was purified using TRIzol Reagent (Life Technologies) 582 following manufacturer´s instructions. Oligo-dT and a MMLV reverse transcriptase kit 583 (Invitrogen) were used for cDNA synthesis. IL-17RA, IL-17RC and IL-17RD transcripts 584 were quantified by real-time quantitative PCR on an ABI PRISM 7700 Sequence Detector 585 (Perkin-Elmer Applied Biosystems) with Applied Biosystems predesigned TaqMan Gene 586 Expression Assays and reagents according to the manufacturer’s instructions. The 587 following probes were used: Mm00434214_m1 for IL-17RA, Mm00506606_m1 for IL- 588 17RC and Mm00460340_m1 for IL-17RD. For each sample, the mRNA abundance was 589 normalized to the amount of 18S rRNA (Mm03928990_g1) and is expressed as arbitrary 590 units (AU).

591 For gene-expression analysis, CD8+ T cells were purified by FACS from the spleen of non- 592 infected and 22-day infected WT and IL-17RA KO mice and lysed with TRIzol reagent. 593 Total RNA was extracted with the RNAeasy Mini Kit (Qiagen). RNA quality was verified 594 in an Agilent Bioanalyzer and measured with a Nanodrop 1000 (Thermo Scientific). cDNA 595 was hybridized on Affymetrix Mouse Gene 2.1 ST arrays as described elsewhere.

596

597 Analysis of microarray data.

598 The microarray data from this publication have been deposited to the GEO database 599 (https://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier: GSE104886. Gene

600 expression data was normalized using RMA algorithm on custom Brainarray CDF (v.22.0.0 601 ENTREZG). Plots of differential expressed genes in spleen CD8 T cells at day 0 post 602 infection and day 22 post infection in WT and IL-17RA KO samples were defined using 603 fold change (|log2FC| >log2(1.2)). Bioinformatic analyses were performed with R software 604 environment. Gene Set Enrichment Analyses (GSEA) between the different spleen CD8 T 605 cell samples were done using specific gene sets from the Molecular Signatures DB 606 (MSigDB). Genes induced by the infection in WT and IL-17RA KO mice but showing 607 significantly higher expression in IL-17RA KO mice were uploaded to Ingenuity Pahtway 608 Analysis (Ingenuity® Systems, www.ingenuity.com) for the analysis of “Disease and bio- 609 function”, “Canonical pathway”, and “Up-stream regulators”. It was considered 610 significantly activated (or inhibited) with an overlap p-value≤0.05 and an IPA activation Z- 611 score as defined under each specific analysis category on the IPA website.

612

613 Statistics.

614 Statistical significance of comparisons of mean values was assessed as indicated by a two- 615 tailed Student’s t test, two way ANOVA followed by Bonferroni’s posttest and Gehan- 616 Breslow-Wilcoxon Test using GraphPad software. P < 0.05 was considered statistically 617 significant.

618 Acknowledgements

619 We thank MP Abadie, MP Crespo, F Navarro, D Lutti, V Blanco, A Romero, L Gatica and 620 G Furlán for their excellent technical assistance. We thank Dr Immo Prinz for providing IL- 621 17A/IL-17F double knockout mice. We acknowledge the NIH Tetramer Core Facility for 622 provision of the APC-labeled TSKB20/Kb and TSKB18/Kb tetramers. We thank A. 623 Rapinat and D. Gentien from Genomic Platform, from the translational research department 624 from Institut Curie, Paris, for the transcriptome experiments. 625 626 Author contributions 627 JTB designed and performed most of the experiments, analyzed data, and 628 wrote/commented on the manuscript; CLAF, FFV and CR performed experiments, 629 analyzed data and commented on the manuscript; MCR, MCAV, MGS performed 630 experiments and commented on the manuscript; NGN performed microarray experiment; 631 WR analyzed microarray data; EP participated in microarray design and analysis and 632 provided funding; CLM and AG participated in data analysis, commented on the 633 manuscript and provided funding; EVAR supervised the research, designed experiments, 634 wrote the manuscript, and provided funding. 635

