Neuropilin-1 Is a T Cell Memory Checkpoint Limiting Long-Term Antitumor Immunity

Articles https://doi.org/10.1038/s41590-020-0733-2

Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity

Chang Liu1,2, Ashwin Somasundaram2,3, Sasikanth Manne4, Angela M. Gocher1,2, Andrea L. Szymczak-Workman1, Kate M. Vignali1,2, Ellen N. Scott1,2,5, Daniel P. Normolle6, E. John Wherry 4, Evan J. Lipson 7, Robert L. Ferris2,8,9, Tullia C. Bruno1,2,9, Creg J. Workman1,2 and Dario A. A. Vignali 1,2,9 ✉

Robust CD8+ T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8+ T cell–restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth. Mechanistically, NRP1 cell-intrinsically limited the self-renewal of the CD44+PD1+TCF1+TIM3− progenitor exhausted T cells, which was associated with their reduced ability to induce c-Jun/AP-1 expression on T cell receptor restimulation, a mechanism that may contribute to terminal T cell exhaustion at the cost of memory differentiation in wild-type tumor-bearing hosts. These data indicate that blockade of NRP1, a unique ‘immune memory checkpoint’, may promote the development of long-lived tumor-specific Tmem that are essential for durable antitumor immunity.

lthough immune checkpoint blockade (ICB) with functions11. Notably, it has been identified as a marker for thymi- 12 anti-CTLA4, anti-PD1, anti-PD-L1) has revolutionized can- cally derived murine regulatory T cells (Treg cells) and is crucial cer therapy, only a small proportion (10–30%) of patients for their suppression of antitumor immunity13,14. NRP1 is elevated A 1,2 with cancer exhibit a durable clinical response . Consequently, in Treg cells from patients with cancer and marks a highly suppres- 14–16 there is intense interest in identifying mechanisms of pre-existing sive Treg cell subset , suggesting that NRP1 might be a therapeutic and acquired immune resistance as well as the development of target for cancer immunotherapy. However, the functional role of new therapeutic approaches to prevent relapse. It is also impera- NRP1 on other T cell subsets, in particular CD8+ T cells, remains tive to understand the mechanisms underlying the lack of a durable poorly understood17–19. response seen in patients post-ICB treatment, an issue that can be In this study, we report that NRP1 is induced on tumor-infiltrating associated with impaired formation of a long-lived memory CD8+ CD8+ T cells despite limited expression on naive CD8+ T cells. 3,4 T cell (Tmem) pool capable of conferring durable remission . It has Although NRP1 expression is highly concordant with multiple IRs, been postulated that the restrained and/or limited Tmem pool that its function is distinct in cell-intrinsically limiting Tmem differentia- occurs in tumor-bearing hosts is reminiscent of observations in tion without substantially modulating effector activity. Analysis of chronic viral infection where severe T cell exhaustion manifests at peripheral blood-derived CD8+ T cells from patients with head and the expense of memory differentiation5. As a consequence, memory neck squamous cell carcinoma (HNSCC) and advanced skin malig- precursors fail to accumulate and thus are unable to form an ade- nancies showed that increased NRP1 expression, particularly within + quate Tmem repertoire. Exhausted T cells (TEX) represent an indepen- the effector memory CD8 T cell compartment, was correlated with dent CD8+ T cell lineage with considerable stability that is distinct the reduced size of the memory CD8+ T cell pool, poor disease out- + from CD8 T effector (Teff) or Tmem, and are marked by coexpression come and decreased responsiveness to ICB. Our study uncovers a of multiple inhibitory receptors (IRs), such as PD1, CTLA4, LAG3, previously unappreciated role for NRP1 in CD8+ T cells and sug- TIM3, TIGIT and 2B4 (refs. 6,7). PD1:PD-L1 ICB is insufficient gests that it is primarily an immune checkpoint of T cell memory. 6 to reinvigorate TEX to become durable Tmem (ref. ), however, it is Thus, targeting NRP1 may facilitate the restoration of durable anti- unclear what pathways would be necessary for restoration of robust tumor immunity. and durable antitumor T cell memory. NRP1, originally discovered as a neuronal and endothelial Results cell receptor required for normal embryonic development, axon NRP1 limits CD8+ T cell–mediated antitumor immunity to guidance and vasculature formation8–10, is also expressed by sev- tumor rechallenge and anti-PD1 immunotherapy. NRP1 was eral immune cell types where it participates in critical immune robustly induced on CD8+ tumor-infiltrating lymphocytes (TILs) in

1Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 2Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 3Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 4Department of Systems Pharmacology and Translational Therapeutics, and Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. 5Graduate Program of Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 6Biostatistics Facility, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 7Department of Oncology, Sidney Kimmel Comprehensive Cancer Center (SKCCC), and Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 8Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 9Cancer Immunology and Immunotherapy Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. ✉e-mail: [email protected]

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology the B16.F10 (B16) mouse melanoma model, although it is undetect- NRP1 promotes terminal exhaustion in tumor-infiltrating CD8+ able on naive CD8+ T cells and only expressed on a small fraction T cells. Tumor-primed T cell memory contributes critically to post- of effector or memory CD8+ T cell subsets from unchallenged mice surgical tumor immunity21,22. We thus asked whether NRP1 acts as − − + (Fig. 1a). This was observed primarily in the KLRG1 CD127 Teff a CD8 T cell-intrinsic modulator of this process. At the time of and the KLRG1loCD127hi memory precursor effector cells (MPECs) tumor resection (D12), although the number of intratumoral CD8+ hi lo + + Cre L/L but remained low in the KLRG1 CD127 short-lived effector TCM (CD44 CD62L ) in the E8I Nrp1 mice was not significantly Cre cells (SLECs) and barely detectable in central memory T cells (TCM) altered compared with the E8I control mice, the number of intra- + + − (Fig. 1a and Extended Data Fig. 1a). The upregulation of NRP1 tumoral CD8 Teff (CD44 CD62L ) was significantly increased, + was also found in CD8 Teff subsets from mice infected with lym- resulting in a higher Teff to TCM ratio (Fig. 2a,b). We further examined phocytic choriomeningitis virus (LCMV), in particular higher in MPECs and SLECs, the bifurcation of which represents the first step the MPECs induced by clone 13 infection (D30), a chronic infec- of memory T cell commitment, but did not observe a significantly tion model known to induce T cell exhaustion20, compared to those altered ratio between these two subsets, although both showed induced by Armstrong (D8), a model of acute infection (Extended increased numbers in the E8ICreNrp1L/L mice (Fig. 2a,b). This was also Data Fig. 1b). Moreover, NRP1 was coexpressed with PD1 on CD8+ the case when we examined tumor-specific CD8+ T cells that recog- T cells both in unchallenged and tumor-bearing mice (Extended nized the melanocyte differentiation antigen gp100 (H2-Db gp100+, Data Fig. 1c), suggesting that NPR1 may act as an IR-like molecule designated as Db-gp100+ thereafter) (Extended Data Fig. 4a,b). in CD8+ T cells. Nevertheless, the frequency of Db-gp100+ CD8+ T cells, as well + + NRP1 CD8 TILs exhibited higher expression of markers their TCM phenotype in the spleen of these mice was significantly associated with T cell activation (CD44, CD69, CD25) and cell increased on D12 (Extended Data Fig. 4c,d), consistent with their proliferation (BrdU) compared to NRP1− cells, but lower enhanced protection against tumor rechallenge (Extended Data Fig. expression of CD62L, CD127 and KLRG1, markers associated 4e). Taken together, these data indicated that loss of NRP1 on CD8+ with naive/memory or senescent T cell phenotypes (Extended T cells promoted an intratumoral effector CD8+ T cell pool that Data Fig. 1d). NRP1 expression, as determined by high dimen- was more capable of generating a peripheral memory T cell pool, sional flow cytometry, consistently marked a subset of PD1hi, without any alteration in proportional fate choice between MPECs multi-IR+ CD8+ TILs, which was most prominent on D18 post-B16 and SLECs. inoculation, suggesting that NRP1 may primarily function on TEX Apart from generating long-lived memory T cells, MPECs are cells (Fig. 1b,c). considered to be the same precursors that give rise to TEX follow- To interrogate whether NRP1 specifically affects CD8+ T cell ing prolonged antigen stimulation, which commonly occurs in function in antitumor immunity, we generated mice with (1) CD8+ tumor-bearing or chronic virally infected hosts5. The Nrp1−/− CD8+ T cell–restricted NRP1-deficiency (E8ICreNrp1L/L) (Extended Data TILs exhibited a slight increase in IR expression on D12 and an Fig. 2a,b), and (2) CD8+ T cell–restricted, temporally controlled, increased proportion of 5-IR expressing cells compared to the constitutive NRP1 expression (E8ICreErt2gfpRosa26LSL.Ametrine.2A.Nrp1; GFP Nrp1+/+CD8+ TILs (Extended Data Fig. 4f,g). In contrast, there was marks CD8 expression, Ametrine marks Cre activity and Rosa26 a significant reduction of IR expression and reduced proportion of driven-Nrp1 expression) (Extended Data Fig. 2c). Deletion of Nrp1 5-IR expressing cells on D18 (Fig. 2c,d), when most intratumoral in CD8+ T cells did not substantially alter the composition of the CD8+ TILs were functional exhausted, evidenced by the substan- immune infiltrate in primary (1°) B16.F10 (B16) tumors (Extended tially reduced frequency of polyfunctional cytokine-producing Data Fig. 3a–d), nor did it result in a notable difference in the growth cells (Extended Data Fig. 4h,i). Although the Nrp1−/− CD8+ TILs of 1° B16 tumors (Fig. 1d and Extended Data Fig. 3e). Furthermore, did not show significantly improved polyfunctionality on D18 whereas therapeutic vaccination of B16-Ova tumor-bearing mice (Extended Data Fig. 4h,i), they did exhibit alterations in certain TEX with attenuated Listeria monocytogenes expressing Ova peptide related-features, such as reduced cell turnover indicated by lower (LM-Ova) exhibited a substantive reduction in tumor growth, this levels of Ki67 and cleaved Caspase 3 (Fig. 2e,f). Conversely, they was comparable between E8ICre and E8ICreNrp1L/L mice (Extended showed increased expression of the survival factor Bcl2 and nota- Data Fig. 3f). Collectively, these data indicate that NRP1 loss bly TCF1, the transcription factor associated with programming + 23,24 from CD8 T cells has no effect on primary tumor growth and the both memory precursor and progenitor TEX (pTEX) . Moreover, effector T cell response to primary tumors following therapeutic TCF1 is also required for the maintenance of intratumoral pTEX vaccination. pool by driving a transcriptional program that confers stem-like, To assess the effect of NRP1 on the recall response to a second- self-renewal properties25. Indeed, while the number of termi- + + − + ary tumor challenge, the primary tumor was resected at day (D) nally TEX (tTEX, CD44 PD1 TCF1 TIM3 ) did not significantly 12, mice left for 1 or 2 months, and then injected with B16 on the differ between the two genotypes on D18, the number of pTEX contralateral side (Fig. 1d). E8ICreNrp1L/L mice exhibited notably (CD44+PD1+TCF1+TIM3−) was significantly increased in the enhanced protection against a secondary (2°) B16 challenge 30 or E8ICreNrp1L/L mice (Fig. 2g). This was particularly evident for the Cre b + + 60 d following 1° tumor resection, relative to E8I mice (Fig. 1e tumor antigen-specific (D -gp100 ) CD8 Teff from the B16-g100 and Extended Data Fig. 3g). In contrast, 2° B16 tumor growth was tumor-bearing E8ICreNrp1L/L mice, which contained a higher fre- CreErt2gfp LSL.Nrp1 slightly exacerbated in E8I Rosa26 mice in which NRP1 quency and number of pTEX on D18 but also D12 (Fig. 2h). The was constitutively expressed on CD8+ T cells following treatment early time point (D12) observation is interesting, as it coincided with tamoxifen before 1° tumor challenge (Fig. 1f and Extended Data with the increased frequency of peripheral Db-gp100+CD8+ in the Fig. 3h). The E8ICreNrp1L/L mice also showed significantly improved E8ICreNrp1L/L mice on D12 (Extended Data Fig. 4c). Altogether, our sensitivity to anti-PD1 checkpoint blockade, when administered at data indicated that loss of NRP1 in late-stage CD8+ effector T cells a suboptimal dose (100 μg per mouse) in the 1° MC38 tumor model, restricts terminal exhaustion by reducing cell turnover and impor- resulting in 70% complete regression compared to 25% complete tantly, imposing a stem-like self-renewing phenotype that may regression in the E8ICre control mice (Fig. 1g–i). Taken together, improve differentiation toward functional, long-lived memory cells. these data indicate that loss of NRP1 in CD8+ T cells substantively enhances immunity against secondary exposure to tumors, includ- NRP1 cell-intrinsically limits the in vivo persistence of tumor ing relatively ‘cold’ tumors such as B16, and improves sensitivity to antigen-specific CD8+ T cells. To accurately track CD8+ T cell dif- anti-PD1 immunotherapy, albeit with little impact on the growth of ferentiation, persistence and fate in vivo, as well as assess if NRP1 untreated primary tumors. functions in a cell-intrinsic or cell-extrinsic manner, congenically

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

a Homeostatic b (naive mice, intestine) NRP1 PD1 LAG3 TIM3 TIGIT 2B4 + 100 20 7 20 7 20 7 20 7 20 7 20 7 75 10 6 10 6 10 6 10 6 10 6 10 6 0 0 5 0 5 5 0 5 0 5 0 5 50 –10 –10 4 –10 4 4 –10 4 –10 4 –10 4 25 –20 –20 3 –20 3 3 –20 3 –20 3 –20 3

bh-SNE2 –30 0 –30 –20 –10 0 10 20 30 –30 –20 –10 0 10 20 30 –30 –20 –10 0 10 20 30 –30 –20 –10 0 10 20 30 –30 –20 –10 0 10 20 30 –30 –20 –10 0 10 20 30 Percentage of NRP1 T eff T RM T CM bh-SNE1 naive

