Enhanced Phenotype Definition for Precision Isolation of Precursor Exhausted Tumor-Infiltrating CD8 T Cells

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.


INTRODUCTION
Sustained antigenic stimulation of CD8 T cells in cancer and chronic infection leads to an exhausted state characterized by high expression of multiple inhibitory receptors, reduced cytotoxicity, and re-expansion capacity (1)(2)(3). Far from being a "failed" immune response, this Tox-driven exhaustion epigenetic program is essential for the persistence of CD8 T cells in the context of sustained antigenic stimulation (4)(5)(6). If CD8 T cells fail to activate the exhaustion program (e.g., when Tox is disrupted), persistent antigenic stimulation erodes immunodominant clones by mechanisms such as activation-induced cell death (4,6).
Recent studies have shown that the pool of exhausted CD8 tumor-infiltrating lymphocytes (TILs) is maintained within the tumor by a precursor subset (memory-like or precursor exhausted, Tpe) that expresses the transcription factor T cell factor 1 (Tcf1), encoded by Tcf7 (7,8). Tpe cells can self-renew and differentiate into exhausted cells (Tex). Tex exert (limited) cytotoxic functions while avoiding overstimulation but they lack proliferative potential (8)(9)(10).
In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo.
To attain tumor-specificity, CD8 T cells are typically isolated from tumor samples. However, not all tumor-infiltrating CD8 lymphocytes (TILs) are tumor-specific (11), and the isolation of tumor-specific TILs is far from trivial. Different studies have shown that tumor-specific TILs can be enriched by means of surface expression of PD-1, Tim3, and CD39 among others (11)(12)(13)(14)(15). However, these markers are highly expressed by terminally differentiated Tex, and hence TIL isolation based on these markers enriches in cells with very limited proliferation potential. Therefore, novel strategies to isolate tumor-specific TILs with high proliferative potential, such as Tpe, are needed for improved in vivo persistence in ACT. Here we aimed at defining an improved selection strategy to increase the purity of tumorspecific Tpe from total TILs.

Mice
C57BL/6 (B6) mice were purchased from ENVIGO (Gannat, France) and CD45.1 OT1 mice were bred at the animal facility (Epalinges) of the University of Lausanne. Mice were all females, 7-10 weeks old, and maintained in conventional facilities of the University of Lausanne. This study was approved by the Veterinary Authority of the Swiss Canton of Vaud (authorization VD3360.c) and performed in accordance with Swiss ethical guidelines.
Tumors were harvested 21 days post tumor engraftment and dissociated with the Tumor Dissociation Kit (130-096-730, Miltenyi Biotec) following manufacturer's instructions before antibody staining for flow cytometry. Tumors of 6-10 individual mice were pooled before marker based cell sorting and transfer to secondary hosts.
Seven days post-infection, spleens from infected mice were collected, smashed through a 70 µm diameter cell strainer (352350, Falcon) and red blood cells were lysed with RBC lysis buffer (158904, Qiagen) before antibody staining for flow cytometry analysis of OT1 cells.

Ex vivo Stimulation of OT1 Cells
Mouse splenocytes were stimulated with 10 µg/ml SIINFEKL peptide or 10 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin as positive control (Sigma-Aldrich) for 4 h at 37 • C in complete DMEM. Unstimulated cells were used as negative control. BD GolgiPlug and GolgiStop (BD Biosciences) were added to the cells 30 min upon starting the stimulation assay.

Data and Statistical Analysis
Flow cytometry data were analyzed with FlowJo (TreeStar). Graphs and statistical analysis were made with Prism (GraphPad Software) except scRNAseq data which was analyzed with R (described above). Specific statistical analyses are described in figures' captions. P-values are coded as * p > 0.05, * * p > 0.01, * * * p > 0.001, and * * * * p > 0.0001.