636 Conflict of interest

637 The authors declare that they have no conflict of interest.

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813 814

815 Figure Legends

816 Fig 1. Absence of IL-17RA signaling results in increased tissue parasitism and a 817 reduced magnitude of parasite-specific CD8+ T cell responses. (A-C) Relative amount 818 of T. cruzi satellite DNA in spleen (A), liver (B) and heart (C) of infected WT and IL- 819 17RA KO (KO) mice determined at the indicated dpi. Murine GAPDH was used for 820 normalization. Data are presented as mean ± SD, N=5 mice. P values calculated with two- 821 tailed T test. (D) Representative plots of CD8 and TSKB20/Kb staining in spleen, liver and 822 blood of WT and KO mice at 22 dpi (WT-I and KO-I, respectively). A representative plot 823 of the staining of splenocytes from non-infected WT mice (WT-N) is shown for 824 comparison. (E) Percentage and cell numbers of TSKB20/Kb+ CD8+ T cells and (F) cell 825 numbers of total CD8+ T cells determined in spleen of WT and IL-17RA KO mice at 826 different dpi. Data shown as mean ± SD, N=5-8 mice. P values calculated using two-way 827 ANOVA followed by Bonferroni‘s post-test. (G) Experimental layout of IL-17 828 neutralization in infected WT mice injected with control isotype or anti-IL-17 Abs (WT- 829 cAb and WT-aIL-17, respectively). (H) Representative plots of CD8 and TSKB20/Kb 830 staining in spleen and liver infected WT treated as indicated in G. (I) Percentage of total 831 and TSKB20/Kb+ CD8+ T cells in mice treated as depicted in G. Results from non-infected 832 WT mice (WT-N) and infection-matched KO mice (KO-I) are shown for comparison. Data 833 are representative of five (A-F), and two (G-I) independent experiments. 834 835 Fig 2. IL-17RA signaling during T. cruzi infection promotes survival of CD8+ T cells. 836 (A) Representative dot plots of CD8 and TSKB20/Kb staining in spleen of WT and KO 837 mice at 0 dpi (WT-N) and 20 dpi (WT-I and KO-I) and representative histograms of BrDU 838 staining in the indicated total and TSKB20/Kb+ CD8+ T cell gate. Numbers indicate the 839 frequency of BrDU+ cells from the corresponding colored group. Histograms are 840 representative of one out of five mice. Bars in the statistical analysis represent data as mean 841 ± SD, N=5 mice. P values were calculated with two-tailed T test. (B) Representative 842 histograms of TMRE staining in total (left) and TSKB20/Kb+ (right) CD8+ T cells from 843 the spleen of WT and IL17RA KO mice at 10 dpi (top) and 20 dpi (bottom). Numbers 844 indicate the frequency of TMRE- (apoptotic) cells in total and TSKB20/Kb+ CD8+ T cells 845 from the corresponding colored group. Grey tinted histogram show staining in CD8+ T

846 cells from non-infected WT mice (WT-N). Histograms are representative of one out of five 847 mice. Bars in the statistical analysis represent data as mean ± SD, N=5 mice. P values were 848 calculated with two-tailed T test. (C and D) Representative histograms of TMRE (C) and 849 BAD (D) stainings in cultures of purified CD8+ T cells activated with coated anti-CD3 and 850 anti-CD28 in the presence of the indicated cytokines. Statistical analysis in (C) shows the 851 frequency of TMRE- (apoptotic) cells in each biological replicate (N=4 mice) and the mean 852 ± SD. P values calculated with paired two-tailed T test (ns: not significant). Statistical 853 analysis in (D) represent protein expression as the geometric mean of fluorescence intensity 854 in each replicate (N=4 mice). Lines link paired samples. P values calculated with paired 855 two-tailed T test. Data in A-C and D are representative of three and two independent 856 experiments, respectively.

857 Fig 3. Substantial differences in the gene-expression profile of CD8+ T cells from T. 858 cruzi-infected IL-17RA KO and WT mice. (A-F) Microarray analysis of purified CD8+ T 859 cells from infected WT (WT-I) and IL-17RA KO (KO-I) mice (22dpi) and non-infected 860 counterparts (WT-N and KO-N). N=3 mice per group. (A) Dot plots displaying the number 861 of genes that show a significant ≥1.2-fold difference in fold change expression of WT-I and 862 KO-I relative to non-infected counterparts. Colors indicate sets of genes with different 863 expression patterns. (B) Top two enrichment plots in KO-I (p<0.001) determined by 864 supervised analysis of all infection induced genes in WT-I and KO-I using GSEA26495 865 and GSEA41867 (MSigDB C5BP) signature gene sets. (C) Heat maps of expression of 866 selected genes according to categories relevant to CD8+ T cell biology. Each gene 867 expression value was represented by the median value from 3 mice. (D-F) IPA of genes 868 induced by the infection but showing significantly higher expression in IL-17RA KO mice 869 (red genes in A). Top 20 “Diseases and Bio-functions” (D), “Canonical pathways” (E) and 870 “Up-stream regulators” (F) that were most significantly altered (P-value < 0.05 with 871 Fischer’s exact test) in KO mice are shown. The activation Z-score was calculated to 872 predict activation (red), inhibition (blue), categories where no predictions can be made and 873 unknown results (with Z-score close to 0). Categories significantly activated or inhibited 874 according to Z-score are marked with a star.