+ B16.F10 (TILs, D12) 5 10 35.6 33.9 4.3 20.1 8.9224.2 9.714.9 9.6 9.1 100 **** 4 NS P = 0.002 10 75 3 10 2B4 50 TIGIT

0 LAG3 (PE) TIM3 (APC)

–3 (PerCP-e710) (APC-Vio770) 25 PD1 (PE-Cy7) 10 0 11.8 8.9 7.7 12.9 0 10–3 0103 104 105 10–3 0103 104 105 10–3 01103 104 105 10–3 0103 104 105 10–3 0 03 104 105 Percentage of NRP1 eff naive T CM NRP1 (BV421) Early TMPECsSLECs

c D9 D12D18 d Primary Rechallenge: 5× Tamoxifen Resection (B16.F10) B16.F10

D–5~D–1 D0 D12 T1: +30 d (D42)T2: +60 d (D72)

E8ICreErt2gfp E8ICre E8ICreErt28I Cre L/L No. of IRs 65432 10 E8I Nrp1 .R26LSL.Nrp1 Arcs PD1TLAG3 IM32B4 TIGIT NRP1

e 1° B16F10 (B16) +30 d rechallenge +60 d rechallenge+f 1° B16 30 d rechallenge (B16)

E8I Cre (10) Sham (5) E8I Cre (11) Sham (5) E8I Cre (6) E8ICreErt2gfp (5) Sham (4) E8ICreErt2gfp (13) E8I Cre Nrp1L/L (11) E8I Cre Nrp1L/L (17) E8I Cre Nrp1L/L (9) E8ICreErt2gfp.Rosa26LSL.Nrp1 (5) E8ICreErt2gfp.Rosa26LSL.Nrp1 (11) 100 100 100 100 100 P = 0.0693 75 75 58.8% 75 75 75 55.6% 50 50 50 50 50 * P = 0.0024 25 25 9.1% * 25 16.7% 25 25 Percentage survival Percentage survival 0 0 0 0 0 Percentage tumor-free Percentage tumor-free 0102030 01020304050 Percentage tumor-free 01020304050 0102030 0510 15 20 Days post injection (B16) Days post rechallenge (B16) Days post rechallenge (B16) Days post injection (B16) Days post rechallenge (B16)

g h i E8I Cre + IgG E8I Cre + IgG (8) E8I CreNrp1L/L + IgG (6) E8I CreNrp1L/L + IgG E8I Cre + anti-PD1 (8) E8I CreNrp1L/L + anti-PD1 (10) E8I Cre + anti-PD1 CR:2/8 E8I CreNrp1L/L + anti-PD1

) CR:7/10 ) 3 Primary Anti-PD1 3 100 3,000 PR:1/8 3,000 PR:1/10 (MC38) (100 µg/i.p.) PD:5/8 PD:2/10 75 D0 D9D6 D12 2,000 2,000 P = 0.004 50 * 1,000 * * 1,000 * 25 * E8ICre *

Cre L/L 0 0 Percentage survival 0

E8I Nrp1 Tumor volume (mm Tumor volume (mm 010203040 01020304050 01020304050 Days post injection Days post injection Days post injection (MC38) (MC38) (MC38)

Fig. 1 | NRP1 limits CD8+ T cell–mediated antitumor immunity to tumor rechallenge and anti-PD1 immunotherapy. a, Expression of NRP1 measured by flow cytometry in subsets of CD8αβ+ T cells isolated from the intestine of naive mice (n = 7, upper) and B16.F10 (B16) tumors recovered on day 12 + + (D12) postinoculation (n = 9, lower); bar graphs tabulating the percentage of NRP1 cells within the indicated CD8αβ T cell subpopulations (TRM, tissue resident memory; NS, not significant). b, Top, Barnes–Hut Stochastic Neighbor Embedding (bh-SNE) visualization of the expressions of indicated markers by B16 intratumoral CD8+ cells recovered from E8ICre (WT) mice, D18 postinoculation. Bottom, representative flow plots depicting coexpression of NRP1 with other IRs in the same sample shown in the bh-SNE plots. c, SPICE (simplified presentation of incredibly complex evaluations) plots illustrating the coexpression of NRP1 and IRs by B16 tumor-infiltrating CD8+ T cells on D9, D12 and D18 postinoculation. d, Experimental scheme for induction of postsurgical tumor immunity with B16 tumors. e, E8ICre and E8ICreNrp1L/L mice subjected to B16 tumor model were monitored for tumor growth at primary (1°) and rechallenge phases, respectively. Left, survival curve for the 1° B16 tumor growth; middle and right, Kaplan–Meier curves for the percentage of tumor-free mice with the +30-d (middle) and +60-d (right) rechallenge schemes, respectively. f, E8ICreErt2gfp (E8ICreErt2gfp) and E8ICreErt2gfpRosa26LSL.mAmetrine.2A.Nrp1 (E8ICreErt2gfpRosa26LSL.Nrp1) mice were treated with tamoxifen (1.5 mg) by i.p. injection for 5 consecutive days before the B16 tumor model. Left, survival curve for the 1° B16 tumor growth; right, Kaplan–Meier curve for the percentage of tumor-free mice with the +30-d rechallenge scheme. g, Experimental scheme for anti-PD1 immunotherapy with MC38 tumor model. h,i, Tumor growth curve (h) and survival curve (i) of MC38 tumor implanted in the E8ICreNrp1L/L (n = 10) and E8ICre mice (n = 8). All data were pooled from three independent experiments, with n = 5 − 17 mice per group (as specified in the legend for each panel). Error bars, mean ± s.e.m.; statistical significance was determined by one-way ANOVA (a); log-rank test (e,f,i) or two-way ANOVA (h) with correction for multiple comparisons (****P < 0.0001 and other P values as indicated). marked Nrp1−/− and Nrp1+/+ pMel transgenic CD8+ T cells that are D-1 (Fig. 3a and Extended Data Fig. 5a). On D0, mice were injected specific for premelanosome protein gp100 (ref. 26) were mixed at with B16-gp100 cells, the tumor resected on D12 and mice rechal- a 1:1 ratio and cotransferred into CD45.1 C57BL/6 recipients on lenged on D42 with B16-gp100 cells. While Nrp1−/− and Nrp1+/+

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology pMel-T cell numbers were comparable at early stages of both the recovered from B16-gp100 tumors (D12 and D21 posttransfer), 1° and 2° tumor, Nrp1−/− cells outnumbered their wild-type (WT) along with draining (DLN) and non-draining lymph nodes (NdLNs) counterparts over time (Fig. 3b,c). The ratio between Nrp1−/− ver- (D12, D21, D35 and D63 posttransfer) (sampling scheme, Fig. 4a). sus Nrp1+/+ donor pMel-T cells positively correlated with the tumor Principal component analysis (PCA) of pooled datasets demon- size, consistent with the notion from the polyclonal CD8+ T cell strated that tumor-derived and peripherally derived T cells segre- + setting that loss of Nrp1 led to a survival advantage in CD8 Teff gated into two distinct clusters, in line with the effector-dominating under prolonged antigen exposure (Extended Data Fig. 5b). As a and memory-dominating phenotype of cells present in these two result, Nrp1−/− pMel-T cell number was increased on D12 and locations (Fig. 4b). The peripherally derived datasets further segre- exhibited delayed contraction compared with their WT counter- gated into four clusters (C1–C4), primarily based on the stage of cell parts (Extended Data Fig. 5c). Additionally, a roughly two to one activation, with the C1 and C2 consisting of cells recovered from the ratio of Nrp1−/− to WT pMel-T cells was consistently observed in primary phase (D12 and D21), C3 with cells from the late exhaus- the peripheral lymphoid tissues, but not in the blood during the tion/early memory phase (D35) and C4 with cells from recall/ 1° tumor phase, which was maintained long-term (Fig. 3b,c and secondary memory phase (D63). Loss of NRP1 had a greater Extended Data Fig. 5d,e). These data indicate that NRP1 restrains impact on the transcriptome of cells in the primary phase (C1 and in vivo persistence of tumor antigen-specific CD8+ T cells in a C2) and recall/secondary memory phage (C4), which was driven cell-intrinsic manner. by biosynthesis (C1), cell division (C2) and the effector T cell

Although NRP1 was highly induced on intratumoral pMel-Teff, activity and T or B cell interaction in the secondary effectors (C4), it was downregulated in early MPECs cells found in the periphery respectively (Supplementary Table 1). When compared to CD8+ on D21 and undetectable in established TCM on D56 (Extended T cell-specific gene signatures derived from LCMV infection mod- Data Fig. 5f), aligned with the observation from polyclonal CD8+ els, the loss of NRP1 in the tumor-infiltrating effector cells resulted T cells that NRP1 is primarily expressed during the effector phase. in a significant enrichment of gene signatures associated with a

Therefore, we hypothesized that the impact of NRP1 on Tmem fate naive or quiescent phenotype and reciprocally a reduction with may be selective for effector-to-memory (E→M) transition, rather exhaustion gene signatures, particularly at D21 (Fig. 4c,d). A simi- than Tmem pool maintenance, which occurs later. lar pattern was observed in the corresponding peripherally derived Consistent with our observations in E8ICreNrp1L/L mice, intra- dataset (cluster C2), suggesting that enhanced quiescence retention −/− tumoral Nrp1 pMel cells contained higher frequency of pTEX during E→M transition might contribute to increased memory + + + − −/− + (CD44 PD1 TCF1 TIM3 ) compared to their WT counterparts on differentiation in Nrp1 CD8 Teff cells (Fig. 4e). Furthermore, D12, supporting the notion that intratumoral pTEX phenotype were effector-, exhaustion- and memory-associated gene signatures were better preserved in the absence of NRP1 (Fig. 3d). Furthermore, enriched in Nrp1−/− pMel-T cells recovered on D63 (C4), indica- from D12 to D42, during which E→M transition occurs, the per- tive of enhanced recall activity and memory cell generation in the centage of Nrp1−/− MPECs within the donor cell pool (CD45.2+) absence of NRP1 (Fig. 4f). was preferentially sustained compared to Nrp1+/+ MPECs (Fig. Gene set enrichment analysis (GSEA) revealed that peripheral +/+ 3e and Extended Data Fig. 5g). Consequently, this contributed to Nrp1 pMel-T cells (mostly MPECs or Tmem) from D21 B16-gp100 −/− an increase in the Nrp1 TCM pool during this period, in con- tumor-bearing hosts exhibited substantive overlap with the gene +/+ − − trast to a smaller, unchanged Nrp1 TCM pool. Consistent with expression profile of Tcf7-GFP P14 cells (‘chronic TCF1 cells’, polyclonal CD8+ T cells, the NRP1+ fraction within the WT pMel GSE83978, ref. 24: NB, the gene Tcf7 encodes the protein TCF1) from donor-derived MPECs exhibited a higher percentage of Ki67+ cells a LCMV C13 infected host (Fig. 4g), which were reportedly defec- compared to their NRP1− counterparts (Extended Data Fig. 5h). tive in long-term persistence following chronic infection compared Additionally, this was associated with a Bcl2loIRF4hi short-lived to the ‘chronic TCF1+ cells’24. Conversely, the Nrp1−/− pMel-T cells effector phenotype, while the NRP1− counterparts had a predomi- possess a phenotype that resembles TCF1+ cells, evidenced by the nantly Bcl2hiIRF4lo long-lived cell phenotype27. Furthermore, abla- association with a gene signature corresponding to Tcf7-GFP+ tion of Nrp1 resulted in a higher ratio of Bcl2hi versus Bcl2lo cells P14 cells from B16-gp33 tumor-bearing host (GSE114631, ref. 25) within MPECs during E→M transition, particularly on D21 (Fig. 3f (Fig. 4g). These peripheral Nrp1−/− pMel-T cells also exhibited sig- and Extended Data Fig. 5i). Taken together, NRP1 appears to limit nificant enrichment for a subset of Id3-dependent genes, which proliferative quiescence in antigen-specific cells during the E→M were downregulated in Id3−/− pMel-T cells recovered from mice transition, leading to reduced cell survival and inefficient commit- that were infected with vaccinia virus encoding the cognate antigen ment to a memory T cell fate. gp100 (gp100-VV) (GSE23568, ref. 28) (Fig. 4h). Coincident with To identify downstream target(s) of NRP1 in CD8+ T cells, we this finding, Id3 expression (measured by an Id3-gfp reporter29) performed transcriptomic analysis using bulk-population RNA was enhanced in intratumoral Nrp1−/− pMel-T cells compared sequencing (bpRNA-seq) of Nrp1+/+ and Nrp1−/− donor pMel-T cells with WT counterparts on D18 (Extended Data Fig. 6a,b). Within

+ + Fig. 2 | NRP1 promotes terminal exhaustion in tumor-infiltrating CD8 T cells. a,b, Numeration of CD8 T cell subsets (TCM, Teff, MPECs, SLECs) infiltrating B16-gp100 tumor on D12, from the E8ICre (n = 7) or E8ICreNrp1L/L (n = 10) mice. Data presented as absolute cell number per gram tumor mass. Data were pooled from two independent experiments with consistent observations. c, Left, representative flow plots for the expression of IRs on CD8+ TILs of 1° B16 tumors on D18. Right, SPICE plots visualization for coexpression of multiple IRs. d, Numeration of 5-IR-coexpressing cells (5-IR+) on D18 (n = 8), depicted as the percentage within CD8+ TILs (left) and absolute number per gram tumor mass (right). Bar represents mean value. e, Representative flow cytometry plot for the expressions of cleaved Caspase 3 (cCasp3), Bcl2, Ki67 and TCF1 on CD8+ TILs recovered from 1° B16 tumors on D18 postinoculation. f, Bar graphs tabulating the mean percentage of cells of indicated phenotype within CD8+ TILs, that is, total cCasp3+ (n = 5), Bcl2hicCasp3− (n = 6), Ki67+ (n = 7) and TCF1+ (n = 8), respectively, on D12 and D18 of 1° B16 tumors. g, Representative flow cytometry plots depicting the subsets within the exhausted CD8+ + + + − T cell (TEX) pool of B16-gp100 tumors, harvested on D12 and D18, segregated by progenitor TEX (pTEX, CD44 PD1 TCF1 TIM3 ) and terminally TEX (tTEX, + + – + CD44 PD1 TCF1 TIM3 ). Bar graphs tabulating the number (mean value) of pTEX and tTEX per gram tumor mass are shown on the right. n = 8 for D12 and + n = 5 for D18, pooled from two independent experiments. h, The same pTEX and tTEX analysis as in f, but within the CD8 TILs specific for gp100-tetramer (Db-gp100+). Only samples with >50 intratumoral Db-gp100+ cells gated for flow cytometry analysis were included. All symbols represent individual mice; error bars, mean ± s.e.m.; statistical significance was determined by two-tailed unpaired Student’s t-test (a,b,d) or two-way ANOVA (f,g) with correction for multiple comparisons; all P values are indicated.

