Author response: SLAMF6 deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 -/- mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 -/- CD8+ T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1 x SLAMF6 -/- cells, and upon activation, they acquired an effector-memory phenotype. Adoptive transfer of Pmel-1 x SLAMF6 -/- T cells to melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. LAG-3 expression was elevated in the SLAMF6 -/- cells, and the addition of the LAG-3-blocking antibody to the adoptive transfer protocol improved the SLAMF6 -/- T cells and expedited the antitumor response even further. The results from this study support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8+ T cells to eradicate tumors. eLife digest The immune system helps to protect our bodies from illnesses and infections. Immunotherapies are medicines designed to treat diseases, such as cancer, by boosting the immune system against the condition. This is a powerful approach but so far immunotherapies have only had partial success and there is a need for further improvements. One protein called SLAMF6 is found on cells from the immune system that attack and kill cancer cells. Immunotherapies that suppress SLAMF6 on immune cells called killer T cells could increase immune system activity helping to treat cancers, particularly melanoma skin cancers. So far the potential for SLAMF6 as a target for immunotherapy has not been fully explored. Hajaj et al. created mice with killer T cells that recognized skin cancer cells and lacked SLAMF6. These modified cells were better at fighting cancer, producing more anti-cancer chemicals called cytokines and killing more cancer cells. The modified cells had a lasting effect on tumors and helped the mice to live longer. The effects could be further boosted by treating the mice in combination with other immunotherapies. SLAMF6 is a possible new target for skin cancer immunotherapy that could help more people to live longer following cancer diagnosis. The next step is to create a drug to target SLAMF6 in humans and to test it in clinical trials. Introduction The SLAM family of receptors (SFRs) is a set of six receptors expressed on hematopoietic cells (Wu and Veillette, 2016; Cannons et al., 2011; Veillette, 2010; Calpe et al., 2008). All SFRs, except 2B4, are homotypic binders, that is they engage the same ectodomain sequence, either in cis (same cell) or in trans (adjacent cell) configuration. Most hematopoietic cell types express 3–5 members of the SLAM family. SFRs generate signals via a bi-phasic mechanism of recruitment to tyrosines in the immunoreceptor tyrosine-based switch motifs (ITSMs) in their cytoplasmic domain. SLAM associated protein (SAP), a small protein containing the Src homology 2 (SH2)-domain, was shown to be the default adaptor of the SFRs, interchanging with protein tyrosine phosphatases, mainly SHP-1, but also SHP-2, inositol phosphatase SHIP-1 and protein tyrosine kinase Csk (Wu and Veillette, 2016; Cannons et al., 2011; Veillette, 2010; Calpe et al., 2008). SLAMF6, also known as CD352, LY108, or NTB-A, is a homotypic SFR expressed on T cells, NK cells, B cells, and dendritic cells (Bottino et al., 2001; Zhong and Veillette, 2008). Kageyama et al. linked SLAMF6 to the anchoring of T cells to their target cells, and subsequent cytolysis of the target (Kageyama et al., 2012). According to these authors, functional SAP is critical for SLAMF6 activity. In mice lacking SAP, SLAMF6 was shown to inhibit T cell function (Kageyama et al., 2012; Zhao et al., 2012; Bottino et al., 2001). The role of SLAMF6 in healthy T cells expressing normal SAP levels was generally inferred from contextual data and is not yet clear. There are indications that SLAMF6 plays an activating role in double-positive thymocytes (Dutta et al., 2013) along with evidence that it plays an inhibitory role in iNKT cells and CD8+ T cells (Lu et al., 2019; Eisenberg et al., 2018). Gene expression profiles of T cell subsets link SLAMF6 to the progenitor-exhausted state (Miller et al., 2019) and to the tuning of the critical number of T cells required for proper differentiation (Polonsky et al., 2018). To elucidate the net function of SLAMF6, we generated