CD39-Tim3-Slamf6+ PD-1+ CD8 TILs Show the Highest Enrichment for Tcf1+ Cells in Mouse and Human Melanoma Tumors
To identify surface markers of tumor-specific Tcf1+ PD-1+ Tpe that would allow their selection for ACT, we performed a differential gene expression analysis of Tcf7+ Pdcd1+ (Tpe) vs. Tcf7-Pdcd1+ (Tex) from scRNAseq data of CD8 TILs from murine B16 (18) and human primary melanoma tumors [publicly available data from Sade-Feldman et al. (16)] (Supplementary Tables 1, 2). In both mouse and human melanoma tumors we observed that Tcf7+ Pdcd1+ Tpe were enriched in Slamf6 and depleted of Havcr2 and Entpd1, which encode Slamf6, Tim3, and CD39 cell surface proteins, respectively ( Figure 1A). Thus, we hypothesized that the expression patterns of Slamf6, Tim3, and CD39 may help discriminating Tpe from Tex among tumor-specific TILs. First, we explored the plausibility of this hypothesis in silico. A previous report already showed that tumor-specific (identified by tetramer staining) Tim3-Slamf6+ cells were enriched in Tcf7+ cells compared to Tim3+ Slamf6-counterparts (7). Here we used Pdcd1 expression (which encodes for PD-1) as a surrogate marker of tumor specificity (14) and observed that indeed, Pdcd1+ Havcr2-Slamf6+ cells were enriched in Tcf7+ cells compared to bulk Pdcd1+ or Pdcd1+ Havcr2-cells ( Figure 1B). However, among Pdcd1+ Tim3-Slamf6+ cells we found heterogeneous expression of Entpd1, a marker of Tex, both in murine and human melanoma tumors ( Figure 1C).
Next we sought to verify whether the surface expression of these markers could be used to identify Tpe cells by flow cytometry. To this end, we analyzed Tcf1 expression on total PD-1+ endogenous and transferred OT1 CD8 TILs from B16.OVA tumors before and after marker-based cell selection. We confirmed that CD39-Tim3-Slamf6+ PD-1+ cells showed the highest enrichment for Tcf1+ cells in both endogenous and transferred tumor-specific OT1 cells (Figure 1E), which accounted for the ∼3.5% of total endogenous or OT1 TILs ( Figure 1F). CCR7 was also highly expressed in Tpes compared to Tex cells (Supplementary Tables 1, 2) but antibody staining of this chemokine receptor was poor and did not select Tcf-1+ CD8 T cells in B16.OVA tumors (Supplementary Figure 2A).

CD39-Tim3-Slamf6+ PD-1+ CD8 TILs Show Increased Re-expansion Capacity and Functionality Compared to CD39+ Counterparts or Bulk PD-1+ TILs
Tpe cells have been described as those cells that maintain antigen-specific responses in the context of chronic infection and tumors (8,9). Therefore, highly homogenous Tpe cell products would be ideal in the context of ACT to improve persistence of transferred cells in the hosts. Thus, we investigated whether our newly described CD39-Tim3-Slamf6+ PD-1+ TIL subset display better recall responses and persistence in vivo compared to bulk TIL populations. To this end, either 300 PD-1+, PD-1+ Tim3-Slamf6+ CD39-, or PD-1+ Tim3-Slamf6+ CD39+ OT1 CD8 TILs cells were isolated from B16.OVA tumors 21 days post-tumor engraftment following the gating strategy of Supplementary Figure 3A and transferred to a secondary host that was infected on the same day with OVA-expressing Listeria monocytogenes (Lm-OVA). Naïve OT1 cells were also transferred as control (Figure 2A). Seven days post-infection (PI), we observed increased frequencies of transferred OT1 cells in mice receiving PD-1+ Tim3-Slamf6+ CD39-cells compared to those receiving their CD39+ counterparts or total PD-1+ OT1 CD8 TILs (Figure 2B), indicating their enhanced recall response and persistence. At 7 days PI, most transferred cells were Tcf1- (Figure 2C), consistent with previous reports showing that tumor-specific Tcf1+ cells differentiate into Tcf1-negative cells (7,8).
In addition to the enhanced expansion capacity, PD-1+ Tim3-Slamf6+ CD39-cells showed enhanced functionality in the recall response compared to their CD39+ counterparts, with increased frequency in IFNγ, TNFα and IFNγ+TNFα double expressing cells (Figure 2D) as well as increased expression of these molecules (Figure 2E).
Altogether, selection of CD39-Tim3-Slamf6+ cells among PD-1+ TILs led to an enhanced persistence and functionality of transferred TILs in a recall response compared to their CD39+ counterparts or bulk PD-1+ TILs.