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles the intratumoral CD8+ T cells, Id3 expression primarily marks quiescence trait in Nrp1−/− pMel-T cells, as determined by the previ- a subset of pTEX, rather than TCM, with TCF1 serving as the key ous global GSEA analysis, but also suggested that TCF1 controlled lineage-defining transcription factor (Extended Data Fig. 6c). this transcription program, of which Id3 is known to be one of its Moreover, this Id3-dependent gene set exhibited considerable key targets24,30–32, that may primarily account for the alterations overlap with the genes that are differentially modulated between observed in Nrp1−/− pMel-T cells. + − −/− chronic TCF1 versus TCF1 cells in a subset of cell-cycle regulation In contrast, the intratumoral Nrp1 pMel-Teff cells exhibited a genes (for example, Ccnb2, Ccnb1, Nek2 and Prc1) (Supplementary significantly enriched signature comprising genes upregulated in Table 2). This observation not only supported an enhanced naive or CXCR5+CD8+ T cells (GSE76279, ref. 33) (Fig. 4i), a PD1-responsive

a E8ICre E8ICreNrp1LlL b E8ICre E8ICreNrp1LlL T TCM eff MPECs SLECs ) ) ) 4 12 4 9 P = 0.004 8 8 8 6 NS ) ) P = 0.02 CM

4 4 P = 0.04 P = 0.04 9 6 6 6 6 4 6 4 4 4 versus T Ratio eff SLECs 3 MPECs 2 3 2 2 2 per g tumor (×10

per g tumor (×10 per g tumor (×10 per g tumor (×10 eff CM

T 0 0 0 0 0 Ratio (T 0 T (MPECs versus SLECs)

c 1° tumor_D18 d

LAG3 TIM3 TIGIT 2B4 Multi-IR (PE) (APC) (PerCP-e710) (APC-Vio770)

105 7.4 25.6 7.3 26.2 9.0 24.0 11.5 20.6 Cre Cre LlL 104 E8I E8I Nrp1 E8I Cre +

40 ) 20 3 19.0 16.9 17.2 16.4 4 10 P = 0.0008 P = 0.0004 CD8 + 0 + 30 15

–3 45.2 44.8 42.8 44.9 CD8

10 + 20 10

5 5-IR 10 4.1 9.4 5.7 8.2 4.1 8.9 7.2 4.1 10 5 per g tumor (×10 104 Percentage 5-IR 0 0 20.4 22.6 20.4 20.6 103 E8I Cre .Nrp1L/L 0 –3 60.4 59.0 57.0 60.6 No. of IRs 54 32 10 PD1 (PE-Cy7) 10

10–3 0 103 104 105 Arcs PD1LAG3TIM32B4 TIGIT IR (indicated on top)

e f Cre Cre LlL 1° tumor_D18 E8I E8I Nrp1

+ 100 100 P = 0.008 5 hi 10 48.3 27.2 33.6 26.0 30.8 24.2 75 P = 0.001 75 104 P = 0.008 50 50 E8I Cre 103 25 25

0 Percentage Bcl2 –3 9.5 15.8 36.0 4.4 5.21 39.8 Percentage cCasp3 10 0 0 D12D18 D12D18 5 10 66.8 11.2 33.1 11.5 49.2 23.8 100 P = 0.031 100 P = 0.0007 + 104 + 75 75 103 Cre E8I 0 Ki67 50 50 . L/L Nrp1 10–3 12.4 9.6 53.5 1.9 6.5 20.5 TCF1 (PcBlue) Bcl2 (PE) (PerCP-Cy5.5) 25 25 Percentage Ki67 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 Percentage TCF1 0 0 cCasp3 (APC) cCasp3 (APC) Ki67 (PerCP-Cy5.5) D12D18 D12D18

g Cre h + + b + Cre CD44+PD1+ E8I CD44 PD1 D -gp100 E8I Cre L/L E8I CreNrp1L/L E8I Nrp1 D12 D18

D12 D18 ) 5 3 100 P = 0.0002 9 10 ) P = 0.003 3.6 13.0 105 1.5 5.0 3 EX 4 75

10 4 pT 6 10 + P = 0.053 3 50 E8I Cre 10 3 10 tumor (×10

-gp100 3 0 25 b D per g tumor (×10 0

–3 per g 10 70.3 61.1 EX 10–3 83.9 78.8 0 pT 0 D12 D18 D12D18 5 5 10 10 2.3 9.2 2.3 17.5 ) 600 100 3 ) 104 104 3 P = 0.0001

EX 75

400 tT E8I Cre 103 103 + . L/L 50 Nrp1 0 0

200 tumor (×10 –3 –3 62.1 -gp100 84.4 46.2 86.4 b

10 per g tumor (10 10 25 TCF1 (PcBlue) TCF1 (PcBlue) D EX per g 10–3 0 103 104 105 10–3 0 103 104 105 tT 0 10–3 0 103 104 105 10–3 0 103 104 105 0 TIM3 (BV605) D12 D18 TIM3 (BV605) D12D18

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

a pMel CD8+ T cell cotransfer model pMel-1x E8ICre + + + Contraction and Recall and (CD45.2 Thy1.1 Thy1.2 ) Priming Cotransfer at 1:1 1° memory formation 2° memory Nrp1+/+ pMel-T ratio, 4 × 105 total D–1 D0 D12 D21 D42D56–70 pMel-1 mice

Nrp1–/– pMel-T B6(CD45.1) First challenge Tumor Second challenge (B16-gp100) resection (B16-gp100) pMel-1x E8ICre Nrp1L/L (CD45.2+ Thy1.2+)

b 1° TILs c 2° TILs +/+ –/– +/+ –/– Nrp1 Nrp1 D9 D12 D15 D21 Nrp1 Nrp1 D14 D28 + + P = 0.0016 P = 0.0005 P = 0.0072 100 100 P = 0.004 105 105 49.2 40.6 31.7 23.6 47.3 12.7 104 104 50 50 103 103 0 50.8 59.4 68.3 76.4 0 52.2 84.9 10–3 10–3 0

0 Percentage in CD45.2 Percentage in CD45.2 10–3 0 103 104 105 9121521 10–3 0 103 104 105 14 28 Days post 1° tumor Days post 2° 1° NdLN 2° NdLN tumor +/+ –/– +/+ –/– Nrp1 Nrp1 D9 D12D21 D42 Nrp1 Nrp1 D14 D28 + + P = 0.0025 P = 0.0034 P = 0.0003 P = 0.0031 P = 0.035 100 100 P = 0.0013 105 105

104 51.8 43.7 24.5 26.3 104 29.5 33.3 50 50 103 103 0 0 70.5 62.6 48.2 56.3 75.5 73.7 10–3 10–3 Thy1.1 (PerCP-Cy5.5) Thy1.1 (PerCP-Cy5.5) 0 0 Percentage in CD45.2 –3 3 4 5 Percentage in CD45.2 –3 3 4 5 10 0 10 10 10 0912 15 21 42 10 0 10 10 10 14 28 Thy1.2 (BV605) Days post 1° tumor Thy1.2 (BV605) Days post 2° tumor

d Intratumoral CD44+PD1+ e Periphery, CD44+ +/+ –/– Nrp1 Nrp1 CD62L– CD62L+ +/+ –/– 5 Nrp1 Nrp1 10 4.8 8.0 (MPECs enriched) (TCM) + + T 105 MPECs CM 104 30 80 P = 0.003 104 P = 0.001 3 60 P = 0.021 10 20 0 103 40 P < 0.0001 TCF1 (PcBlue) –3 10 10 83.3 74.8 0 20 T P < 0.0001 CD27 (PE-Cy7) –3 eff 10–3 0 103 104 105 10 0 0 –3 3 4 5

10 0 10 10 10 Percentage in CD45.2 TIM3 (PE) Percentage in CD45.2 D12 D21 D35 D42 D12 D21 D35 D42 CD62L (APC) f Nrp1+/+ Nrp1–/– NdLN CD44+CD27+CD62L– NRP1– NRP1+ Nrp1+/+ Nrp1–/– P = 0.02 10.0 125 105 105 P = 0.003 34.0 0 ) 16 P = 0.048 lo 4 4 7.5 100 10 10 12 /Bcl2 3 21.8 3 hi 5.0 75 10 78.2 10 8 Bcl2 (PE) percentage percentage 0 0 99.0

EX 58.8 2.5 EX 50 4 CD8 (APC-Cy7) –3 10–3 tT

pT 10 0.0 25 10–3 01103 104 105 10–3 0103 104 105 10–3 0 03 104 105 Ratio (Bcl2 0 D12D21 NRP1 (BV421) IRF4 (Alexa647)

Fig. 3 | NRP1 cell-intrinsically limits the in vivo persistence of antigen-specific CD8+ T cells. a, Experimental scheme for pMel-T cell cotransfer model. b,c, Representative flow cytometry plots depicting the Nrp1+/+ and Nrp1−/− pMel-T cell donors detected in the B16-gp100 tumors (upper), and the matched NdLNs (lower) during primary phase (n = 5 for D9 and D15, n = 7 for D12 and D21) (b) and recall phase (n = 7 for both time points) (c). Bar graph on the side tabulates the mean percentage of cells of either genotype within the donor cell pool (CD45.2+). Replicates were derived from individual recipients, and three independent time course experiments with consistent observations were pooled. d, Top, representative flow cytometry plots showing the expression of TCF1 and TIM3 by intratumoral Nrp1+/+ or Nrp1−/− pMel-T donors (gated on CD44+PD1+), from B16-gp100 tumor harvested on D12. Bottom, +/+ −/− the percentage of pTEX and tTEX within the Nrp1 or Nrp1 pMel-T donors were tabulated (n = 8). Each pair of connected dots represented donor cells recovered from the same individual recipient; data were pooled from two independent experiments. e, Left, gating scheme used to identify subsets + enriched for MPECs or TCM within the CD44 donor-derived pMel-T cells found in the periphery between D12 and D42 of the primary phase. Right, +/+ −/− + − + + + frequencies of Nrp1 (n = 5) or Nrp1 (n = 5) -derived donor cells of the CD27 CD62L (MPECs), or CD27 CD62L (TCM) phenotype within CD45.2 compartment over time, recovered from NdLN. Each replicate was derived from an individual recipient, and three independent time course experiments with consistent observations were pooled. f, Left, representative flow cytometry plots showing the expression of Bcl2 and IRF4 in the NRP1− and NRP1+ fractions of Nrp1+/+ pMel-T cells of MPECs phenotype, recovered from NdLN on D21 of the primary phase. Right, ratio between the Bcl2hiIRF4lo and Bcl2loIRF4hi subset within the Nrp1+/+ or Nrp1−/− pMel-T cells (n = 3, from individual recipients) recovered from NdLN of CD45.1 recipient, on D12 and D21 of the primary phase. Bar represents mean value. Error bars, mean ± s.e.m.; statistical significance was determined by two-tailed paired Student’s t-test (b–d and f) or two-way ANOVA (e) with correction for multiple comparisons (P values as indicated). subset with stem cell-like, self-renewal properties34. This was con- observation that E8ICreNrp1L/L mice exhibited enhanced sensitivity + + sistent with the role of TCF1 in programming CXCR5 CD8 T cells to PD1 blockade-induced tumor regression and the enhanced pTEX during chronic LCMV infection34. This was also consistent with our phenotype in the Nrp1−/− TILs. The latter notion was particularly

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

a B6(CD45.1) D21

D–1 D0 Experimental Nrp1+/+ pMel-T D12 D35 D42D63 manipulation Nrp1–/– pMel-T Sampling for RNA-seq 1° B16-gp100 Resection 2° B16-gp100

TissueTumor Peripheral (DLN)Peripheral(NDLN)

Time point D12 (1°) D21 (1°) D12 (1°) D21 (1°) D63 (2°) D12 (1°) D21 (1°) D35 (1°) D63 (2°)

Genotype Nrp1–/– versus Nrp1+/+ Nrp1–/– versus Nrp1+/+ Nrp1–/– versus Nrp1+/+

b All samples DLN and NdLN samples g GSE83978_Chr_TCF1–_UP +/+ 200 Nrp1 GSE114631_TCF1+_UP 100 C2 Tumor –/– GSE GSE DLN C1 Nrp1 GSE83978 83978 114631 and NdLN NDLN GSE114631 100 NES 1.72 1.29 DLN 0.6 0 P <1 × 10–5 0.038 Nrp1+/+ 0.4 –5