875 Fig 4. CD8+ T cells from T. cruzi infected IL-17RA KO mice show features of 876 exhausted cells. (A-B) Representative histograms and statistical analysis of the geometric 877 mean of expression or percentage of cells expressing several inhibitory (A) and death 878 receptors (B) in total and TSKB20/Kb+ spleen CD8+ T cells from infected WT (WT-I) and 879 IL-17RA (KO-I) mice (22 dpi). Staining of non-infected WT mice (WT-N) is showed as 880 gray tinted histograms for comparison. (A-B) Data in statistical analysis are presented as 881 mean ± SD, N=4-6 mice. (C) Percentage of spleen CD8+ T cells from infected WT and KO 882 mice (22 dpi) that exhibit polyfunctional effector function denoted by expression of 883 CD107a, IFNγ and/or TNF upon 5 h of the indicated stimulation. Data shown as mean ± 884 SD, N=5 mice. Data were background subtracted. All P values were calculated using two- 885 tailed T test. Data are representative of at least three independent experiments.

886 Fig 5. Checkpoint blockade reinvigorates parasite-specific CD8+ T cell immunity and 887 enhances parasite control in infected IL-17RA KO mice. (A) Layout of the checkpoint 888 blockade experiment. (B) Representative plots and statistical analysis of CD8 and 889 TSKB20/Kb staining in spleen and liver of the experimental groups indicated in (A). 890 Numbers within plots indicate the frequency of total CD8+ cells (up) and TSKB20-specific 891 CD8+ T cells (down) at 21 dpi. Bar graphs represent data as mean ± SD, N=7 mice. (C) 892 Relative amount of T. cruzi satellite DNA in the spleens of the indicated experimental 893 groups at 21 dpi. Murine GAPDH was used for normalization. Data are presented as mean 894 ± SD relative to WT-cAb group, N=7 mice. (D) Enzymatic activity (UI/L) of alanine 895 transaminase (ALT), aspartate transaminase (AST), creatinine kinase-MB and creatinine 896 phosphokinase (CPK) on plasma of the experimental groups indicated in (A) at 21 dpi. N=7 897 mice. All P values were calculated with two-tailed T test. Data are representative of two 898 independent experiments.

899 Fig 6. Intrinsic IL-17RA-signaling modulates the maintenance, phenotype and 900 function of CD8+ T cells during T. cruzi infection. (A) Concentration of IL-21 in plasma 901 of infected WT and IL-17RA KO mice at 20 dpi. Data are presented as mean ± SD, N=4 902 mice. (B) Amounts of Il17ra, Il17rc and Il17rd transcript determined in spleen CD8+ T 903 cells purified from non-infected WT mice, normalized to 18S RNA. Data are presented as 904 mean ± SD, N=4 mice (C) Representative histograms and statistical analysis of the

905 expression of IL-17RA (protein) on different CD8+ T cell subsets identified according to 906 CD44 and CD62L staining in spleen cell suspensions from non-infected (N) and infected 907 (I) WT mice (22 dpi). Staining of IL-17RA on spleen CD8+ T cells from IL-17RA KO 908 mice is showed as negative control. Data in statistical analysis are presented as mean ± SD, 909 N≥6. (D) Representative plots and statistical analysis of CD8 and TSKB20/Kb staining and 910 of CD45.1 and CD45.2 staining within Total and TSKB20/Kb+ CD8+ T cells in the blood 911 and spleen of infected F1 CD45.1/CD45.2 WT recipient mice (20dpi) non-injected 912 (control) or injected with equal numbers of CD45.1+ WT and CD45.2+ KO CD8+ T cells. 913 Numbers in the plots indicate the frequency of the correspondent cell subset. Bar graphs 914 display the frequency of CD45.1+ WT cells and CD45.2+ KO cells within the indicated 915 populations upon gating only in the injected cells. (E) Representative plots and statistical 916 analysis of CD8 and TSKB20/Kb staining and of CD45.1 and CD45.2 staining within Total 917 and TSKB20/Kb+ CD8+ T cells in the spleen of infected CD8α-/- recipients (17dpi) 918 injected with equal numbers of CD45.1+ WT and CD45.2+ KO CD8+ T cells. Pie charts 919 display the frequency of CD45.1+ WT and CD45.2+ KO cells within the indicated gates. 920 Bar graph shows the ratio between the frequencies of TSKB20/Kb+ CD8+ T cells and the 921 total CD8+ T cells within CD45.1 WT and CD45.2 IL-17RA KO populations. (F) 922 Representative histograms and statistical analysis of the geometric mean of PD-1 923 expression in total and TSKB20/Kb+ CD8+ T cells within CD45.1+ WT and CD45.2+ IL- 924 17RA KO CD8+ T cells from the spleen of CD8α-/- recipient mice. Data in statistical 925 analysis are presented as mean ± SD, N=4-6 mice. (G) Percentage of polyfunctional 926 effector cells denoted by expression CD107a, IFNγ and/or TNF upon 5h of the indicated 927 stimulation on CD45.1+ WT and CD45.2+ IL-17RA KO CD8+ T cells from the spleen of 928 CD8α-/- recipient mice. Data shown as mean ± SD, N=4 mice. Data were background 929 subtracted. All P values were calculated using two-tailed T test. Data are representative of 930 two independent experiments.

