1. Cancer Biology
  2. Immunology and Inflammation
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SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint

  1. Emma Hajaj  Is a corresponding author
  2. Galit Eisenberg
  3. Shiri Klein
  4. Shoshana Frankenburg
  5. Sharon Merims
  6. Inna Ben David
  7. Thomas Eisenhaure
  8. Sarah E Henrickson
  9. Alexandra Chloé Villani
  10. Nir Hacohen
  11. Nathalie Abudi
  12. Rinat Abramovich
  13. Jonathan E Cohen
  14. Tamar Peretz
  15. Andre Veillette
  16. Michal Lotem
  1. Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Israel
  2. Wohl Institute for Translational Medicine, Hadassah Medical Organization, Israel
  3. Lautenberg Center for Immunology and Cancer Research, Faculty of Medicine, Hebrew University, Israel
  4. Broad Institute of MIT and Harvard, United States
  5. Boston Children's Hospital, Department of Pediatrics, United States
  6. Center for Cancer Research, Massachusetts General Hospital, United States
  7. Department of Medicine, Harvard Medical School, United States
  8. Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, United States
  9. Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Israel
  10. IRCM, Montreal Clinical Research Institute, Canada
Research Article
Cite this article as: eLife 2020;9:e52539 doi: 10.7554/eLife.52539
6 figures, 1 table and 1 additional file

Figures

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
Figure 2 with 1 supplement
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.

Figure 2—figure supplement 1
Gating strategy.

Initially, non-single cells were excluded using FSC-A and FSC-H axes. Then, based on the morphology of the cells in the FSC-A SSC-A axes, the live cell population was gated. In this population, only cells that stained positively for CD8+ expression were subjected to further analysis.

Figure 3 with 1 supplement
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.

Figure 3—figure supplement 1
Characterization of Pmel-1 x SLAMF6 -/- mice.

(A) Immunohistochemistry staining of Pmel-1 and Pmel-1 x SLAMF6 -/- spleen sections using anti CD4 and anti-CD8+ antibodies (X10 magnification). (B) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were labeled with CFSE and activated; at the indicated time points the cells were stained for CD8+ expression, and CFSE level gated on the CD8+ population was measured using flow cytometry. (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes activated for 7 days followed by 7 days maintenance with IL-2 (30 IU/ml) or without its addition. Percentage apoptotic and dead cells was measured by PI-Annexin V. Summary of two experiments shown. No Tx, no treatment. (D) After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 -/- CD8+ T cells were stained with antibodies against SLAM family receptors. The expression level of each receptor in CD8+ cells is presented.

Figure 4 with 1 supplement
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, tetramer. Student t test. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 4—figure supplement 1
Additional results regarding Pmel-1 x SLAMF6 -/- T cell superiority.

(A, B) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml). Cells were then incubated for 6 hr with B16-F10/mhgp100 melanoma cells. IFN-γ production was detected by flow cytometry. One representative experiment (A) and a summary of triplicates (B) are shown. (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days and lysed. RNA was extracted and quantitative RT-PCR for cytokine expression was performed. Data were normalized to Hprt expression for each mouse strain. Pmel-1 x SLAMF6 -/- values for each gene were normalized to Pmel-1 values. (D) Photographs from days 42 and 58 post-tumor inoculation of a mouse that developed vitiligo following ACT with Pmel-1 x SLAMF6 -/- cells. Vitiligo spots are marked. (E) Immunohistochemistry staining of tumors from mice receiving ACT of Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes, harvested 7 days post-ACT. Tumor sections were stained with anti-CD8+ Ab (X20 magnification).

The contribution of cis and trans SLAMF6 interactions to CD8+ T cell function.

(A) Separated CD8+ splenocytes from Pmel-1 and Pmel-1 x SLAMF6 -/- were co-cultured (1 × 105) overnight at the indicated ratios with non-T splenocytes from both mice splenocytes. IFN-γ secretion was measured by ELISA. (B) SLAMF6 expression on EL4 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 pulsed EL4 cells (empty or SLAMF6 transfected), at a 1:1 effector-to-target ratio. IFN-γ secretion was measured by ELISA. IFN-γ values are normalized to the results of EL4-empty for each mouse splenocytes. One-way ANOVA test. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 6 with 1 supplement
Mechanism associated with the inhibitory function of SLAMF6.