0 Dim.1 FDR <1 × 10 0.186 t -SNE2 Nrp1–/– 0.2 Tumor –100 NDLN –100 C4 0 5,000110,000 5,000 DLN C3 NdLN, D21 –0.2

Enrichment score (ES) –0.4 –400 –2000 200 100500–50–100 Nrp1+/+ Nrp1–/– t-SNE1 Dim.2

h GSE23568_ID3_KO_down c d Tumor_D12 Tumor_D21 0.6 NES: 1.55 P = 0.0025 Naive Naive –/– –/– 0.4 FDR: 0.006 2 Nrp1 versus 2 Nrp1 versus +/+ +/+ 1 Nrp1 1 Nrp1 0.2 0 0

–1 –1 0 NdLN, D21 –2 –2 5,00010,00015,000 –0.2

EFF ESG EFF ESG Enrichment score (ES) Nrp1–/– Nrp1+/+

i GSE76279_CXCR5_positive D12D21 D12 D21 MEM MEM NES 1.25 1.41 0.6 P 0.03 <1 × 10–5 e –5 Periphery_C2 f Periphery_C4 0.4 FDR 0.02 <1 × 10

Naive –/– Naive –/– 0.2 2 Nrp1 versus 2 Nrp1 versus +/+ +/+ 1 1

Nrp1 Nrp1 Tumor 0 10,000 20,000 30,000 0 0 –0.2 –1 –1

–2 –2 Enrichment score (ES) –0.4 EFF ESG EFF ESG Nrp1–/– Nrp1+/+

MEM MEM

Fig. 4 | Impact of NRP1 on the transcriptome of antigen-specific CD8+ T cells in vivo. bpRNA-seq was performed on donor-derived Nrp1+/+ (CD45.2+Thy1.1+Thy1.2+) and Nrp1−/− (CD45.2+Thy1.2+) pMel-T cells, sorted on D12, D21 and D35 of the primary phase, as well as D21 post-rechallenge (D63) from tumor (D12 and D21 only), DLN and NdLN, respectively. A total of 34 datasets were generated and grouped into five clusters (one tumor-derived and four periphery-derived), by unbiased clustering (described in Methods) for subsequent analysis. Genotype comparison (Nrp1−/− versus Nrp1+/+) was performed at pathway level using the GSEA within each cluster against the MSigDB C7. a, Sampling scheme for Nrp1−/− and Nrp1+/+ pMel-T cells subjected to bpRNA-seq. b, Left, t-distributed stochastic neighbor embedding (t-SNE) plots illustrating the segregation between tumor- and periphery-derived datasets; right, subclusters within the periphery-derived datasets by PCA. c–f, Radar plots depicting the enrichment for CD8-associated phenotypes, that is, naive, effector (EFF), exhausted (ESG) and memory (MEM), that corresponded to the Nrp1–/– pMel-T cell in the tumor-derived cluster (D12 and D21) and periphery-derived cluster nos. 2 and 4, by using the CD8-specific GSEA analysis (described in Methods). g–i, The GSEA plots assessing the enrichment of the gene signatures of chronic TCF1− P14 cells (GSE83978) or of TCF1+ P14 cells (GSE114631) (g); the targets of Id3 (GSE23568) between the peripherally derived Nrp1−/− and Nrp1+/+ pMel-T cells (h) and the signature of CXCR5+CD8+ T cells (GSE76279) between the tumor-derived Nrp1−/− and Nrp1+/+ pMel-T cells (i).

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

a Day 0–2: primary stimulation Day 2–8: chronic stimulation

Bulk CD8+ Negative selection Restimulation for CD8+ T cells

123456 123456789101112 A A B CD3/CD28 CD3/CD28 CD3/CD28 B C beads beads beads D C E F D G H

Cre +/+ Anti-CD3/CD28 (plate-bound) • E8I → Nrp1 –1 2° Restim D2–D4 D4–D6 Cre L/L 100 U ml rhIL-2 • E8I Nrp1 → Nrp1–/– 3° Restim D2–D4 D4–D6 D6–D8

TCF1+TIM3– b Restim. None 2° 3° Re-stim None 2° 3° c 60 Nrp1+/+

5 5 + 10 34.2 13.3 10.92.3 1.7 18.0 10 68.0 28.4 18.9 47.8 4.3 55.0 P = 0.019 –/– 40 Nrp1

4 4 PD1 10 10 + 20 3 3

+/+ 10 10 CD44

Nrp1 Percentage in 0 0 0 –3 33.8 74.0 78.4 –3 0.5 24.9 38.7 10 10 Restim. None 2° 3° 5 105 9.3 54.0 + – 10 35.2 10.8 16.03.3 4.7 33.8 74.0 21.5 38.6 31.1 100 Ly108 TIM3 4 4 + 10 10 75 PD1 + P = 0.014 Nrp1–/– 103 103 50 0 0 CD44

Percentage in 25 –3 34.6 62.1 56.3 –3 0.2 16.2 33.3 10 Ly108 (PE) 10 TCF1 ( PcBlue) 0 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 Restim. None 2° 3° TIM3 (BV605) TIM3 (Alexa647)

e d CD44+PD1+ P = 0.023 105 100 Isotype 2,500 Nrp1+/+ 80 Non-stim 2,000 Nrp1–/– 104 60 Ly108+TIM3– 1,500 103 40 Ly108+TIM3+ 1,000 Ly108 (PE)

0 c-Jun (gMFI) 20 Ly108–TIM3+ 500 10–3 Normalized to mode 0 0 –3 3 4 5 –3 3 4 5 10 0 10 10 10 10 0 10 10 10 – + + TIM3 (BV605) c-Jun (Alexa647) + TIM3 + TIM3 – TIM3

Ly108 Ly108 Ly108 f CD44+PD1+

Ly108+TIM3– Ly108+TIM3+ Ly108–TIM3+ E8ICre 4,000 P = 0.018 100 E8ICre E8ICreNrp1L/L P = 0.006 80 E8ICreNrp1L/L 3,000 60 P = 0.053 2,000 40

c-Jun (gMFI) 1,000 20

Normalized to mode 0 0 – + + 10–3 0 103 104 105 10–3 0 103 104 105 10–3 0 103 104 105 c-Jun (Alexa647) + TIM3 + TIM3 – TIM3

Ly108 Ly108 Ly108

Fig. 5 | NRP1 inhibits c-Jun/AP-1 activation in chronically stimulated CD8+ T cells. a, Experimental scheme for in vitro chronic stimulation of CD8+ T cells. b, Representative flow cytometry plots showing the expression of TCF1, TIM3 and Ly108 in the in vitro chronically stimulated Nrp1+/+ or Nrp1−/− CD8+ T cells. c, The percentage of TCF1+TIM3− or Ly108+TIM3− within the CD44+PD1+ gate, from Nrp1+/+ or Nrp1−/− CD8+ T cells cultured under the indicated conditions. d, Left, representative flow plot depicting the gating strategy for measuring c-Jun expression. Right, representative histogram illustrating the expression of c-Jun by indicated subsets in the CD8+ T cells subjected to chronic stimulation. e,f, Expression of c-Jun, quantified by genomic mean fluorescence intensity (gMFI) by flow cytometry, within the indicated subsets from chronically stimulated Nrp1+/+ or Nrp1−/− CD8+ T cells (n = 5 for each group) (e), or CD8+ TILs from B16-gp100 tumor implanted in the E8ICre (n = 6) or E8ICreNrp1L/L (n = 5) mice (f). Representative histograms for the genotype comparison within the indicated subsets are shown. Error bars, mean ± s.e.m.; data in a–e were aggregated from five independent in vitro stimulation assays and data in f were from two independent mice cohorts. Statistical significance was determined by two-tailed unpaired Student’s t-test (c) or two-way ANOVA (e,f) with correction for multiple comparisons. All P values are indicated. relevant considering that CXCR5 signature enrichment was more quiescence (Extended Data Fig. 6d). Taken together, our transcrip- significant on D21 compared to D12 (Fig. 4i), which indicated that tomic analysis strongly suggested that the absence of NRP1 drives the upregulated CXCR5 signature was associated with a reduced a TCF1-dependent transcriptional program that promotes memory exhaustion phenotype in the Nrp1−/− CD8+ TILs from late-stage fate choice in MPECs under settings associated with prolonged anti- tumors. This was validated by increased CXCR5 protein expression gen stimulation. on Nrp1−/− pMel-T cells recovered from D21 tumors, compared to their WT counterparts. This elevated CXCR5 expression was pref- NRP1 inhibits c-Jun/AP-1 activation in chronically stimulated erentially enriched in the Ki67− fraction, supporting the notion that CD8+ T cells. To better determine the CD8+ T cell-intrinsic target a gain of ‘stemness’ was associated with retention of proliferative that is directly modulated by NRP1, we sought to model chronic

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

ab c Healthy donor Early Advanced Early versus advanced disease 01.2 1.01.5 09.5 P = 0.016 Healthy donor 80 104 P = 0.045 Early Advanced 60 P = 0.0037 104 Naive 48.4 1.08 3 40 TEM 10 P = 0.0009 NS TCM 103 20 NS 0 98.8 0 95.71.8 89.51.0 Surface NRP1(%) 0 3 4 5 3 4 5 3 4 5 CCR7 0 0 10 10 10 0 10 10 10 0 10 10 10 TEM TEMRA T T 44.2 EM EMRA (PE-Dazzle 594) 6.20 0.95 0.95 1.3 15.4 0.7 38.1 d Arcs No. of IRs 104 10–3 0 103 104 PD1 5 CD45RA (BV520) 4 T LAG3 EMRA 3 3 10 CD39 2 TIGIT 0 97.50.5 82.11.3 60.90.3 1 PD1(PerCP-Cy5.5) NRP1 0 103 104 105 0 103 104 105 0 103 104 105 0 NRP1(FITC)

e fgh Stable (SD) versus NRP1(%)<3 (n = 14) ) NRP1(%)<9 (n = 16) + progressive disease (PD) NRP1(%) 3 (n = 9) NRP1(%) 9 (n = 7) T) ≥ ≥ + NS 80 SD 80 P = 0.004 100 100 100 40 PD r = –0.4141 60 P = 0.005 NS P = 0.034 50 40 10 50 * 50 20 Effector memory

Surface NRP1 (%) 0 0 0 0 0 Percentage survival Percentage survival

0 5 10 15 (percentage of total CD8 SD PD TEM TEMRA 0 20 40 60 0 20 40 60 EM (percentage of total CD8

(Months) (Months) Surface NRP1 (%) T

i j P = 0.006 ) RESP Anti-PD1 + PROG Anti-PD1 P = 0.018 P = 0.004 Anti-PD1 50

50 ) 50 +anti-CTLA4 + NS Anti-PD1 40 Anti-PD1 40 40 +anti-CTLA4 r = –0.4327 +anti-LAG3 30 Anti-PD 30 NS 30 NS 20 +anti-LAG3 20 r = –0.6393

( % in CD8 20 10 P = 0.024

EM 10 (percentage in CD8 T

Surface NRP1 (%) 0 10 EM 0 10 20 30 40 50 T Pre Pre Pre Pre Surface NRP1 (%) Post Post Post Post RESP PROG RESP PROG

k P = 0.005 l RESP Anti-PD1 P = 0.024 PROG Anti-PD1 30 NS 15 P = 0.026 Anti-PD1 12.0 +anti-CTLA4 Anti-PD1 Anti-PD1 20 10 +anti-CTLA4 + 8.0 r = –0.2022 +anti-LAG3 NS (%) + Anti-PD1 P = 0.3926 10 5 +anti-LAG3 TCF1 4.0

TCF1 r = –0.4237 0 P = 0.063 Surface NRP1 (%) 0 0 0310 20 0 Pre Pre Pre Pre Post Post Post Post Surface NRP1 (%) RESP PROG RESP PROG

+ + Fig. 6 | Elevated NRP1 TEM in patients with cancer is associated with poor survival and decreased response to ICB therapy. a–h, CD8 T cell subsets were analyzed on banked PBL of healthy donors (n = 10), and patients with advanced HNSCC (n = 48, cohort A, Supplementary Table 3). a, Gating scheme of CD8 subsets (naive, TEM, TEMRA) in human peripheral blood (PB) (TEM, effector memory T cells; TEMRA, effector memory RA T cells). b, Representative + + flow cytometry plots for surface NRP1 and PD1 expression on CD8 TEM and TEMRA. c, Bar graphs tabulating the percentage of surface NRP1 cells within + PBL-derived CD8 TEM or TEMRA of healthy donors (n = 10) and the patients with HNSCC stratified by disease stage (early, n = 23 versus advanced, n = 25). Bar represents mean value. d, SPICE plots depicting coexpression of surface NRP1 with other IRs (PD1, LAG3, CD39 and TIGIT) by PBL CD8+ T cells from + + the advanced patient cohort. e, Bar graphs tabulating the percentage of surface NRP1 cells within PBL-derived CD8 TEM or TEMRA, stratified by recurrence (stable disease versus progressive disease). SD, stable disease (n = 16); PD, progressive disease (n = 9). Bar represents mean value. f, Survival curve + + stratified by high or low (threshold indicated) surface NRP1 expression (by percentage of NRP1 cells) on CD8 TEM and TEMRA, respectively. + *P = 0.0454. g, Correlation between percentage of TEM within CD8 T cells and their surface NRP1 expression across all the patients with HNSCC (n = 48). + h, The percentage of TEM within CD8 T cells in subgroups of advanced patients stratified by disease recurrence (SD, n = 16; PD, n = 9). i–m, PBL were isolated from patients with advanced skin cancers both before (pre) or after (post) the start of ICB therapies (n = 40; cohort B, Supplementary Table 4). + Patients were stratified by responsiveness to treatment (responder (RESP) versus progressor (PROG)). i, Surface NRP1 expression on CD8 TEM (left) + + and percentage of TEM within PBL CD8 T cells (right). j, Correlation between percentage of TEM in CD8 T cells and TEM-NRP1 expression, both from + post-ICB measurements, stratified by response. k,l, Within each CD8 TEMRA subset, the percentage of cells expressing NRP1 (left) and TCF1 (right) (k) and the correlation between post-ICB TCF1 versus surface NRP1 expression (l), stratified by response; error bars, mean ± s.e.m.; statistical significance was determined by one-way ANOVA (c), two-tailed unpaired Student’s t-test (e,h), Wilcoxon test (i,k), and the log-rank test (f) and Pearson correlation analysis (g,j,l). Coefficient r was calculated and a t-test was performed to assess linear association (all P values are indicated).