a transgenic mouse with the Pmel-1 melanoma-specific T-cell receptor (TCR) expressed in CD8+ T cells, in which the SLAMF6 gene was knocked out. In this report, we show for the first time that SLAMF6 -/- CD8+ T cells display improved anti-melanoma activity and prevent melanoma growth more effectively than CD8+ T cells with intact and functional SLAMF6. Since SLAMF6 is constitutively expressed on T cells, it acts as an inhibitory checkpoint receptor whose absence allows the eradication of established tumors by CD8+ T cells. Results SLAMF6 is constitutively expressed on T cells and increases upon activation SLAMF6 is an immune receptor constitutively expressed on non-activated and activated T cells (Eisenberg et al., 2018). The level of SLAMF6 transcription and receptor expression, however, is dynamic, changing with time and activation states. To record SLAMF6 expression in a longitudinal manner, human tumor-infiltrating lymphocytes (TILs) were activated for 5 days, and SLAMF6 transcript and protein expression were measured (Figure 1A–C). After 1 day of activation, there was an initial decrease in the SLAMF6 transcript that switched to over-expression (Figure 1C). From 3 days after activation onward, SLAMF6 receptor expression consistently increased (Figure 1A and B). Interestingly, the increased expression was most pronounced in T cells activated in the absence of IL-2 (Figure 1D). A similar pattern was observed for the expression of the murine SLAMF6 receptor on Pmel-1 CD8+ T cells (Figure 1E). Figure 1 Download asset Open asset SLAMF6 is constitutively expressed on T cells and increases upon activation. (A–C) SLAMF6 expression in human TIL412 cells, activated for five days. (A) Flow cytometry at the indicated time points. (B) Median fluorescence intensity (MFI) of SLAMF6, days 1–5. (C) Quantitative RT-PCR for SLAMF6. RNA was extracted at the indicated time points. Data normalized to HPRT expression at each time point and to the basal expression level on day 0. One-way ANOVA. **, p<0.01, ***, p<0.001. (D) SLAMF6 expression by flow cytometry in human TIL412 cells activated for 5 days with anti-CD3 or with anti-CD3 plus IL-2, at the indicated time points. (E) SLAMF6 expression by flow cytometry in Pmel-1 mouse splenocytes activated for 6 days, at the indicated time points. (F) Row normalized expression of immune-related genes from RNAseq, clustered according to similar expression patterns. CD4+ T cells from two donors were stimulated with anti-CD3 plus anti-CD28 for 72 hr, RNA was extracted and sequenced. Numbers in the top panel indicate hours. (G) Magnification of cluster C. SLAMF6 is marked. Figure 1—source data 1 RNA sequencing of healthy donors CD4 T cells along activation. https://cdn.elifesciences.org/articles/52539/elife-52539-fig1-data1-v2.csv Download elife-52539-fig1-data1-v2.csv To identify other immune-related genes that may cluster with SLAMF6, longitudinal RNA sequencing data were generated from CD4 T cells from two healthy human donors. Five groups of genes (clusters A-E) were identified (Figure 1F). Cluster A represents genes highly expressed in non-activated cells, and downregulated upon activation, such as BCL2 and JAK1. Cluster B represents fast-rising genes undergoing transcription as early as 3–6 hr following activation, but down-regulated after that; genes in this cluster include the activation marker CD69. Clusters C and D include intermediate genes, upregulated after 6 hr (C) or between 12 and 24 hr (D), and downregulated later. Lastly, cluster E includes late-rising genes, such as LAG3. The SLAMF6 transcript appears in cluster C, rising at 6 hr of activation and staying high after that (Figure 1G). Other genes in cluster C are CD44, encoding a glycoprotein that takes part in T cell activation (Huet et al., 1989), and CD28 and ICOS, which encode co-stimulatory immune receptors (Turka et al., 1990; Dong et al., 2001). The increase in the transcription of receptors that are stably expressed at all times may hint at enhanced recruitment and degradation of these receptors during activation, possibly in the immune synapse (Onnis and Baldari, 2019). SLAMF6 expressed in trans by a melanoma target inhibits antitumor T cell reactivity Because SLAMF6 