DISCUSSION
ACT therapy of in vitro expanded TILs has shown promising clinical efficacy in melanoma patients (20). However, poor in vivo persistence of transferred cells remains a major limitation. TIL culture conditions can be optimized to obtain cell products with longer in vivo persistence (21)(22)(23). However, these protocols bypass selection of tumor-specific T cells, limiting the anti-tumor capacity of expanded T cells. Here we propose a flow cytometry gating strategy that enables the selection of tumor-specific TILs with higher in vivo persistence and functionality in recall responses compared to bulk populations.
In the tumor, high affinity T cells show increased PD-1 expression compared to low affinity counterparts (19). Therefore, isolation of PD-1+ cells may allow selection of high affinity T cells, which show enhanced tumor control than low affinity T cells (19). Moreover, high affinity T cells may include those recognizing mutated self-antigens (neoantigens) which have been shown to respond to vaccination (24,25) and anti-PD-1 therapy (24).
We propose the use of PD-1 as a marker of tumor specificity as previously described (14) while simultaneously gating out Tim3+ and CD39+ cells. Even though Tim3 and CD39 can further enrich in tumor-specific TILs (11,13,15), this is achieved at the expense of a dramatic loss of in vivo recall capacity. Recent insights into the heterogeneity of tumor antigen-specific CD8 TILs revealed the existence of a small subset of memory-like PD-1+ CD8 TIL with high proliferation potential and selfrenewal capacity, the Tpes.
By transcriptomic and flow cytometry analyses of CD8 TILs from murine B16 and human primary melanoma, we found that Tpes can be efficiently captured by gating on Tim3-Slamf6+ CD39-cells. Indeed, this CD8 T cell subset yielded maximum enrichment for Tcf1+ cells among PD-1+ TILs. Furthermore, adoptively transferred CD39-Tim3-Slamf6+ TILs exhibited increased persistence and functionality in recall responses to the Lm-OVA acute infection, compared to the CD39+ counterparts. This is consistent with previous reports associating CD39 with terminally differentiated exhausted CD8 T cells (16,26,27). Previous reports (28)(29)(30) described CXCR5 as a marker of CD8 T cells with stemcell like properties as Tpes. However, in the analyzed mouse and human melanoma RNAseq datasets CXCR5 was not enriched in Tpes (Supplementary Tables 1, 2), neither allowed identification of Tcf1+ cells by flow cytometry in B16.OVA tumors (Supplementary Figure 2B). Thus, the ability of CXCR5 staining to identify cells with stem-cell like properties may be context dependent.
Hence, we conclude that our proposed gating strategy represents an optimal trade-off between enriching for tumor-specific TILs with high-affinity and preserving recall capacity.
Altogether, we show that selection of CD39-Tim3-Slamf6+ PD-1+ TILs enables enrichment of tumor specific Tcf1+ PD-1+ TILs Tpe that may enhance efficacy of adoptive cell transfer therapies in cancer patients.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by Veterinary Authority of the Swiss Canton of Vaud (authorization VD3360.c).

AUTHOR CONTRIBUTIONS
AM-U, SC, SL, and PR: conception and design. AM-U and SC: development of methodology. AM-U, SL, CG, and CY: acquisition of data. AM-U, SC, and SL: analysis and interpretation of data. AM-U, SC, SL, CG, and PR: writing review and/or revision of the manuscript. SC and PR: study supervision.