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology antigen stimulation in vitro. Naive Nrp1+/+ or Nrp1−/− CD8+ T cells stages (Fig. 5f). Taken together, these data indicate that NRP1 sup- activated with plate-bound anti-CD3 plus anti-CD28 for 48 h were presses the activation of c-Jun, a mechanism known to counteract either rested in IL-2-containing medium (acute activation, 1°), or the T cell exhaustion transcriptional program39, thereby promot- subjected to two- (2°) or three- rounds (3°) of restimulation by ing progression to terminal exhaustion in chronically stimulated anti-CD3/anti-CD28-coated beads (chronic stimulation, Fig. 5a). CD8+ T cells. The NRP1 ligand Semaphorin-4A is highly expressed on CD8+ + T cells and thus available for NRP1 ligation in cis or trans in this Elevated NRP1 TEM in patients with cancer is associated with assay system (Extended Data Fig. 7a)13,35. Compared to acute acti- poor survival and decreased response to ICB therapy. Last, we vation, repetitive stimulation was sufficient to drive some of the interrogated the clinical relevance of NRP1 expression and its + hallmarks of in vivo TEX, such as the coexpression of multiple IRs modulation of CD8 memory T cell differentiation in patients with (for example PD1 and LAG3), and altered cytokine production (a cancer. We assessed a cohort of peripheral blood lymphocyte (PBL) switch from IFN-γ+TNF-α+ to partial IFN-γ+GzmB+ producers), samples from treatment-naive patients with HNSCC, consisting of which were increased by the end of the 3° compared to 2° restimu- 23 early stage disease (T1, T2 and/or N0) and 25 advanced disease lation (Extended Data Fig. 7b,c). Surface expression of NRP1 was cases (T3, T4 and/or N1, N2B, N2C) (Cohort A, Supplementary more robustly maintained in the chronically stimulated cells com- Table 3). Increased surface NRP1 expression was significantly pared to the acutely activated cells, consistent with the notion that associated with advanced disease, which was observed in both the + NRP1 expression requires continuous antigen stimulation, similar CD8 effector memory (TEM) and terminally differentiated effec- to many known IRs (Extended Data Fig. 7a). tor (TEMRA) subsets (Fig. 6a–c). Consistent with the NRP1 expres- Although there was no significant difference in the overall sion pattern observed on the TILs from the B16 mouse model, multi-IR or cytokine expression between genotypes at any time NRP1 expression correlated with PD1 expression on CD8+ T cells point with chronically stimulated cells, loss of Nrp1 resulted in an in patients with HNSCC along with enhanced expression of other + − increase in the percentage TCF1 TIM3 pTEX-like cells, as well as IRs, raising the possibility that this also correlated with exhaus- + − + an increase in the percentage of Ly108 TIM3 cells (Ly108, encoded tion (Fig. 6d). NRP1 expression on CD8 TEM, but not TEMRA, by Slamf6, is considered a reliable surface marker for TCF1+ cells24). was enhanced in patients with advanced disease and correlated − + This was rapidly replaced by the TCF1 TIM3 tTEX-like pheno- with disease recurrence and decreased overall survival (Fig. 6e,f), +/+ + type within Nrp1 pMel-T cells (Fig. 5b,c). Retention of TCF1 suggesting that the NRP1 expression on CD8 TEM may be a pre- and Ly108 expression in Nrp1−/− cells was more prominent under dictor for poor prognosis. NRP1 may also play a role in limiting

2° than 3° restimulation, indicating that NRP1 acts as one of the the peripheral Tmem pool in the patients with HNSCC, indicated by early regulators that promotes TCF1 loss in chronically activated the inverse correlation between NRP1 expression and the size of CD8+ T cells. the memory CD8+ T cell compartment in the periphery (Fig. 6g). It is known that signaling downstream of the T cell recep- Consistent with this notion, patients with advanced, progressive tor (TCR) and costimulation is dampened in T cells subjected to HNSCC and elevated NRP1 expression on their TEM cells exhibited + prolonged antigen stimulation, resulting in an inadequate pairing an even greater reduction in peripheral TEM percentage within CD8 between nuclear factor of activated T cells (NFAT) and the canonical T cells (Fig. 6h). AP-1 family transcription factor (c-Jun/c-Fos heterodimer), thereby The impact of CD8+ T cell expression of NRP1 on responses driving a gene transcription program that promotes T cell dysfunc- of patients with cancer to ICB immunotherapy was interro- tion36,37. Thus, we next assessed NFAT1 nuclear translocation and gated in a second cohort (cohort B, Supplementary Table 4) of induction of c-Jun, a key member of the AP-1 family, in response 40 patients with advanced skin cancers who received single agent to TCR restimulation. NFAT1 translocated to the nucleus in ~20% anti-PD1 or combinatorial ICB therapies ((1) anti-PD1 alone; (2) of both Nrp1+/+ and Nrp1−/− pMel-T cells that were chronically anti-PD1 + anti-CTLA4 and (3) anti-PD1 + anti-LAG3). PBL taken stimulated compared to 80% of naive CD8+ T cells (Supplementary before and after 12 weeks of therapy was collected from 20 ICB Fig 7d,e). The transcriptional activation of c-Jun, an event that responders and 20 ICB progressors, stratified based on RECIST requires concerted signaling through the TCR in conjunction with 1.1 criteria. The significant increase in surface NRP1 expression 38 + costimulation , was also reduced in the chronically stimulated on CD8 TEM post-ICB therapy was only observed in patients with cells, the extent of which correlated with the progression from the tumors that progressed, although their baseline NRP1 expression Ly108+TIM3− to the Ly108−TIM3+ stage (Fig. 5d). Therefore, the before treatment was comparable (Fig. 6i). By contrast, the size of three subsets of chronically stimulated cells, stratified by Ly108 the TEM pool was significantly reduced in the ICB progressors post- (surface surrogate for TCF1 expression) and TIM3 expression, were treatment (Fig. 6i), which inversely correlated with their surface not only phenotypically distinct but also associated with progressive NRP1 expression (Fig. 6j), suggesting the inability to maintain a signaling impairment. c-Jun activation was significantly increased sizable TEM pool may be associated with an unfavorable response in chronically stimulated Nrp1−/− pMel-T cells, in particular for the to ICB therapy. While these observations aligned with the synergy Ly108+TIM3− and Ly108+TIM3+ cell subsets (Fig. 5e). These data observed between NRP1 deficiency and anti-PD1 treatment in indicated that NRP1 may directly modulate the pairing between mouse models (Fig. 1g–i), they further suggested that high NRP1

NFAT and AP-1 by downmodulating c-Jun. expression by TEM may contribute to acquired resistance to ICB Prompted by this observation, we asked whether NRP1 also mod- immunotherapy. Of note, a reduction in TCF1 expression by CD8+ + ulates c-Jun induction in tumor-infiltrating CD8 T cells on restim- TEMRA was also significantly associated with ICB therapy resistance ulation. Indeed, c-Jun activation in the CD8+CD44+PD1+ Nrp1+/+ (Fig. 6k), in line with recent reports suggesting that intratumoral TILs was only preserved in the Ly108+TIM3− subset, progressively TCF1+CD8+ T cells are preferentially targeted for reinvigoration declined as cells differentiated through the Ly108+TIM3+ and and correlate with better survival40. In addition, a trend toward an Ly108−TIM3+ subsets, whereas their peripheral CD8+CD44+PD1+ inverse correlation between expression of TCF1 and NRP1 by CD8 + − T cell counterparts (predominantly Ly108 TIM3 ) uniformly TEMRA was found in the tumor progressors, which was consistent upregulated c-Jun on TCR restimulation (Extended Data Fig. 7f). with the notion that NRP1 promotes terminal exhaustion by nega- This indicated that decreased c-Jun activation also correlated with tively modulating TCF1 (Fig. 6l). Taken together, these data indicate −/− the progression toward exhaustion in vivo. In contrast, the Nrp1 that NRP1 was highly expressed on the human TEX in patients with CD8+CD44+PD1+ TILs showed improved c-Jun activation on cancer and negatively associated with the size of the memory T cell restimulation, particularly at the Ly108+TIM3+ and Ly108−TIM3+ pool, disease prognosis and responsiveness to ICB therapy.

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Discussion 3. Reading, J. L. et al. Te function and dysfunction of memory CD8+ T cells in In summary, our findings highlight NRP1 as a previously unchar- tumor immunity. Immunol. Rev. 283, 194–212 (2018). 4. Fares, C. M., Van Allen, E. M., Drake, C. G., Allison, J. P. & Hu-Lieskovan, S. acterized immune checkpoint that affects the development and Mechanisms of resistance to immune checkpoint blockade: why does + function of intratumoral CD8 T cells in a cell-intrinsic manner checkpoint inhibitor immunotherapy not work for all patients? Am. Soc. Clin. and selectively affects the generation of memory precursors during Oncol. Educ. Book 39, 147–164 (2019). an antitumor immune response. This may distinguish NRP1 from 5. Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell other IRs, such as PD1, CTLA4 and LAG3, that primarily affect exhaustion. Nat. Rev. Immunol. 15, 486–499 (2015). 6. Pauken, K. E. et al. Epigenetic stability of exhausted T cells limits durability effector T cell development and function. Although loss of NRP1 of reinvigoration by PD-1 blockade. Science 354, 1160–1165 (2016). did not enhance the antitumor response to a primary tumor, it had 7. Sen, D. R. et al. Te epigenetic landscape of T cell exhaustion. Science 354, a substantive effect on the development of T cell memory to subse- 1165–1169 (2016). quent tumor challenge. Nevertheless, CD8+ T cell–restricted NRP1 8. Kawasaki, T. et al. A requirement for neuropilin-1 in embryonic vessel deletion in combination with ICB therapy did result in enhanced formation. Development 126, 4895–4902 (1999). 9. Kitsukawa, T. et al. Neuropilin–semaphorin III/D-mediated chemorepulsive tumor clearance suggesting that targeting NRP1 may serve to signals play a crucial role in peripheral nerve projection in mice. Neuron 19, enhance priming of the antitumor immune response in conjunction 995–1005 (1997). with ICB. 10. Gu, C. et al. Neuropilin-1 conveys semaphorin and VEGF signaling during Mechanistically, NRP1 may affect memory T cell development in neural and cardiovascular development. Dev. Cell 5, 45–57 (2003). 11. Roy, S. et al. Multifaceted role of neuropilins in the immune system: potential three ways. First, NRP1 expression correlates with Bcl2 loss, leading targets for immunotherapy. Front. Immunol. 8, 1228 (2017). + to substantive restraint on the frequency of antitumor CD8 T cells 12. Bruder, D. et al. Neuropilin-1: a surface marker of regulatory T cells. Eur. J. over time, suggesting that NRP1 contributes to antigen-dependent Immunol. 34, 623–630 (2004). maintenance of CD8+ T cell exhaustion and impaired memory 13. Delgofe, G. M. et al. Stability and function of regulatory T cells is maintained differentiation41. Second, the absence of NRP1 sustains a TCF1+ by a neuropilin-1–semaphorin-4a axis. Nature 501, 252–256 (2013). 14. Overacre-Delgofe, A. E. et al. Interferon-γ drives Treg fragility to promote pTEX subset that originates from phenotypically defined MPECs anti-tumor immunity. Cell 169, 1130–1141.e11 (2017). hi lo (CD127 KLRG1 ) but are transcriptionally and epigenetically dis- 15. Battaglia, A. et al. Metastatic tumour cells favour the generation of a tinct from conventional memory precursors42. While intratumoral tolerogenic milieu in tumour draining lymph node in patients with early + TCF1 pTEX exhibit enhanced in vivo persistence, it is unclear cervical cancer. Cancer Immunol. Immunother. 58, 1363–1373 (2009). whether they are capable of converting to bona fide memory T cells 16. Battaglia, A. et al. Neuropilin-1 expression identifies a subset of regulatory Cre L/L T cells in human lymph nodes that is modulated by preoperative in vivo. Our data support this notion as E8I Nrp1 mice exhibit chemoradiation therapy in cervical cancer. Immunology 123, enhanced protection from secondary tumor challenge, which 129–138 (2008). was associated with an enlarged peripheral TCM cell pool follow- 17. Jackson, S. R., Berrien-Elliott, M., Yuan, J., Hsueh, E. C. & Teague, R. M. ing primary tumor resection on D12. Future lineage-tracing or Neuropilin-1 expression is induced on tolerant self-reactive CD8+ T cells but in situ labeling approaches specifically targeting pT would help to is dispensable for the tolerant phenotype. PLoS ONE 9, e110707 (2014). EX 18. Fleissner, D., Hansen, W., Gefers, R., Buer, J. & Westendorf, A. M. Local definitively address this model. Finally, the increased proliferation induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid observed in the presence of NRP1 may serve as a mechanism to tissues. PLoS ONE 5, e15373 (2010). compensate for exhaustion-driven CD8+ T cell dysfunction but at 19. Leclerc, M. et al. Regulation of antitumour CD8 T-cell immunity and the expense of memory differentiation, which requires proliferative checkpoint blockade immunotherapy by Neuropilin-1. Nat. Commun. 10, quiescence (slower turnover) while gaining stem cell-like proper- 3345 (2019). 43,44 20. Kalia, V., Sarkar, S. & Ahmed, R. CD8 T-cell memory diferentiation during ties (self-renewal and multipotency) . Given the heterogeneity acute and chronic viral infections. Adv. Exp. Med. Biol. 684, 79–95 (2010). observed within TEX, NRP1 may identify a TEX subset that exhibits 21. Bursuker, I. & North, R. J. Immunological consequences of tumor excision: inflexibility in terms of potential for memory differentiation and from active immunity to immunological memory. Int. J. Cancer 37, reinvigoration following ICB. The apparent impact of NRP1 on 275–281 (1986). CD8+ T cells in patients with advanced cancer is consistent with our 22. Zhang, P., Cote, A. L., de Vries, V. C., Usherwood, E. J. & Turk, M. J. Induction of postsurgical tumor immunity and T-cell memory by a poorly mouse models of cancer wherein increased NRP1 expression cor- immunogenic tumor. Cancer Res. 67, 6468–6476 (2007). related with a reduced TEM pool and decreased patient survival. Our 23. Wu, T. et al. Te TCF1-Bcl6 axis counteracts type I interferon to repress findings identify NRP1 as a new immune checkpoint that affects exhaustion and maintain T cell stemness. Sci. Immunol. 1, eaai8593 (2016). the development of T cell memory to tumor antigens, suggesting 24. Utzschneider, D. T. et al. T cell factor 1-expressing memory-like CD8+ T cells sustain the immune response to chronic viral infections. Immunity 45, that combinatorial blockade of PD1 and NRP1 may lead to more 415–427 (2016). durable, systemic antitumor immunity and long-term remission in 25. Siddiqui, I. et al. Intratumoral Tcf1+PD-1+CD8+ T cells with stem-like patients with cancer. properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50, 195–211 (2019). Online content 26. Overwijk, W. W. et al. Tumor regression and autoimmunity afer reversal of a functionally tolerant state of self-reactive CD8+ T cells. J. Exp. Med. 198, Any methods, additional references, Nature Research report- 569–580 (2003). ing summaries, source data, extended data, supplementary infor- 27. Man, K. et al. Transcription factor IRF4 promotes CD8+ T cell exhaustion mation, acknowledgements, peer review information; details of and limits the development of memory-like T cells during chronic infection. author contributions and competing interests; and statements of Immunity 47, 1129–1141 (2017). data and code availability are available at https://doi.org/10.1038/ 28. Ji, Y. et al. Repression of the DNA-binding inhibitor Id3 by Blimp-1 limits the formation of memory CD8+ T cells. Nat. Immunol. 12, 1230–1237 (2011). s41590-020-0733-2. 29. Yang, C. Y. et al. Te transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8+ T cell subsets. Nat. Immunol. 12, Received: 12 May 2019; Accepted: 9 June 2020; 1221–1229 (2011). Published: xx xx xxxx 30. Wang, Y. et al. Te transcription factor TCF1 preserves the efector function of exhausted CD8 T cells during chronic viral infection. Front Immunol. 10, 169 (2019). References 31. Jadhav, R. R. et al. Epigenetic signature of PD-1+TCF1+CD8 T cells that act 1. Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 as resource cells during chronic viral infection and respond to PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454 (2012). blockade. Proc. Natl Acad. Sci. USA 116, 14113–14118 (2019). 2. Khalil, D. N., Smith, E. L., Brentjens, R. J. & Wolchok, J. D. Te future of 32. Gullicksrud, J. A. et al. Diferential requirements for Tcf1 long isoforms in cancer treatment: immunomodulation, CARs and combination CD8+ and CD4+ T cell responses to acute viral infection. J. Immunol. 199, immunotherapy. Nat. Rev. Clin. Oncol. 13, 273–290 (2016). 911–919 (2017).