931

932 Supporting Information

933 S1 Figure . Complementary evaluation of parasite-specific CD8+ T cell responses and 934 tissue parasitism in T. cruzi-infected IL-17A/IL-17F DKO mice. (A) Relative amount of

935 T. cruzi satellite DNA in spleen and liver of infected WT and IL-17A/IL-17F DKO mice 936 determined at 22 dpi. Murine GAPDH was used for normalization. (B) Representative plots 937 and statistical analysis of CD8 and TSKB20/Kb staining in spleen of WT and IL-17A/IL- 938 17F DKO mice at 22 dpi. Numbers on plots represent the frequency of TSKB20/Kb+ CD8+ 939 T cells. (C-D) Representative histograms and statistical analysis of the geometric mean 940 expression of the inhibitory receptor PD-1 (C) and the death receptor CD95/Fas (D) in total 941 and TSKB20/Kb+ spleen CD8+ T cells from WT and IL-17A/IL-17F DKO mice at 22 dpi.. 942 Grey tinted histogram show staining in CD8+ T cells from non-infected WT mice. Data in 943 statistical analysis (A-D) are presented as mean ± SD, N=4-6 mice. P values calculated with 944 two-tailed T test. (A-D) Data are representative of at least three independent experiments.

945 S2 Figure. Complementary studies of proliferation and apoptosis in total and 946 parasite-specific CD8+ T cells in WT and IL-17RA KO infected mice. (A and B) 947 Representative histograms and statistical analysis of Ki-67 (A) and Annexin V (B) staining 948 within the 7ADD- gate in total (left) and TSKB20/Kb+ (right) CD8+ T cells from the 949 spleen of WT and IL17RA KO mice at 10 dpi (B) and/or 20 dpi (A-B). Grey tinted 950 histogram show staining in CD8+ T cells from non-infected (N) WT mice. Histograms are 951 representative of one out of five mice. Numbers indicate the frequency of Ki-67+ (A) and 952 Annexin V+ (B) cells from the corresponding colored group. Bar graphs represent data as 953 mean ± SD, N=4 mice. P values calculated with two-tailed T test. (C) Representative 954 histograms of TMRE staining in total (left) and TSKB20/Kb+ (right) CD8+ T cells from 955 the spleen of infected WT mice treated with isotype control or anti-IL-17 as described in 956 Figure 1G. Grey tinted histogram show staining in CD8+ T cells from non-infected WT 957 mice. Numbers indicate the frequency of TMRElow (apoptotic) cells from the 958 corresponding colored group. Histograms are representative of one out of seven mice. Bar 959 graphs in the statistical analysis represent data as mean ± SD, N=7 mice. P values 960 calculated with two-tailed T test. (D) Representative histograms of the expression of Bcl-2, 961 Bim and Bax in cultures of purified CD8+ T cells activated during 24h with coated anti- 962 CD3 and anti-CD28 in the presence of medium or IL-17A (100ng/mL) as indicated. (A-D) 963 Data are representative of two independent experiments.

964 S3 Figure. Representative flow cytometry data plots of the evaluation of CD8+ T cell 965 effector function. Representative plots and analysis strategy of the frequency of spleen 966 CD8+ T cells from infected WT and IL-17RA KO mice (22dpi) showing a combination of 967 three and two effector function including expression of CD107a, IFNγ and/or TNF upon 5 968 h of culture with the indicated stimulation. Plots are representative of one out of five mice. 969 Data are representative of two independent experiments.

970 S4 Figure. Increasing parasite doses does not diminished the frequency of parasite- 971 specific CD8+ T cells cells or upregulated inhibitory receptors on CD8+ T cells. (A) 972 Parasitemia at 22 dpi determined in the blood of WT mice infected with increasing doses of 973 parasites (500, 5000 and 50000 tripomastigotes). (B) Representative plots and statistical 974 analysis of CD8 and TSKB20/Kb staining in spleen of WT infected as described in A. (C 975 and D) Statistical analysis of the geometric mean of expression of inhibitory (C) and death 976 (D) receptors in total and TSKB20/Kb+ CD8+ T cells from WT mice infected as described 977 in A. Data in statistical analysis are presented as mean ± SD, N=4-6 mice. P values 978 calculated with two-tailed T test. (A-D) Data are representative of at least 2 independent 979 experiments.