is constantly present on T cells, it is difficult to decipher its effect when it acts as a ligand, introduced in trans. To solve this problem, we generated a cell line derived from B16-F10/mhgp100 melanoma, over-expressing SLAMF6 (Figure 2A). Compared to the wild-type cell line, the SLAMF6-expressing melanoma cells co-cultured with Pmel-1 CD8+ T cells led to decreased IFN-γ secretion by the lymphocytes (Figure 2B and C). To evaluate the effect of SLAMF6 expression on melanoma rejection in vivo, SLAMF6-expressing melanoma was compared to the parental B16-F10/mhgp100 line, in an adoptive T cell transfer regimen, using activated Pmel-1 CD8+ T cells (Figure 2D). The B16-F10/mhgp100/SLAMF6+ tumors grew more aggressively: on day 23, the mean tumor volume was 431 mm3 in the SLAMF6-expressing melanomas, compared to 137 mm3 in the non-modified tumors (p=0.04, Student's t-test) (Figure 2E). Figure 2 with 1 supplement see all Download asset Open asset SLAMF6 expressed in trans by a melanoma target inhibits anti-tumor T cell reactivity. (A) SLAMF6 expression on B16-F10/mhgp100 parental or transfected (SLAMF6 or empty) melanoma cells. (B) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells at the indicated effector-to-target ratios. IFN-γ secretion was measured by ELISA. (C) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells. IFN-γ production was detected by intracellular staining and flow cytometry (gated on CD8+). Three replicates. The gating strategy is illustrated in Figure 2—figure supplement 1. (D, E) Pmel-1 splenocytes were expanded with gp10025-33 peptide (1 µg/ml) and IL-2 (30 IU/ml) for 7 days. On day 7, cells were transferred i.v. into irradiated C57Bl/6 mice bearing palpable (1 week) B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 tumors. IL-2 (0.25 × 106 IU) was administered i.p. twice a day for 2 days. Tumor growth was measured twice a week. Mice were sacrificed when the tumor reached 15 mm in diameter. (D) Scheme showing experimental layout. (E) Spider plot showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. One-way ANOVA. *, p<0.05, **, p<0.01, ***, p<0.001. These experiments show that trans-activation of SLAMF6 on lymphocytes, which in this system was achieved with the SLAMF6-expressing melanoma, inhibits the melanoma-specific CD8+ T cell response and allows rapid tumor growth. Establishment of Pmel-1 x SLAMF6 -/- mice as a source of SLAMF6-/- antigen specific lymphocytes To evaluate the role of SLAMF6 in melanoma-cognate T cells, we generated a new mouse strain by breeding Pmel-1 mice with SLAMF6 -/- mice. The offspring of this cross represented a new strain, which could serve as a source of CD8+ T cells lacking SLAMF6 and expressing the transgenic TCR against the H-2Db gp100: 25–33 peptide (Figure 3A). Evaluation of the lymphocyte subsets in these mice showed a lower percentage (15%) of CD8+ cells in Pmel-1 x SLAMF6 -/- mouse spleens compared to Pmel-1 splenocytes (24%) (Figure 3B and Figure 3—figure supplement 1A). Despite the smaller percent of CD8+ cells in the spleens of the SLAMF6-deficient mice, the ratio of CD8+ subpopulations (naive, effector, effector memory, and central memory) was similar in both mouse strains (Figure 3C). Figure 3 with 1 supplement see all Download asset Open asset Establishment of Pmel-1 x SLAMF6 -/- mice as a source of SLAMF6-KO antigen-specific lymphocytes. (A) SLAMF6 and Vβ13 expression in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes measured by flow cytometry. (B) Percent CD8+, CD4, and CD19 cells in spleens from Pmel-1 or Pmel-1 x SLAMF6 -/- untreated mice. (C) Pmel-1, and Pmel-1 x SLAMF6 -/- CD8+ untreated splenocytes were stained with anti-CD44 and anti-CD62L. One representative experiment is shown. (D) Percent CD8+ cells in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 7 days of in vitro activation with gp10025-33 peptide and IL-2 (30 IU/ml). (E) Flow cytometry for activation markers (CD25, CD69, CD137) in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 3 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. (F) Expression of PD-1 in Pmel-1 or Pmel-1 x SLAMF6 -/- CD8+ T cells after 7 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. (G, H) After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 -/- CD8+ T cells were stained with anti-CD44 and anti-CD62L. CD8+ subpopulations were defined for each mouse strain. (G) One representative experiment and (H) summary of subpopulations identified by flow cytometry in five experiments is shown. EM, effector memory, CM, central memory. Student t-test. *, p<0.05, **, p<0.01, ***, p<0.001. In the initial in vitro activation assays, it was already clear that the Pmel-1 x SLAMF6 -/- T cells have improved functional capacity. Their proliferative response to peptide stimulation was preserved, which was mandatory to produce ample numbers of CD8+ lymphocytes for the adoptive T cell transfer regimen (Figure 3D). CFSE dilution curves were identical in the two mouse strains, as were the activation-induced cell death (AICD) rates (Figure 3—figure supplement 1B,C). After 3 days of activation, the -/- mice had higher expression levels of CD25 and CD137 (4-1BB) activation markers (Figure 3E). In parallel, higher PD-1 expression was detected on day 7 (Figure 3F), which in this experimental context was initially taken as an indicator of activation, but was later attributed to SAP deficiency in the SLAMF6 -/- lymphocytes (see below in Results and Figure 5A and B). PD-1 overexpression was also noted by Lu et al. in iNKT cells from SFR -/- mice (Lu et al., 2019). The expression of other SLAM family members and ligands (CD48, LY9, CD244, CD8+4, CD319) on T cells during activation was similar in Pmel-1 and Pmel-1 x SLAMF6 -/- cells (Figure 3—figure supplement 1D). Lastly, we phenotyped the T cells following 3-day and 7-day activation to assess subset ratios based on CD44 and CD62L differentiation markers. While initially, all Pmel-1 T cells were naive, only a negligible number of the activated Pmel-1 x SLAMF6 -/- cells remained in the naïve CD62Lhigh/CD44low state compared to 10% of the Pmel-1 cells. The complete shift of the activated Pmel-1 x SLAMF6 -/- lymphocytes towards effector and effector memory phenotypes indicates the strength of their response to activation (Figure 3G and H). Pmel-1 x SLAMF6 -/- lymphocytes, generated to evaluate the effect of SLAMF6 on antigen-specific activated T cells, showed stronger activation and global acquisition of an effector phenotype. We attribute these results, in contrast to those previously obtained with lymphocytes from SLAMF6 -/- mice, to the role of SLAMF6 in the immune synapse, as the results could only be obtained if a synapse formation had been initiated via cognate TCRs. Pmel-1 X SLAMF6 -/- T cells have a better functional capacity In the previous experiments, the effect of SLAMF6 deletion was evaluated in the activation and proliferation phases. To test if this superior activation also affects anti-tumor immunity, we characterized Pmel-1 x SLAMF6 -/- melanoma specific T cells in the effector phase. Comparing IFN-γ secretion in response to melanoma cells by Pmel-1 versus Pmel-1 x SLAMF6 -/- lymphocytes showed that cytokine production by the Pmel-1 x SLAMF6 -/- lymphocytes was significantly higher at all effector-to-target ratios (p=0.05, Figure 4A and B and Figure 4—figure supplement 1A and B). In addition to IFN-γ, higher secretion of GM-CSF and lower levels of IL-10 and IL-13 were measured in the SLAMF6 -/- T cells (Figure 4C and D). Since GM-CSF is a strong recruiter of innate immune cells, while IL-10 and IL-13 drive suppressor traits, this secretion profile supports autocrine and paracrine immune activation. mRNA data validated the secretion assays (Figure 4—figure supplement 1C). Importantly, Pmel-1 x SLAMF6 -/- T cells produced higher levels of granzyme B in response to melanoma compared to Pmel-1 T cells, which are already strong killers due to their TCR design (Figure 4E and F). These results indicate that in the absence of the SLAMF6 modulatory effect, even strong cytolysis can be further enhanced. Figure 4 with 1 supplement see all Download asset Open asset Pmel-1 x SLAMF6 -/- T cells have a better functional capacity. (A–D) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated overnight with B16-F10/mhgp100 melanoma cells. (A) The cells were incubated at a 1:1 effector-to-target ratio. IFN-γ secretion was measured by ELISA. Each point