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

33. Leong, Y. A. et al. CXCR5+ follicular cytotoxic T cells control viral infection 41. Wherry, E. J., Barber, D. L., Kaech, S. M., Blattman, J. N. & Ahmed, R. in B cell follicles. Nat. Immunol. 17, 1187–1196 (2016). Antigen-independent memory CD8 T cells do not develop during chronic 34. Im, S. J. et al. Defini g CD8+ T cells that provide the proliferative burst afer viral infection. Proc. Natl Acad. Sci. USA 101, 16004–16009 (2004). PD-1 therapy. Nature 537, 417–421 (2016). 42. Miller, B. C. et al. Subsets of exhausted CD8+ T cells diferentially mediate 35. Ito, D. et al. mTOR complex signaling through the SEMA4A-plexin B2 axis is tumor control and respond to checkpoint blockade. Nat. Immunol. 20, required for optimal activation and diferentiation of CD8+ T cells. J. 326–336 (2019). Immunol. 195, 934–943 (2015). 43. Kaech, S. M., Hemby, S., Kersh, E. & Ahmed, R. Molecular and 36. Singh, N. J. Self-reactivity as the necessary cost of maintaining a diverse functional profling of memory CD8 T cell diferentiation. Cell 111, memory T-cell repertoire. Pathog. Dis. 74, fw092 (2016). 837–851 (2002). 37. Martinez, G. J. et al. Te transcription factor NFAT promotes exhaustion of 44. Kaech, S. M. & Ahmed, R. Memory CD8+ T cell diferentiation: initial activated CD8+ T cells. Immunity 42, 265–278 (2015). antigen encounter triggers a developmental program in naive cells. 38. Su, B. et al. JNK is involved in signal integration during costimulation of T Nat. Immunol. 2, 415–422 (2001). lymphocytes. Cell 77, 727–736 (1994). 39. Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 40. Jansen, C. S. et al. An intra-tumoral niche maintains and diferentiates published maps and institutional affiliations. stem-like CD8 T cells. Nature 576, 465–470 (2019). © The Author(s), under exclusive licence to Springer Nature America, Inc. 2020

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Methods approximately 7–10 mm in diameter. Mice were anesthetized with isoflurane and Cell lines and reagents. Te B16.F10 melanoma cells (referred to as B16) were tumors were removed with a 2-mm perimeter of healthy skin. Incisions were obtained from M.J. Turk (Dartmouth College, New Hampshire), the B16-gp100 closed with steel wound clips, and mice were given carprofen containing MediGel cells from A.L. Rakhmilevich (University of Wisconsin, Madison, Wisconsin) and (ClearH2O) 24 h before and after surgery for pain management. Mice with the B16-OVA cells from G. Delgofe (University of Pittsburgh). Te MC38 colon recurrent primary tumor after surgery (<5%) were removed from study. For tumor 5 adenocarcinoma cells were obtained from J.P. Allison (M.D. Anderson Cancer challenge, 1.25 × 10 B16 or B16-gp100 cells were inoculated in the left flank on Center, Houston, Texas). Tumor cell lines were cultured in RPMI1640 (for B16, D30 or D60 posttumor resection. B16-gp100 and B16-OVA) or DMEM (for MC38) supplemented with 10% fetal + bovine solution (FBS), 100 units per ml penicillin, 100 μg ml−1 streptomycin, pMel-T cell adoptive transfer. Bulk CD8 T cells were purified from naive pMel- Cre + + Cre L/L + 2 mM glutamine, 1 mM pyruvate, 5 mM HEPES, 100 μM nonessential amino acids 1×E8I Thy1.1 Thy1.2 and pMel-1×E8I Nrp1 Thy1.2 mice by negative and 2-ME). Te B16-gp100 and B16-OVA cells were cultured in the presence of selection. Briefly, single cell suspensions from pooled spleen and lymph nodes were 0.8 mg ml−1 geneticin (TermoFisher Scientific). ll cell lines and assay cultures incubated with a cocktail containing biotinylated antibodies against CD4 (GK1.5), were maintained at 37 °C and 5% CO2. CD25 (PC61), CD49b (DX5), γδT (GL3), B220 (RA3-6B2), Gr1(RB6-8C5), CD19 (6D5), CD11b (M1/70), CD11c (N418), Ter119 (TER-119), IAb (KH74), CD16/32 Human patients and specimen processing. Patients were seen in the Department (93) and CD105 (MJ7/18). Non-CD8 cells were removed by mixing the labeled cell of Otolaryngology at University of Pittsburgh Medical Center (UPMC) (cohort suspension with the streptavidin-coated magnetic beads (Pierce) at 4 °C for 20 min, A), and the Department of Oncology at the SKCCC and Bloomberg–Kimmel followed by separation in a magnetic field. The unbound CD8+ pMel-T cells Institute for Cancer Immunotherapy at Johns Hopkins University School of (of purify >90%) were washed in sterile PBS (1×), with purity determined on / / Medicine (cohort B). Cohort A consisted of a cohort of banked PBL samples from a flow cytometer. The Nrp1+ + and Nrp1− − pMel-T cells were mixed at 1:1 ratio 5 patients with HNSCC who had early stage (n = 23) or advanced (n = 25) disease for (postpurity correction, 4 × 10 in total) and injected (i.v.) into the CD45.1 recipient, 5 correlation with disease progression and survival. Cohort B from SKCCC consisted followed by B16-gp100 tumor inoculation (1.25 × 10 , i.d.) within around 18–24 h of banked PBL samples from patients with advanced skin cancers that were ICB posttransfer. therapy responders (complete or partial responsers) (n = 20), or progressors (PD) 5 (n = 20). LCMV infection. Mice were infected with 2 × 10 PFU of LCMV Armstrong (i.p.) 6 Patients diagnosed with HNSCC electing to undergo treatment were offered or 4 × 10 PFU of Clone 13 (i.v.) at 8 weeks of age. Lymph nodes and spleens were the option to participate in the University of Pittsburgh Cancer Institute protocol taken at D8 post-infection(p.i.) for Armstrong, and at D30 p.i. for Clone 13 and for research. Patients signed an informed consent that was approved by the processed for flow cytometry. Institutional Review Board of the University of Pittsburgh. Blood samples were obtained from patients at time of surgery or at time of Flow cytometry and image flow cytometry. Single-cell suspensions were prepared clinic visit. Whole blood was centrifuged, and serum/plasma was immediately from mouse spleens or tumors as previously described. Briefly, tumor-infiltrating aliquoted in 5 × 1-ml amounts into sterile cryovials. Peripheral blood mononuclear leukocytes (TILs) were isolated by digesting B16 or MC38 tumors with Collagenase 1 cells are then separated via Ficoll Paque Plus (GE Healthcare). Viable peripheral IV (1 mg ml− ) at 37 °C for 30 min. After red blood cell lysis, live/dead cell blood mononuclear cells were frozen in FBS with 10% DMSO and stored in liquid discrimination was performed using Live/Dead Fixable Aqua Dead Cell Stain Kit nitrogen. Once the patient sample cohort was identified, cells were thawed and (Life Technologies). Fc block was performed by staining with anti-CD16/32 to stained with designated antibodies followed by flow cytometric analysis. avoid nonspecific binding. Surface staining was performed at 4 °C for 30 min in fluorescence-activated cell-sorting staining buffer (1× PBS/5% FBS/0.5% sodium Mice. The E8ICre mice were obtained from I. Taniuchi (Rikagaku Kenkyūsho, azide) containing designated antibody cocktails. For transcription factor (Ki67, Japan). The Nrp1L/L mice were obtained from D. Cheresh (University of California, TCF1) and intracellular proteins (Bcl2, cleaved Caspase 3) staining, cells were San Diego). The Id3-GFP reporter mice (Id3tm2.1Cmu; MGI, 5305600) were obtained fixed and permeabilized using with Foxp3 Transcription Factor Buffer Set (catalog from L. D’Cruz (University of Pittsburgh). pMel-1 mice (B6.Cg-Thy1a/Cy 00–5523, Life Technologies), following the manufacturer’s instructions. To detect 1 Tg(TcraTcrb)8Rest/J, stock No: 005023) and CD45.1 mice (B6.SJL-Ptprca Pepcb/ the cytokine-producing cells, cells were stimulated with PMA (100 ng ml− ) and 1 BoyJ, stock no: 002014) were purchased from the Jackson Laboratory and bred in Ionomycin (500 ng ml− ) for 5 h in the presence of Monensin (catalog 00-4505-51, house. The E8ICreErt2GFP and Rosa26LSL.mAmetrine.2A.Nrp1 mice were generated in house. Life Technologies) before cell surface staining, followed by standard intracellular To generate E8ICreERT2.GFP transgenic mice, the plasmid used to generate the staining procedure as above. All flow cytometry data were acquired on a BD LSR E8ICre.GFP mouse (kindly provided by I. Taniuchi)45 was altered by removing the Fortessa analyzer (BD Biosciences) and analyzed by FlowJo software (v.10.5.3, Cre and inserting iCreERT2. To generate the Rosa26LSL.mAmetrine.2A.Nrp1 mice, the Treestar, Inc.). For NFAT1 imaging cytometry, cells were fixed with 1.5% PFA Rosa26.LSL.mAmetrine.2A.Nrp1 targeting construct was made using Rosa26 plasmids, for 10 min at room temperature and permeabilized in fluorescence-activated pROSA26-PA (Addgene no. 21271) and pBigT (Addgene no. 21270)46, and cell-sorting staining buffer containing 0.1% Triton X-100. Cells were then stained mAmetrine-2A-Neuropilin-1 inserted in the ATG start codon within the in staining buffer with anti-NFAT1 (NFAT1 (D43B1) XP, rabbit mAb no. 5861, Cell Rosa26 locus downstream of the lox-stop-lox cassette. The linearized targeting Signaling Technology) for 45 min, washed and then stained with an Alexa Fluor construct was electroporated into JM8A3.N1 embryonic stem cells and neomycin 647-conjugated antirabbit secondary antibody (goat antirabbit IgG (H + L) secondary resistant clones were screened by Southern blot analysis using EcoRV and MscI Ab, Alexa Fluor Plus 647, ThermoFisher Scientific) for 30 min, and the nuclei digestions for the 5′ and 3′ ends, respectively. Clones that were correctly targeted were stained with DAPI. Data were collected on an Amnis ImageStream X Mark II and greater than 85% normal diploid by karyotype analysis were injected Imaging Flow Cytometer and analyzed with IDEAS software (EMD Millipore). into C57BL/6J blastocysts. Chimeric mice were mated to C57BL/6J mice and transmission of the targeted allele verified by PCR. BrdU (5-bromo-2’-deoxy-uridine) incorporation. To evaluate cell proliferation All animal experiments were performed in the American Association for in vivo, mice implanted with B16.F10 tumors were injected with 2 mg BrdU 12 h the Accreditation of Laboratory Animal Care-accredited, specific-pathogen-free before analysis. Tumors and lymph nodes were harvested, processed to yield facilities (temperatures of ~18–23 °C (65–75 °F) with 40–60% humidity, 12-h single-cell suspension. Cells were stained with surface markers and Ki67 as light/12-h dark cycle) in the Division of Laboratory Animal Resources, University described above, and then fixed and permeabilized with Cytofix/Cytoperm (BD of Pittsburgh School of Medicine (UPSOM). Female and male mice of all the Biosciences) and permeabilization buffer, respectively. After DNase I treatment strains mentioned were used at 4–8 weeks of age. Animal protocols were approved at 37 °C for 45 min, cells were stained with allophycocyanin anti-BrdU for 45 min by the Institutional Animal Care and Use Committees of University of Pittsburgh. before analyzing on a flow cytometer.