980 S5 Figure. Representative flow cytometry data plots of the evaluation of CD8+ T cell 981 effector function in mice adoptively transferred. Representative plots and analysis 982 strategy of the frequency of CD45.1+ WT and CD45.2+ IL-17RA KO CD8+ T cells from 983 the spleen of CD8α-/- mice adoptively transferred and infected as indicated in figure 7E. 984 The plots show a combination of three and two effector function including expression of 985 CD107a, IFNγ and/or TNF upon 5 h of culture with the indicated stimulation. Plots are 986 representative of one out of four mice. Data are representative of two independent 987 experiments.

Fig 1

AbioRxiv preprintSpleen doi: https://doi.org/10.1101/314336B ; thisLiver version posted May 11, 2018.C The copyrightHeart holder for this preprint (which was not 8 1.5 60 certified by peer review) is theWT-I author/funder, who has** p=0.004 granted bioRxivWT-I a license to display the preprint*** p=0.0001 in perpetuity.WT-I It is made available under KO-I aCC-BY-ND 4.0KO-I International license. KO-I 6 *** p=0.005 1.0 40 4 0.5 20 2 * p=0.029 Tc satellite DNA Tcsatellite Tc satellite DNA Tcsatellite DNA Tcsatellite (relative expression) (relative (relative expression) (relative (relative expression) (relative 0 0.0 0 14 22 130 22 22 Days pi Days pi Days pi

D Spleen Liver Blood

WT-NI 0,066% WT-I 1,67% KO-I 0,603% WT-I 4,13%KO-I 1,76% WT-I 3,85% KO-I 0,979% CD8 CD8 CD8 TSKB20/Kb TSKB20/Kb TSKB20/Kb

E F 2.0 p<0.01 8 80 P<0.001 p<0.001 WT-I p<0.001 WT-I WT ** p<0.01 *** *** KO-I KO-I KO 1.5 6 *** ** 60

1.0 4 40 CD8+Cells 6 0.5 2 10 20 CD8+TSKB20+Cells 6 %CD8+TSKB20+Cells

0.0 10 0 0 0 10 20 30 40 80 0 10 20 30 40 80 0 10 20 30 40 80 Days pi Days pi Days pi

***ns **p=0.0087 G H Spleen I 30 * ***ns 3 ***p<0.0001

control Ab (cAb) WT-N T.cruzi anti IL-17A Ab (aIL-17) 20 2,44% 0,66% 2 WT-cAb x WT-aIL-17 KO-I 10 1 %CD8+ cells 0 7 10 13 16 19 21 days pi WT WT-cAb WT-aIL-17 0 0 %CD8+TSKB20/Kb+ cells CD8 TSKB20/Kb Fig 2

A bioRxiv preprint doi: https://doi.org/10.1101/314336; this version postedB May 11,Total 2018. CD8 The copyrightTSKB20/Kb+ holder for thisTotal preprint CD8 (which TSKB20/Kb+ was not certified byWT-N peer review) is theWT-I author/funder, whoKO-I has granted bioRxiv a license to display the preprint in perpetuity. It is made available under 35,1 28,9 80 **p=0.0054 80 **p=0.0029 aCC-BY-ND 4.0 International license70,2. 76,3 60 60

WT-N 40 40 d10 pi WT-I 20 20

KO-I % TMRE- cells % TMRE- cells % TMRE- 0 0 WT-N WT-I KO-I WT-I KO-I

Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ *p=0.0158 41,0 47,8 60 * p= 0.0026 80 p= 0.1464 51,8 63,0 80 80 **p=0.0083 54,4 63,8 72,7 72,8 60 60 60 40 WT-N 40 40 40 d20 pi d20 pi 20 WT-I 20 20 20

KO-I cells % TMRE- % BrDU+ % cells BrDU+ % BrDU+ % cells BrDU+ % TMRE- cells % TMRE- 0 0 0 WT-N WT-I KO-I WT-I KO-I WT-N WT-I KO-I WT-I KO-I BrDU TMRE

C 80

p=0.0057 p=0.0024p=0.007 ns p=0.0472p=0.0225 Medium IL-17A IL-17F IL-17C IL-17E IL-21 IL-17A+21 60 ** * ** * *

40,5% 31,1% 54,0% 58,2% 42,9% 32,8% 33,8% 40

%TMRE- cells %TMRE- 20

0 A F C E 1 1 7 7 7 7 2 2 um 1 1 1 1 - di - - - - IL A+ IL IL IL IL 7 TMRE Me -1 IL D 3000 *p=0.0366 Isotype Medium 2500 IL-17A 2000