(A) RNA expression of Sh2d1a transcript (SAP) in WT and SLAMF6 -/- splenocytes. (B) Immunoblot analysis of expression of SLAMF6, SAP and SHP-1 in WT and SLAMF6 -/- splenocytes. (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were activated with gp10025-33 peptide for the indicated time points. At the end of the activation, cells were fixed and stained for phosphorylated S6. (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 18 hr or kept only with IL-2 for 18 hr (non-activated). After 18 hr, the cells were lysed, RNA was extracted, and quantitative RT-PCR for transcription factors expression was performed. Data was normalized to Hprt expression for each mouse strain. Values for each condition were normalized to Pmel-1 non-activated values for each gene. (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 expansion phase, the cells were kept for an additional 5 days without supplements. Expression of exhaustion markers was measured in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes. (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 10 μg/ml anti-LAG-3 or isotype control 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. (G–I) 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 Anti-Lag3 or Isotype control. On day 7, Isotype or Anti-Lag3 activated cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. Anti-Lag3 or Isotype control were injected i.p. five times in the 2 weeks post-transfer. 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 30 post-tumor inoculation. (I) Tumor volume on day 16 post-tumor inoculation. *, p<0.05, **, p<0.01.

Figure 6—figure supplement 1
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 18 hr or only kept with IL-2 (non-activated).