In vivo mouse tumor models. The E8ICre, Nrp1L/LE8ICre, E8ICreErt2GFP and Tamoxifen treatment. Tamoxifen working solution (10 mg ml−1) was prepared by E8ICreErt2GFPRosa26LSL.mAmetrine.2A.Nrp1 mice were inoculated with B16 cells (1.25 × 105, dissolving the tamoxifen in the 5% ethanol-sunflower seed oil (v/v) by shaking intradermally) or MC38 cells (5.0 × 105, subcutaneously). Tumors were measured overnight at 37 °C and stored at −20 °C, light protected. Mice were given 1.5 mg every 3 d with a digital caliper in two dimensions (width and length) and presented tamoxifen solution (approximately 75 mg kg−1 body weight) by i.p. injection daily as tumor volume (mm3, defined as the w2 × l/2). Antimouse PD1 (Clone 29 F.1A12) for 5 consecutive days, before tumor implantation. was injected intraperitoneally (i.p.) on D6, D9 and D12 at 100 μg per mouse. For attenuated Listeria monocytogenes infection, ActA-deficient L. monocytogenes bpRNA-seq. bpRNA-seq on adoptively transferred pMel-T cells was performed was grown in tryptic soy broth (Sigma-Aldrich) supplemented with 50 µg ml−1 following a protocol developed in laboratory based on the Smart-Seq2 technology48. +/+ + + + −/− + + streptomycin (Sigma-Aldrich) at 37 °C until optical density (OD600) of 0.1 Briefly, Nrp1 (CD45.2 Thy1.1 Thy1.2 ) or Nrp1 (CD45.2 Thy1.2 ) pMel-T cells (108 CFU ml−1). Then 107 CFUs in 200 µl phosphate-buffered saline (PBS) were were recovered from tumor, as well as the matched draining and NdLNs of the transferred intravenously into mice. CD45.1 recipient at the designated day(s) post-B16-gp100 tumor challenge. Five hundred cells from either genotype were double sorted (purity >99.5%) directly Surgical tumor excision and challenge. Surgical excision of primary tumors to individual well of a 96-well plate containing 2 μl lysis buffer (0.2% Triton was performed as previously described47. Briefly, intradermal primary tumors X-100 with RNase inhibitor at 2 U μl−1). The plate was spun down at 2,000g for (implanted in the right flank) were excised on D12 postinoculation, at size of 2 min and immediately proceeded with reverse transcription. A mixture (2 μl) of

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology reverse-transcription primers and dNTP (1 mM) was added to each well followed (GEO) and are accessible through the GEO Series accession code GSE151494. by incubation at 70 °C for 3 min. Denatured templates and reverse-transcription Source data are provided with this paper. primers were quickly spun down and added with 6 μl reverse-transcription master mix containing MgCl2 (9 mM), first-strand buffer (5×), Superscript II reverse −1 −1 Code availability transcriptase (10 U μl ), DTT (5 mM), Betaine (1 M), RNase inhibitors (1 U μl ) Computational and mathematical codes used in the RNA-seq data analyses and template switch oligo (1 μM) followed by the reverse transcription reaction on have been described in the article. Additional information is available from the a PCR cycler (42 °C for 90 min, ten cycles of 50/42 °C and 70 °C for 15 min, hold at corresponding author on reasonable and appropriate request. Source data are 4 °C). An addition of 15-cycle complementary DNA amplification was performed provided with this paper. following cDNA synthesis by the KAPA Hot Start II High-Fidelity DNA Polymerase. The amplified cDNA was purified using Ampure XP beads (at 0.6:1 bead to cDNA ratio) and eluted with 17.5 μl elution buffer. cDNA size (peak at roughly 1.5–2 kb) References was verified with TapeStation5000 and quantified by the Qbit. 45. Maekawa, Y. et al. Notch2 integrates signaling by the transcription factors Sequencing libraries were prepared from 1 ng cDNA using the Nextera XT RBP-J and CREB1 to promote T cell cytotoxicity. Nat. Immunol. 9, DNA Library Prep kit (Illumina FC-131-1096), following the manufacturer’s 1140–1147 (2008). instructions. cDNA Libraries were quantified by the KAPA library quantification 46. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP kit (KAPA KK4854) and size (peak at ~400 bp) verified on TapeStation1000. Ten and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001). diluted libraries (2 nM) were pooled and sequenced with the NextSeq 500/550 47. Byrne, K. T. et al. Autoimmune melanocyte destruction is required for robust High Output v.2 kit using 75 bp single read. CD8+ memory T cell responses to mouse melanoma. J. Clin. Invest. 121, 1797–1809 (2011). In vitro T cell culture. Bulk CD8+ T cells were purified by negative selection from 48. Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profling in spleen and lymph nodes of naive mice (purity above 95%). They were initially single cells. Nat. Methods 10, 1096–1098 (2013). plated at 1 × 106 per well in a 24-well plate, in the presence of plated-coated 49. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights anti-CD3 (1 μg ml−1) and anti-CD28 (2 μg ml−1) and cultured in RPMI1640 unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, medium supplemented with hIL2 (25 U ml−1) and 10% heat-inactivated FBS. For R29 (2014). the ‘chronical stimulation’ scheme, cells were washed off the original plate 48 h 50. Ritchie, M. E. et al. limma powers diferential expression analyses for after the first activation, counted and replated at 1 × 105 cells per well in U-bottom RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015). 96-well plate, with 200 μl fresh RPMI containing latex bead-coated anti-CD3 51. Smyth, G. K. Linear models and empirical Bayes methods for assessing (3 μg ml−1) and anti-CD28 (5 μg ml−1), at 1:2.5 cell to beads ratio. This was followed diferential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. by a second and/or third round stimulations every 2 d with fresh batches of https://doi.org/10.2202/1544-6115.1027 (2004). anti-CD3/anti-CD28 beads. In each stimulation step, cells in each well were split 52. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based in half by moving to a new well, with the amount of beads used and the number approach for interpreting genome-wide expression profles. Proc. Natl Acad. of viable cells activated being kept proportionally equal. As a control group, cells Sci. USA 102, 15545–15550 (2005). subjected to initial 48-h activation but without restimulation were rested in IL-2 53. Mootha, V. K. et al. PGC-1α-responsive genes involved in oxidative containing medium for the same duration before analysis. phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267–273 (2003). Transcriptomic (RNA-seq) analysis. RNA-seq reads were aligned to GRCm38/ 54. Doering, T. A. et al. Network analysis reveals centrally connected genes and mm10 build of Mus musculus genome using STAR-2.5.2a. The Unique mapped pathways involved in CD8+ T cell exhaustion versus memory. Immunity 37, reads were normalized using PORT (https://github.com/itmat/normalization/ 1130–1144 (2012). wiki) and counts were converted to log2 counts per million, quantile normalized and precision weighted with the ‘voom’ function of the limma package49,50. A linear model was fitted to each gene, and empirical Bayes moderated t-statistics were Acknowledgements used to assess differences in expression51. Heatmaps were created using R (v.3.5.1) We thank all the current and former members in the Vignali Laboratory (https://www. package pheatmap_1.0.12 and plots for PCA were created using ggplot 2_3.1.0. Vignali-lab.com; @Vignali_Lab) for all their constructive comments and advice during P values from Bayes moderated t-tests were adjusted to control the global false this project. We also thank K. Adams, D. Pardoll, M. Schollenberger and S. Topalian discovery rate. Genes were called differentially expressed if they achieved an false for assistance in procuring biospecimens from patients at Johns Hopkins; L. D’Cruz discovery rate of 0.05 or less. (Univeristy of Pittsburgh) for providing the Id3-GFP reporter mouse strain and helpful discussion; H. Shen, D. Falkner and A. McIntyre from the Immunology Flow Core Gene ontology analysis and gene set tests. Cluster-specific, significantly enriched for cell sorting; C. Schmidt, E. Brunazzi and the staff of the Division of Laboratory biological processes (gene ontology terms) were analyzed with QIAGEN’s Animals for genotyping and animal husbandry; S. Canna (University of Pittsburgh) for Ingenuity Pathway Analysis (Qiagen, www.qiagen.com/ingenuity). GSEA52,53 was the LCMV C13 strain; A. Lund (New York University) for the attenuated LM-Ova; and performed against MSigDB (v.5.1) C7 database from which ID3 ko signature W. Horne, J. Kolls and the University of Pittsburgh HSCRF Genomics Research Core (GSE23568) was identified as one of the enriched signatures. for assistance with sequencing. This work was supported by the National Institutes of Gene signatures were generated for CXCR5 Pos (GSE76279) by identifying Health (grant nos. R01 CA203689 and P01 AI108545 to D.A.A.V.; AI105343, AI117950, differentially expressed genes between CXCR5 positive versus CXCR5 negative AI082630, AI112521, AI115712, AI108545, CA210944 to E.J.W.; and T32 CA082084 and groups from a published dataset33. For CD8-specific gene signature comparison, F32 CA247004 to A.M.G), NCI Comprehensive Cancer Center Support CORE grants GSEA were run against collected datasets corresponding to traits of naive, effector, (nos. P30 CA047904 and P50 CA097190 to R.L.F. and D.A.A.V. and P30 CA006973 to memory and exhaustion, as previously described in the LCMV infection model54. E.J.L.) and the Stand Up 2 Cancer (SU2C) research grant to E.J.W. E.J.W. is supported by The normalized enrichment scores from GSEA for the LCMV genes signatures the Parker Institute for Cancer Immunotherapy that supports the cancer immunology were plotted as a radar plot using R (3.5.1) package fmsb_0.6.3. program at UPenn. E.J.L. is supported by the Bloomberg–Kimmel Institute for Cancer Immunotherapy, the Barney Family Foundation, Moving for Melanoma of Delaware Statistical analysis. Statistics were performed using Graphpad v.8.0.2. The Mantel– and The Laverna Hahn Charitable Trust. This project benefited from a SPECIAL Cox log-rank test was used for comparisons in the survival/tumor incidence ORDER BD LSR FORTESSA (funded by NIH grant no. S10 OD011925-01) and an analysis shown Figs. 1e,f,i and 6f and Extended Data Fig. 4e. Two-way analysis of IMAGESTREAMx MARK II (funded by grant no. NIH 1S10 OD019942-01), used in variance (ANOVA) was used for comparing tumor growth curves, as in Fig. 1h and the UPSOM Unified Flow Core. This project also used the Hillman Cancer Center Extended Data Figs. 3e–h and 4e, and time course data as in Figs. 2f–h, 3e and 5e,f Immunologic Monitoring and Cellular Products Laboratory that is supported in part by and Extended Data Fig. 5g. One-way ANOVA was used for the comparison among award no. P30 CA047904. multiple (>2) groups, as in Fig. 1a; a two-tailed unpaired Student’s t-test was used for comparison between two groups (genotypes, patient subgroups or treatment), Author contributions as in Figs. 2a,b,d, 5c and 6e and Extended Data Figs. 1b, 3d, 4c,d,g,i; whereas a D.A.A.V. conceived, directed and obtained funding for the project. C.L. and D.A.A.V. paired Student’s t-test was specifically used for comparing two genotypes under the conceptualized, designed and analyzed the experiments and wrote the manuscript. C.L. coadoptive transfer setting, such as in Fig. 3b–d,f and Extended Data Figs. 5d,e and performed the experiments. S.M. analyzed the RNA-seq data. A.S., D.P.N. and T.C.B. 6b,d. The Wilcoxon test was used for Fig. 6i,k. processed and analyzed the healthy donor and HNSCC PBL specimens. A.L.S.-W. and K.M.V. generated the Rosa26LSL.mAmetrine.2A.Nrp1 and the E8ICreErt2GFP mice. E.N.S. helped Reporting Summary. Further information on research design is available in the with in vitro CD8+ T cell culture and stimulation. A.M.G. helped with the LCMV Nature Research Reporting Summary linked to this article. model. R.L.F. provided PBL samples from patients with HNSCC. E.J.L. contributed to the acquisition, analysis, interpretation and revision of clinical data. E.J.W. Data availability provided reagents and contributed advice. C.J.W. contributed to experimental design, Bulk RNA-seq datasets of Nrp1+/+ and Nrp1−/− pMel-T cells have been deposited analysis and developing mouse strains. All authors provided feedback and approved in the National Center for Biotechnology Information Gene Expression Omnibus the manuscript.