1500 Geom Mean BAD

1000

IL-17A BAD Medium Fig 3

A B C WT-N WT-I KO-N KO-I WT-N WT-I KO-N KO-I klrg1 il-2 bioRxivDay preprint 22 doi: https://doi.org/10.1101/314336; this version posted May 11, 2018. The copyright holderklre1 for this preprint (whichprf1 was not klra3 gzma E ector certified by peer review)561 is the author/funder, who has granted bioRxiv a license to display markers the preprint in perpetuity.itgax It is made availablegzmk under 1891 aCC-BY-ND 4.0 International licenseActivation/ . cd69 fasl 188 ctla4 ifng lag3 il10 havcr2 cxcl9 cd160 molecules E ctor cxcl10 Death Death

receptors tigit

Inhibitory/ gzmb fas il2ra pdcd1 il2rb eomes il12rb1 tcf7 il12rb2 id3 il21r Log2 Fold Change (KO) (KO) Change Fold Log2 foxo1 267 il18r1 foxo3 1136 il18rap zeb2 244 il15ra tbx21

Cytokines receptors Cytokines ifngr2 prdm1 il7r Log2 Fold Change (WT) id2 sell bcl6 ccr7 Transcription Factors Transcription e2f1 ccr6 irf4 ccr9 batf cxcr6 ccr5 cxcr3 Homing/Migration

D E F

Activated Activated Activated Unknow Inhibited Inhibited Z-score=0 Unknown Fig 4

A bioRxivTotal CD8 preprint doi:TSKB20/Kb+ https://doi.org/10.1101/314336Total CD8 ; thisTSKB20/Kb+ version posted May 11, 2018. The copyright holder for this preprint (which was not certified by peerWT-N review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under *** p=0.0002 aCC-BY-ND*** p=0.0002 4.0 International license. *** p=0.0006 ** p=0.0032 WT-I 800 800 150 150 KO-I 600 600 100 100 400 400 50 Geom Mean Geom 50 Mean Geom 200 200 Geom Mean Geom Geom Mean Geom 0 0 0 0 I -N -I - T T O-I T W K W KO-I PD-1 KO-I TIGIT W WT-NWT-I WT-I KO-I

50 ** p=0.0054 * p=0.0132 50 50 * p=0.0109 50 ** p=0.0037 40 40 40 40 30 30 30 30 20 20 20 20 %Cells %Cells %Cells %Cells 10 10 10 10 0 0 0 0 I I -I -I T- O- T T-N K W KO CTLA-4 W W BTLA WT-NWT-I KO-I WT-I KO-I

B Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ WT-N ** p=0.0022 WT-I 50 50 *** p=0.0001 600 *** p=0.0003 600 ** p=0.0048 KO-I 40 40 400 400 30 30 20 20 200 200 Geom Mean Geom 10 10 Mean Geom Geom Mean Geom Geom Mean Geom 0 0 0 0

KO-I KO-I WT-IKO-I WT-I KO-I FAS WT-NWT-I WT-I CD120b WT-N

C TSKB20 stimulation Polyclonal stimulation 1.5 6 * p=0.0394 * p=0.0122 p=0.2008 *p=0.0186 * p=0.0123 ** p=0.0016 p=0.1624 p=0.0747 WT-I KO-I 1.0 4

0.5 2 %CD8+ cells%CD8+ %CD8+ cells%CD8+

0.0 0 CD107 + + + - + + + - TNF + + - + + + - + IFNg + - + + + - + + Fig 5

Spleen p= 0.8422 ***p= 0.0006 A bioRxiv preprint doi: https://doi.org/10.1101/314336B WT+cAb WT+aPD-L1; this version postedKO+cAb May 11, 2018.KO+aPD-L1 The copyright holder30 for this preprint4 (which was not ***p= 0.0388 certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. p= 0.2272 It is made available under p= 0.8447 3 aCC-BY-ND 4.0 International license. 20 2 control Ab (cAb) or **p= 0.0059 T.cruzi anti PD-L1 (aPD-L1) 10 1 14.6 22.1 14.5 15.1 %CD8+ T Cells T %CD8+ x 1.2 2.41 0.33 0.74

0 Cells T %CD8+TSKB20+ 0 CD8 b 1 b 1 1 A L1 Ab L A L TSKB20/Kb c D c D c DL -cAb D 0 15 18 21 days pi - P P - P P T T a O a W -a KO- W - K - T O-a T O WT W K W K control Ab (cAb) or T.cruzi anti PD-L1 (aPD-L1) ***p= 0.0396 ***p= 0.2238 Liver ***p= 0.0004 x WT-cAb WT-aPDL1 KO-cAb KO-aPDL1 80 ***p= 0.0011 10