represents one mouse. (B) The cells were incubated at the indicated effector-to-target ratios. IFN-γ secretion was measured by ELISA. (C) The cells were incubated at a 1:1 effector-to-target ratio. GM-CSF secretion was measured by ELISA. Each point represents one mouse. (D) Conditioned medium was collected and analyzed with Quantibody mouse cytokine array. (E, F) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated for 16 hr with B16-F10/mhgp100 melanoma cells. Granzyme-B expression was detected by flow cytometry. One representative experiment (E) and a summary of triplicates (F) are shown. (G–J) B16-F10/mhgp100 mouse melanoma cells were injected s.c. into the back of C57BL/6 mice. Pmel-1 or Pmel-1 x SLAMF6 -/- mouse splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml). On day 7, Pmel-1 cells or Pmel-1 x SLAMF6 -/- cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. N = 8 mice per group. Tumor size was measured three times a week. (G) Scheme of the experimental layout. (H) Spider plots showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. CR, complete response. (I) Normalized tumor volume (Mean ± SEM) until day 45, on which the first mouse had to be sacrificed. Tumor dimensions were normalized to the 1st measurement. (J) Kaplan Meier survival curve. (K) Percent T cells specific for gp10025-33 peptide in the spleen or tumor draining lymph nodes (DLN) of mice sacrificed 7 days post-ACT. Tet, Student *, p<0.05, **, p<0.01, ***, p<0.001. To evaluate the antitumor activity of Pmel-1 x SLAMF6 -/- cells, we adoptive cell transfer of 7 day Pmel-1 or Pmel-1 x SLAMF6 -/- CD8+ T cells, transferred into mice bearing palpable B16-F10/mhgp100 melanoma in their back by a of IL-2 (Figure The curves melanoma growth following Pmel-1 versus Pmel-1 x SLAMF6 -/- T cell transfer that in the first 4 tumor growth was in both on tumors with Pmel-1 and grew in six of the mice Pmel-1 x SLAMF6 -/- and three of the mice remained for days (Figure was noted in all mice complete showing in the -/- at the the strength of the response (Figure 4—figure supplement 1D). In a similar Pmel-1 CD8+ T cells or Pmel-1 x SLAMF6 -/- CD8+ T cells were transferred into mice bearing tumors. A mice were and and lymph nodes were extracted and evaluated for the presence of transferred T cells, using flow cytometry to the gp10025-33 A higher of gp10025-33 cells was found in the draining lymph nodes of mice that had Pmel-1 x SLAMF6 -/- cells (Figure compared to those had Pmel-1 cells. from both groups had a similar of lymphocytes (Figure 4—figure supplement 1E). In melanoma-specific T cells lacking SLAMF6 showed improved functional capacity both in vitro and in and longer lasting tumor with longer tumor compared to their wild-type The of cis and trans SLAMF6 to CD8+ T cell function SLAMF6 homotypic may receptors on a in or in between effector T cells and SLAMF6-expressing In Figure we showed that of SLAMF6 in trans on a melanoma target T cell antitumor activity. the Pmel-1 x SLAMF6-/- CD8+ T cells, we then established to the effect of SLAMF6 on the effector T cells and their SLAMF6-/- mice as a source of of SLAMF6, and cells to express SLAMF6 as SLAMF6-expressing (Figure Figure 5A that SLAMF6 in the response of Pmel-1 T cells, to the inhibitory effect of the trans of SLAMF6. A increase in function was observed in the SLAMF6 -/- T cells compared to the T cells when with to the in the absence of cis SLAMF6. when SLAMF6-/- T cells were activated by SLAMF6-/- their cytokine secretion increased even more This that not only the absence of SLAMF6 on the T cells their secretion but also SLAMF6-deficient are This was with the (Figure which the Pmel-1 T cells, as but also the Pmel-1 x SLAMF6-/- CD8+ T cells, which were to be to this Figure 5 Download asset Open asset The of cis and trans SLAMF6 to CD8+ T cell (A) CD8+ splenocytes from Pmel-1 and Pmel-1 x SLAMF6 -/- were co-cultured (1 × overnight at the indicated ratios with splenocytes from both mice IFN-γ secretion was measured by ELISA. (B) SLAMF6 expression on parental or transfected (SLAMF6 or empty) cells. (C) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated overnight with gp10025-33 cells or SLAMF6 at a 1:1 effector-to-target ratio. IFN-γ secretion was measured by ELISA. IFN-γ are normalized to the results of for each mouse One-way *, p<0.05, **, p<0.01, ***, p<0.001. These results show that the inhibitory effect of SLAMF6 from both in cis and in trans the role of SLAMF6 on further associated with the inhibitory function of SLAMF6 The of the next of experiments was to identify the improved effector function of Pmel-1 x SLAMF6 -/- lymphocytes. We initially evaluated the level of SAP, the adaptor required for SLAMF6 by the which was intact in the SLAMF6 -/- mice (Figure SAP is a critical adaptor that kinase to SLAMF6. while SAP transcript was found at similar levels in and SLAMF6 -/- lymphocytes, SAP protein was not in SLAMF6 -/- cells (Figure The between the transcript and the protein could be due to the rapid degradation of cytoplasmic SAP in its SAP protein deficiency also that SLAMF6 is its in non-activated CD8+ T cells, even SAP also the inhibitory activity of PD-1 et al., 2018). shown in Figure PD-1 expression is more than increased in 7 day activated Pmel-1 x SLAMF6 -/- lymphocytes compared to Pmel-1, as we have this not PD-1 overexpression can the of this receptor to generate a in T cells. Figure 6 with 1 supplement see all Download asset Open asset associated with the inhibitory function of SLAMF6. (A) RNA expression of transcript in and SLAMF6 -/- (B) of expression of SLAMF6, SAP and in and SLAMF6 -/- (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were activated with gp10025-33 peptide for the indicated time points. the of the activation, cells were and stained for (D) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were either activated with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) for hr or only with IL-2 for hr After hr, the cells were RNA was and RT-PCR for transcription expression was Data was normalized to expression for each mouse strain. for each were normalized to Pmel-1 non-activated for each (E) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) for 7 days. After the the cells were for an 5 days Expression of exhaustion markers was measured in Pmel-1 or Pmel-1 x SLAMF6 -/- (F) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) and or for 7 days, and then incubated overnight with B16-F10/mhgp100 melanoma cells at a 1:1 effector-to-target ratio. IFN-γ secretion was measured by ELISA. B16-F10/mhgp100 mouse melanoma cells were injected s.c. into the back of C57BL/6 mice. Pmel-1 x SLAMF6 -/- mouse splenocytes were expanded with gp10025-33 peptide and IL-2 (30 IU/ml) in the presence of either or On day 7, or activated cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. or were injected i.p. five times in the 2 N = 5 mice per group. Tumor size was measured three times a week. (G) Scheme of the experimental layout. (H) Tumor volume (Mean ± SEM) until day (I) Tumor volume on day 16 *, p<0.05, **, we measured by flow cytometry the level of protein an of and (Figure in was found in the Pmel-1 x SLAMF6 -/- T cells. we to identify transcription whose activity between the Pmel-1 cells and the Pmel-1 x SLAMF6 -/- cells. The most found was which increased more than in activated Pmel-1 x SLAMF6 -/- by (Figure and Figure supplement T-bet was as the transcription factor 1 T it has been found to a role in the acquisition of effector by CD8+ T cells et al., one signals T-bet expression et al., which is in line with the IFN-γ secretion we observed by the Pmel-1 x SLAMF6 -/- lymphocytes (Figure Lastly, the level of immune receptors that exhaustion was during activation. Five days after the of a 7-day activation Pmel-1 x SLAMF6 -/- T cells displayed similar levels of and to those in the Pmel-1 T cells, but higher expression of LAG-3 (Figure that LAG-3 represents a mechanism for the enhanced activation of Pmel-1 x SLAMF6 -/- T cells, we a antibody against LAG-3 in the activation We then measured IFN-γ secretion by activated T cells in response to B16-F10/mhgp100 melanoma cells. LAG-3 on Pmel-1 x SLAMF6 -/- lymphocytes increased their cytokine secretion it not Pmel-1 cells. SLAMF6 with LAG-3 resulted in a increase in IFN-γ production (Figure To evaluate the combination of SLAMF6 -/- cells and LAG-3 we an experiment in melanoma-bearing mice, using Pmel-1 x SLAMF6 -/- CD8+ T