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Competing interests Surface Oncology and Arsenal Biosciences. E.J.W. has a patent licensing agreement on the D.A.A.V. declares competing financial interests and has submitted patents covering PD1 pathway with Roche/Genentech. E.J.L. consults for Array BioPharma, Bristol-Myers Nrp1 that are licensed or pending and is entitled to a share in net income generated Squibb, EMD Serono, MacroGenics, Novartis, Merck, Regeneron and Sanofi Genzyme. from licensing of these patent rights for commercial development. D.A.A.V. consults for E.J.L. receives research funding from Bristol-Myers Squibb, Merck and Regeneron. and/or is on the scientific advisory board of Tizona, Werewolf, F-Star, Astellas/Potenza, BMS, Oncorus, Innovent Bio, Kronos Bio and G1 Therapeutics; has grants from BMS, Astellas/Potenza, Tizona and TTMS; patents licensed and royalties from Astellas/Potenza, Additional information Tizona and BMS; and stock from TTMS, Tizona, Potenza, Oncorus and Werewolf. Extended data is available for this paper at https://doi.org/10.1038/s41590-020-0733-2. R.L.F. consults for and/or is on the scientific advisory board for Aduro Biotech, Inc., Supplementary information is available for this paper at https://doi.org/10.1038/ Astra-Zeneca/MedImmune, Bristol-Myers Squibb, EMD Serono, GlaxoSmithKline, s41590-020-0733-2. Iovance Biotherapeutics, Inc., MacroGenics, Inc., Merck, Nanobiotix, Numab Therapeutics Correspondence and requests for materials should be addressed to D.A.A.V. AG, Oncorus, Inc., Ono Pharmaceutical Co. Ltd, PPD (Benitec, Immunicum), Regeneron Pharmaceuticals, Inc., TTMS and Torque Therapeutics Inc. R.L.F. receives research Reprints and permissions information is available at www.nature.com/reprints. funding from Astra-Zeneca/MedImmune, Bristol-Myers Squibb, Tesaro and TTMS and Editor recognition statement Zoltan Fehervari was the primary editor on this article has stock from TTMS. E.J.W. has consulting agreements with and/or is on the scientific and managed its editorial process and peer review in collaboration with the rest of the advisory board for Merck, Roche, Pieris, Elstar and Surface Oncology. E.J.W. is a founder of editorial team.

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

Extended Data Fig. 1 | Characterization of NRP1 expression on CD8+ T cells in vivo. a, Gating strategy and phenotyping markers used for the identification of CD8+ cell subsets under conditions of homeostatic (naive mice), or sites of immunological challenge (LCMV infection or B16 tumor). b, Expression of NRP1 measured by flow cytometry in subsets of CD8ab+ T cells from spleens of mice infected by acute LCMV (Armstrong, D8 p.i, n = 7) or chronic LCMV (Clone 13, D30 p.i., n = 7). Bar graph tabulating the mean percentage of NRP1+ cells of indicated subsets and symbols represented individual mouse, 2 independent cohorts pooled. Error bars, mean±s.e.m; two-tailed unpaired Student’s t test. c, Representative flow cytometry plots depicting the + co-expression of NRP1 and PD1 within naive (spleen-derived) and TEFF CD8 cells (spleen or intratumoral) from unchallenged or B16-tumor bearing mice. d, Histograms depicting the correlation of NRP1 expressed by intratumoral CD8+ T cells with known markers of activation (CD44, CD69, CD25), cell proliferation (BrdU), naive/memory (CD62L, CD127) and senescence (KLRG1) phenotypes.

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Extended Data Fig. 2 | Genetic models for the “loss- or gain-of function” manipulations of Nrp1 transcription restrictively in CD8+ T cells. a, b “Loss-of-function” model: Validation for the CD8-restricted Nrp1 deletion in the E8ICreNrp1L/L mice. (a) Representative histograms for NRP1 staining on CD4+ (green) and CD8+ (blue) tumor-infiltrating lymphocytes (TILs) of B16 tumors (D12). (b), Surface NRP1 expression on major immune cell populations derived from naive spleen (upper) or 1° B16 TILs (lower, D15 post implantation) of E8ICre or E8ICreNrp1L/L mice (n = 5 for each group). Loss of NRP1 protein restrictively by CD8+ cells in the E8ICreNrp1L/L mice was highlighted; c, “Gain-of-function” model: (Upper) Schematic structure of the Rosa26LSL.mAmetrine.2A.Nrp1 targeting construct. (Lower) Tamoxifen-induced CD8-specific (marked by GFP reporter) “constitutive” NPR1 expression (surface NRP1 staining) driven by Rosa26 promoter (marked by Ametrine reporter), in CD8+ T cells from peripheral blood (PB) of naïve mice or 1° B16 TILs. Gated on CD8+GFP+ cells.

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

Extended Data Fig. 3 | CD8-expressing NRP1 is dispensable for controlling the growth of primary tumors. a–e, B16 tumors implanted in the E8ICre or E8ICreNrp1L/L mice were harvested on D15, and the composition of major immune cell populations were analyzed by flow cytometry. (a) bh-SNE depiction of intratumoral CD45+ cells (80,233 in total, pooled from E8ICre and E8ICreNrp1L/L mice), projected by cell source. (b) Phenograph depiction of intratumoral CD45+ cells as in (a), and different cell lineages (colored) were identified by the expression of lineage-specific markers. (c) Percentage of major immune cell populations (indicated) within CD45+ TILs. (d) Numeration of CD45+ cells, presented by counts per gram tumor mass; Error bars, mean±s.e.m; Symbol represented individual mouse (n = 5 for each genotype). (e–h) Growth curves for (e) 1° B16 tumor; (f) B16-OVA tumor (Vaccination with attenuated Listeria monocytogenes expressing Ova peptide (LM-Ova) was given on Day 4) (g) 2° B16 tumor in the E8ICre and E8ICreNrp1L/L mice, challenged on Day 30 (left) or 60 (right); (h) 1° and 2° B16 tumors (+30d rechallenge) implanted in the E8ICreErt2gfpRosa26LSL.Nrp1 and E8ICreErt2gfp mice. Data were representative of 2 independent experiments (e, f); or pooled from 4 independent experiments (g, h). Error bars, mean±s.e.m; Statistical significance was determined by two-tailed unpaired Student’s t test (d) or two-way ANOVA with correction for multiple comparisons (e–h).

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Extended Data Fig. 4 | NRP1 modulates the memory differentiation of tumor-primed effector CD8+ T cells but not effector polyfunctionality. a–d, CD8+ T cells from primary B16-gp100 tumors and matched spleens were analyzed on D12 (n = 8 per genotype). (a) Numeration of total Db-gp100+ cells, as well + + + as their subsets (TCM, TEFF, MPECs, SLECs) within intratumoral CD8 T cells; (b) Ratio between intratumoral Db-gp100 CD8 MPECs and SLECs; (c-d) b + + + + Representative flow cytometry plots (c, left) and quantification for the frequencies of D -gp100 CD8 T cells (c, right) and (d) Db-gp100 TCM in CD8 T cells from spleen; e, The incidence (left) and growth (right) of 2° B16-gp100 tumor (+30d rechallenge). f, Representative flow plots for the expression of IRs on CD8+ TILs of 1° B16 tumors on D12 (left) and SPICE plots (right) visualization for co-expression of multiple IRs. g, Numeration of 5-IR-co-expressing cells (5-IR+) on D12 (n = 5), by frequency within CD8+ TILs (left) and absolute number per gram tumor mass (right). Data were representative from 2 independent cohorts. h, i Expression of cytokines (IFN-γ against GzmB, TNF-α and IL2, respectively) by the CD8+ TILs on D18 of 1° tumor. SPICE plots visualization for the multi-cytokine producing cells. i, Percentage of single, dual, or multi-cytokine producing cells within the CD8+ TILs as shown in h, n = 6 per genotype. Error bars, mean±s.e.m; Statistical significance was determined by two-tailed unpaired Student’s t test (a–d, g,i) or log-rank test (e, left) or two-way ANOVA with correction for multiple comparisons (e, right).

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

Extended Data Fig. 5 | NRP1 cell-intrinsically limits the in vivo persistence of antigen-specific CD8+ T cells. a, The level of Db-gp100+ transgenic TCR, activation status (CD44 vs. CD62L); and the ratio within donor pool (CD45.2+, recovered from spleen) of Nrp1–/– and Nrp1+/+ pMel-T cells 12 h after adoptive transfer, prior to B16-gp100 tumor inoculation (D0). b, Correlation between ratio of intratumoral Nrp1–/– vs. Nrp1+/+ donor pMel-T cells with tumor volume (mm3). c, Kinetics of tumor density (counts/mm3 tumor size) of intratumoral Nrp1–/– and Nrp1+/+ pMel-T cells during 1° B16-gp100 growth. (n = 5 for each time point) d, e Frequencies of Nrp1–/– and Nrp1+/+ pMel-T cells within the CD45.2+ donor pool in spleen and peripheral blood (PB) during (d) primary phase and (e) recall phase at the indicated time points (n = 5 for each time point). f, Representative flow cytometry plots for NRP1 expression on +/+ –/– + – pMel-T cells of naïve, effector, MPECs, and TCM phenotypes, respectively. g, Frequencies of Nrp1 or Nrp1 donor-derived cells of CD27 CD62L (MPECs + + + enriched) or CD27 CD62L (TCM) phenotype within CD45.2 compartment over time, recovered from draining lymph nodes (DLN) and spleen from D12 to D42, n = 5 for each time point. h–i, Representative flow cytometry plots depicting the expression of Bcl2 against Ki67 with NRP1+ vs. NRP1– fractions (f) or Bcl2 vs. IRF4 in Nrp1+/+ or Nrp1–/– pMel-T cells (g) recovered from NdLN, on D12 and D21 post 1° B16-gp100 inoculation. Data in c–e and g were pooled from 2 independent time course cohorts. Error bars, mean±s.e.m; Statistical significance was determined by two-tailed paired Student’s t test (d, e) or two-way ANOVA (g), ****p < 0.0001.

Nature Immunology | www.nature.com/natureimmunology NATuRE IMMunology Articles

Cre L/L Extended Data Fig. 6 | Loss of NRP1 upregulated Id3 and CXCR5 expression on pTEX infiltrating primary B16-gp100 tumors. The E8I Nrp1 and E8ICre strains on pMel-1 background were further crossed with an Id3-GFP reporter mouse strain, resulting in one mutant allele carrying the modified Id3 locus with insertion of sequence encoding GFP into the ATG initiation codon (Id3gfp/+). a. Scheme for pMel-T cell adoptive transfer. Briefly, congenically-mismatched bulk CD8+ T cells were purified from the following 3 groups of naive mice (pMel-1×E8ICrexId3+/+(CD45.2+Thy1.1+), pMel- 1×E8ICrexId3gfp/+ (CD45.2+Thy1.1+Thy1.2+) and pMel-1×E8ICreNrp1L/LxId3gfp/+ (CD45.2+Thy1.2+)), co-transferred at 1:1:1 ratio into CD45.1 recipients, followed by B16-gp100 tumor implantation one day later. Tumor-infiltrating CD8+ T cells were analyzed on D12 or D18. b, Frequency of Id3-GFP+ within Nrp1+/+Id3gfp/+ and Nrp1–/–Id3gfp/+ donor cells; c, Representative flow cytometry plots (left) depicting the phenotype of intratumoral Id3-GFP+ vs. Id3-GFP– cells. Bar plot (right) tabulating the composition of Id3-GFP+ and Id3-GFP– fractions, from Nrp1+/+ or Nrp1–/–-derived donors, respectively (n = 6 per group). d, Expression of CXCR5 and Ki67 on B16-gp100 tumor-infiltrating pMel-T cells, recovered on D21 post implantation. Cells shown in the representative plot were gated on Nrp1+/+ and Nrp1–/– donors by the congenic markers. Bar graphs tabulating the genomic mean fluorescence intensity (gMFI) and the percentage of CXCR5+ cells within Ki67+ and Ki67– fraction within each genotype. Data were pooled from 2 independent experiments, with 9 recipient mice per experiment in a-c, 4 replicates per group in d. Error bars, mean±s.e.m; Statistical significance was determined by two-tailed paired Student’s t test (b, d).

Nature Immunology | www.nature.com/natureimmunology Articles NATuRE IMMunology

Extended Data Fig. 7 | Phenotypical analysis of in vitro chronically stimulated CD8+ T cells. a–c, Representative flow cytometry plot depicting the expression of (a) NRP1, and Semaphorin-4a (Sema4a), (b) IRs (PD1 and LAG3) and (c) cytokines (IFN�, Granzyme B and TNF�) on Nrp1+/+ or Nrp1–/– CD8+ cells subjected to chronic antigen stimulation in vitro (scheme described in Fig. 5a); d, e, Visualization of nuclear trans-localization of NFAT1 by image flow cytometry in (d) naive CD8+ T cells or (e) in vitro chronically stimulated (2° re-stim) Nrp1+/+ or Nrp1–/– CD8+ cells, in response to PMA plus Ionomycin (P + I) stimulation; The representative images of co-staining between NFAT1 and nuclear probe (DAPI) in individual cells and the histogram depiction of the localization similarity score between NFAT1 and DAPI were shown. f, (Left) Representative flow plot depicting the gating strategy for measuring c-Jun activation within CD44+PD1+ CD8+ cells from B16-gp100 tumors or matched spleens; (Right) Representative histogram illustrating the expression of c-Jun by indicated cell subsets.

Nature Immunology | www.nature.com/natureimmunology