**p= 0.0162 8 60 0 15 18 21 days pi 6 KO **p= 0.0259 40 4

%CD8+ T Cells T %CD8+ 20 57.0 68.0 42.2 49.8 2 6.49 9.30 2.18 3.85 0 Cells T %CD8+TSKB20+ 0 CD8

b 1 b 1 b 1 b 1 TSKB20/Kb A L A L A L c D c D c DL -cA D - P P - P P T T a a W -a KO- W - KO - T O-a T O W K W K C D ALT AST CPK-MB CPK ** p=0.0089 ns *p=0.0416 p=0.5022 4 **p=0.0028 **p=0.0025 ns *p=0.0211 1500 5000 **p=0.0057 ***p=0.0001 1500 **p=0.0047 6000 **p=0.0038 **p=0.0006 **p=0.0053 **p=0.0027 3 4000 1000 1000 4000 3000 2 2000 500 500 2000 Activity (UI/L) Activity (UI/L) Activity (UI/L) Activity 1 (UI/L) Activity Tc satellite DNA Tcsatellite 1000 (relative expression) (relative

0 0 0 0 0 1 1 b b 1 b L N A L1 A L b 1 b 1 b N 1 b 1 Ab - c D -N A L A -N A L A L1 - Ab L A L -c DL T - -c T -c D -c D T -c D -c D T P PD T-c O P PDL1 P P P P a W -a WT aPD O-c a W T a W T a O a W -a KO-cAb- W K WT - K W -a KO - W - K - T O T T O T O W KO WT-aPD K W KO- W K W K Fig 6

IL-21 WT-N A bioRxiv preprintB doi: https://doi.org/10.1101/314336C ; this version posted May 11, 2018. The copyright holder for this preprint (which was notWT-I certified by peer review)1000 is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.2000 It is made available under **p=0.0029 WT-N Total Naive Memory Effector p=0.5386 *p=0.0368 80 aCC-BY-ND 4.0 International license. WT-N **p=0.0046 p=0.7719 100 1500 60 WT-I 10 KO-I 1000 40 Geom Mean pg/mL 500 20 1

0 0.1 0 Relative expression to 18s IL-17RA WT-I Total Naive Memory Effector WT-N KO-I IL-17RAIL-17RCIL-17RD Injected D Control Total CD8 TSKB20/Kb+

*p=0.0326 **p=0.0042 80 80

Blood 60 60 40 40 20 20 %CD8+ cells 0 0 (witthin injected cells) (witthin injected cells) %CD8+TSKB20+ cells %CD8+TSKB20+

***p=0.0005 **p=0.0038 80 100 80 60

Spleen 60 40 40 20 20 %CD8+ cells 0 (witthin injected cells)

0 cells %CD8+TSKB20+ (witthin injected cells) CD8 CD45.2 CD8 CD45.2 TSKB20/Kb CD45.1 TSKB20/Kb CD45.1

WT (CD45.1)KO (CD45.2) WT (CD45.1)KO (CD45.2) Spleen E CD45.1 (WT) F Live cells Total CD8 TSKB20/Kb+ CD45.2 (KO) 3 ***p=0.0008

Ratio 1

0 (TSKB20kb+/CD8+) CD8+ Total cells CD8+ TSKB20/Kb+ cells CD8 CD45.2 CD45.2 TSKB20/Kb CD45.1 CD45.1 WT (CD45.1)KO (CD45.2) G TSKB20 stimulation Polyclonal stimulation Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ H WT-N 2.0 *p=0.0358 p=0.2268 8 * p=0.0403 p=0.2067 WT-I *p=0.0120 * p=0.0471 2000 ** p= 0.0012 2000 p= 0.0620 1.5 p=0.0959 KO-I 6 1500 1500 1.0

4 1000 1000 %CD8+ T Cells T %CD8+ 0.5 %CD8+ Cells T 2 Geom Mean 500 Geom Mean 500

0 0 0.0 0

-N .1 .1 .2 T 5 5 5 4 4 CD107 + + + - + + + - PD-1 W D D45.2 D C C CD4 C TNF + + - + + + - + IFNg + - + + + - + +

CD45.1 (WT) CD45.2 (KO) Suppl Fig 1

A Spleen Liver B Spleen Spleen 6 bioRxiv preprint doi: https://doi.org/10.1101/314336; this version posted May 11, 2018. The copyright holder for this preprint* p=0.0361 (which was not certified by peer review)3 *** p<0.0001 is the author/funder,WT 10 who*** p<0.0001has grantedWT bioRxiv a license to display the preprint in perpetuity. It is made available under DKO DKO WT 1,76% DKO 0,705% 8 aCC-BY-ND 4.0 International license. 4 2 6

4 1 2 Tc satellite DNA satellite Tc Tc satellite DNA satellite Tc 2 (relative expression) (relative (relative expression) (relative CD8

0 0 cells %CD8+TSKB20/Kb+ 0 22 22 TSKB20/Kb WT-I DKO-I Days pi Days pi

C Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ D Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+

WT-N WT-N WT-I * p=0.0007 ** p=0.0037 WT-I 40 80 80 * p=0.1005 250 ** p=0.0001 DKO-I DKO-I 30 60 60 200 150 20 40 40 100 10 20 20 Geom Mean Geom Geom Mean Geom

