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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 TOX 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 T cell receptor (TCR) stimulation leads to expression of the transcription factor 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 cytotoxic T cell 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) protein, 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 genes
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 proteins
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 signal. 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 line 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).
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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