After 18 hr, the cells were lysed, RNA was extracted, and quantitative RT-PCR for transcription factors expression was performed. Data was normalized to Hprt expression for each mouse strain. Values for each condition were normalized to Pmel-1 non-activated values for each gene.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background Mus musculus Female)C57BL/6Harlan laboratoriesC57BL/6
Strain, strain background Mus musculus Female)Pmel-1This paperPmel-1Kind gift from M. Baniyash
Strain, strain background Mus musculus Female)SLAMF6 -/-This paperSLAMF6 -/-Kind gift from I. Shachar
Strain, strain background Mus musculus Female)Pmel-1 x SLAMF6 -/-This paperPmel-1 and SLAMF6-/- mice were bred to generate Pmel-1 X SLAMF6-/- mice according to the ethics requirements (Authority for biological and biomedical models, Hebrew University, Jerusalem, Israel).
Genetic reagent Mus musculusSLAMF6SINO biologicalHG11945-UTpCMV3-mSLAMF6
Cell line Mus musculusB16-F10/mhgp100This paperKind gift from Ken-ichi Hanada, Surgery Branch, NCI, NIH
Cell line Mus musculusEL4This paperKind gift from Lea Eisenbach, Weizmann Institute, Israel
Cell line (Homo sapiens)TIL412This paperThe cells were maintained in Lotem’s laboratory
Biological sample (Homo sapiens)PBMCsThis paperBlood drawn from donors recruited from the Boston community as part of the Phenogenetic Project and ImmVar Consortium
AntibodyMonoclonal Rat anti mouse CD16/32 (93)Biolegend, San Diego, CA1013020.2 μg/100 μl
AntibodyMonoclonal mouse anti mouse SLAMF6 (330-AJ)Biolegend, San Diego, CA1346100.2 μg/100 μl
AntibodyMonoclonal rat anti mouse TNFα (MP6-XT22)Biolegend, San Diego, CA5063140.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD19 (6D5)Biolegend, San Diego, CA1155210.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD44 (IM7)Biolegend, San Diego, CA1030160.2 μg/100 μl
AntibodyMonoclonal mouse anti mouse TIM3 (RMT3-23)Biolegend, San Diego, CA1197060.2 μg/100 μl
AntibodyMonoclonal rat anti mouse LAG3 (C9B7W)Biolegend, San Diego, CA1252100.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD3 (145–2 C11)Biolegend, San Diego, CA1003021 μg/ml
AntibodyMonoclonal mouse anti human CD3 (UCHT1)BD Biosciences, San Jose, CA550368
AntibodyMonoclonal mouse anti human CD28 (CD28.2)BD Biosciences, San Jose, CA556620
AntibodyMonoclonal rat anti mouse IFNγ (XMG1.2)Biogems, Westlake Village, CA808120.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD8 (53–6.7)Biogems, Westlake Village, CA101220.2 μg/100 μl
AntibodyMonoclonal rat anti mouse GZMB (NGZN)Biogems, Westlake Village, CA722120.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD4 (GK1.5)Biogems, Westlake Village, CA061120.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD25 (PC61.5)Biogems, Westlake Village, CA073120.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD62L (MEL-14)eBioscience, CA25-0621-810.2 μg/100 μl
AntibodyMonoclonal mouse anti mouse Vb13 (MR12-3)eBioscience, CA17-5797-820.2 μg/100 μl
AntibodyMonoclonal Armenian hamster anti mouse CD69 (H1.2F3)eBioscience, CA14-0691-820.2 μg/100 μl
AntibodyMonoclonal Armenian hamster anti mouse CD279 (J43)eBioscience, CA12-9985-820.2 μg/100 μl
AntibodyMonoclonal rat anti mouse CD244 (eBio244F4)eBioscience, CA14-2441-820.2 μg/100 μl
AntibodyMonoclonal Syrian hamster anti mouse CD137 (17B5)eBioscience, CA12-1371-820.2 μg/100 μl
AntibodyMonoclonal anti human SLAMF6 (REA339)Miltenyi Biotec, Bergisch Gladbach, GermanyCd3520.2 μg/100 μl
AntibodyMonoclonal rabbit anti pS6 (D57.2.2E)Cell Signaling Technology, Danvers, MA48580.2 μg/100 μl
AntibodyMonoclonal rat anti mouse LAG-3 (C9B7W)InVivoMab, BioXcell, NHBE017410 μg/1 ml
AntibodyMonoclonal rat anti-Ly108 (3E11)Merck, Kenilworth, NJMABF9191:1000
AntibodyMonoclonal rat anti-SAP (1A9)Biolegend, San Diego, CA6907021:1000
AntibodyMonoclonal mouse anti b-actin (sc-47778)Santa Cruz Biotechnology, TXC41:1000
AntibodyMonoclonal rabbit anti-SHP1This paper1:1000
Generated in Andre’ Veillette laboratory
AntibodyMonoclonal rabbit anti mouse CD4 (ab183685)AbcamEPR19514For immunohistochemistry
AntibodyMonoclonal rabbit anti mouse CD8+ (ab203035)Abcamab203035For immunohistochemistry
Sequence-based reagentPrimersAll primers are listed in the primers table in the Materials and methods section
Peptide, recombinant proteinMART-126–35Biomer Technology, Cheshire, UK
Peptide, recombinant proteingp10025-33Genscript biotech, NJ
Commercial assay or kitIFN-γ ELISABiolegend430801
Commercial assay or kitGM-CSF ELISABiolegend432201
Commercial assay or kitGenElute Mammalian Total RNA kitSigma Aldrich, MARTN70RNA production
Commercial assay or kitqScript cDNA Synthesis kitQuantabio, Beverly, MA95047RNA transformed to cDNA
Commercial assay or kitRNeasy 96 kitQiagen, Hilden, Germany74181RNA production
Commercial assay or kitAnnexin V apoptosis detection kiteBioscience88-8007-74Survival assay
Commercial assay or kitMouse CD8 T cell isolation kitStemcell technologies, Vancouver, CAEasySep 19853ACD8 isolation from total splenocytes
Commercial assay or kitQuantibody mouse cytokine arrayRayBiotech, Peachtree Corners, GAQAM-CYT-1
Chemical compound, drugIL-2Chiron, CArecombinant human IL-2
Software, algorithmFCS express five flow research editionDe Novo software
Software, algorithmCellSens Entry 1.8Olympus Life ScienceAcquisition software for immunohistochemistry
Otherlyse/fix bufferBD BiosciencesCat: 558049
OtherPermII bufferBD BiosciencesCat: 558052

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