Geom Mean Geom 50 Geom Mean Geom 0 0 0 0 PD-1 WT-I WT-I FAS WT-I WT-I WT-N DKO-I DKO-I WT-N DKO-I DKO-I A Suppl Fig2 C B D d20 pi Bcl-XL d20 pi d10 pi TMRE AnnexinV Ki-67 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint Total CD8 TSKB20/Kb+ Total CD8 oa D TSKB20/Kb+ Total CD8 26,6 24,3 51,6 29,7 51,6 29,7 74,5 70,5 doi: TSKB20/Kb+

1200 Geom1400 Mean1600 Bcl-XL1800 2000 https://doi.org/10.1101/314336 Medium **p=0.0069 32,7 24,2 60,6 37,4 60,6 37,4 74,9 63,5 IL-17A KO-I WT-I WT-N WT+aIL-17 WT+cAb WT-N KO-I WT-I WT-N Bcl-2

% AnnexinV+ cells % AnnexinV+ cells % Ki67+ cells 20 40 60 80 20 40 60 80 10 20 30 40 20 40 60 80 0 0 0 WT-N 0 TNW- KO-I WT-I WT-N TNW- KO-I WT-I WT-N TNW- KO-I WT-I WT-N oa D TSKB20/Kb+ Total CD8 Total CD8

WT- cAb Total CD8

WT- aIL-17A **p=0.0087 *p=0.0167 a *p=0.0130 ; p=0.1119 CC-BY-ND 4.0Internationallicense this versionpostedMay11,2018. Bim

ns KO TSKB20/Kb+ TSKB20/Kb+

% Ki67+ cells 100 100 20 40 60 80 10 20 30 40 20 40 60 80 20 40 60 80 % AnnexinV+ cells %AnnexinV+ cells 0 0 0 WT- cAb 0 **p=0.0015 * p=0.0470 * TIKO-I WT-I TIKO-I WT-I TIKO-I WT-I *p=0.0241 WT- aIL-17A *p=0.0385 BAX ns

KO IL-17A Medium WT-N The copyrightholderforthispreprint(whichwasnot . Suppl Fig 3

AbioRxiv preprint doi:Medium https://doi.org/10.1101/314336; this version postedTSKB20 May 11, 2018. The copyright holder for thisPMA/IONO 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 WT-I aCC-BY-ND 4.0 International license.

CD107 CD107 CD107 TNF TNF TNF IFN-g IFN-g IFN-g KO-I

CD107 CD107 CD107 TNF TNF TNF IFN-g IFN-g IFN-g Suppl Fig 4

bioRxiv preprint doi: https://doi.org/10.1101/314336; this version posted May 11, 2018. The copyright holder for this preprint (which was not A certified by peer review)B is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.**p=0.020 It is made available under 3 2.5 p=0.1107

6 500tp aCC-BY-ND5000tp 4.0 International500000tp license. **p=0.036 2.0 2 1.5

1.0 1

Parasites/mL (10 ) Parasites/mL 0.5 %CD8+TSKB20+ cells 0 0.0 CD8 CD8 CD8 TSKB20/Kb TSKB20/Kb TSKB20/Kb 500tp 500tp 5000tp 5000tp 50000tp 500000tp

C D Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+ Total CD8 TSKB20/Kb+

p=0.0552 *p=0.0115 p=0.4204 p=0.0650 p=0.3989 p=0.6897 *p=0.0310 *p=0.0348 *p=0.0159 *p=0.0312 p=0.8505 p=0.8673 400 300 *p=0.5106 p=0.1313 500 **p=0.0040 800 ***p=0.0004 2000 p=0.3813 2000 p=0.7385 400 300 600 1500 1500 200 300 200 400 1000 1000 200 Geom Mean Geom Mean Geom Mean Geom Mean Geom Mean Geom Mean 100 100 200 500 500 100

0 0 0 0 0 0

500tp 500tp 500tp 500tp 500tp 500tp 5000tp50000tp 5000tp50000tp 5000tp50000tp 5000tp50000tp 5000tp50000tp 5000tp50000tp

PD-1 TIM-3 FAS Suppl Fig 5

bioRxiv preprint doi: https://doi.org/10.1101/314336; this versionTSKB20 posted May 11, 2018. The copyright holder for this preprint (whichPMA/IONO 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 CD8+ T cells aCC-BY-ND 4.0 International license. CD8+ T cells

WT (CD45.1) KO (CD45.2) WT (CD45.1) KO (CD45.2)

CD45.1

CD45.1 CD45.2 CD45.2

SSC

CD107 CD107 CD107 CD107 CD107- CD107+ CD107- CD107+ CD107- CD107+ CD107- CD107+

TNF

TNF IFN-g IFN-g IFN-g IFN-g