Tumor resident memory CD8 T cells and concomitant tumor immunity develop independently of CD4 help

Abstract

Tissue resident memory (Trm) CD8 T cells infiltrating tumors represent an enriched population of tumor antigen-specific T cells, and their presence is associated with improved outcomes in patients. Using genetically engineered mouse pancreatic tumor models we demonstrate that tumor implantation generates a Trm niche that is dependent on direct antigen presentation by cancer cells. However, we observe that initial CCR7-mediated localization of CD8 T cells to tumor draining lymph nodes is required to subsequently generate CD103⁺ CD8 T cells in tumors. We observe that the formation of CD103⁺ CD8 T cells in tumors is dependent on CD40L but independent of CD4 T cells, and using mixed chimeras we show that CD8 T cells can provide their own CD40L to permit CD103⁺ CD8 T cell differentiation. Finally, we show that CD40L is required to provide systemic protection against secondary tumors. These data suggest that CD103⁺ CD8 T cell formation in tumors can occur independent of the two-factor authentication provided by CD4 T cells and highlight CD103⁺ CD8 T cells as a distinct differentiation decision from CD4-dependent central memory.

Figure 1

Characterization of tumors in KPC-LSIY mice. (A) In vivo luciferase expression in Pdx1-cre + /LSIY + by MuriGlo-assisted CT scan. (B) Survival curves of KPC mice compared to KPC-LSIY mice. (C) Representative histology of premalignant progression in KPC-LSIY mice. (D) Representative histology of moderately differentiated and sarcomatoid PDAC phenotypes, liver metastasis, lung metastasis, as well as thymic lymphoma and in KPC-LSIY mice. (E) Representative histology of orthotopic and subcutaneous tumors using KPC (PK5) and KPC-LSIY (PK5L)-derived tumor cell lines.

Figure 2

Direct interaction of tumor antigen specific T cells with antigen in tumors. (a) Analysis of CD8 T cells in tumor and peripheral lymphoid organs. PK5L1940 tumors were implanted sc. into C57BL/6 mice and tumors, tumor-draining lymph node (TDLN), non-draining lymph node (NDLN), and spleen were analyzed for SIY-specific CD8 T cells at d14. Subplots show CD62L and Ly6C expression on SIY-specific CD8 T cell populations in the lymphoid organs versus the tumor. (b) PK5L1940 tumors were implanted sc. into Nur77-GFP mice and tumor infiltrating T cells were analyzed by flow cytometry. (i) representative flow plots. The SIY-specific T cell population of infiltrating CD8 T cells was identified by pentamer staining, and CD103 + and CD103 + CD39 + subpopulations were identified and Nur77-GFP expression was determined in each cell type. Graphs show (ii) Percent Nur77-GFP + and (iii) MFI of Nur77-GFP in each group. (c) PK5L1940 cancer cells were deleted of B2M expression using CRISPR/Cas9. (i) MHCI expression in cancer cells was detected by flow cytometry compared to isotype control antibody (gray), with cells untreated (green) or pre-treated with IFNg (red). (ii) PK5L1940 or PK5L1940-B2M^−/− were co-cultured with SIY-specific 2C cells or Ova-specific OT1 cells and assessed for expression of the activation marker CD69. (iii) PK5L1940 or PK5L1940-B2M^−/− were implanted sc. into C57BL/6 mice and harvested at d14 and analyzed for infiltrating CD8 T cells and SIY-specific CD8 T cells by flow cytometry. Key: NS = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Results are representative of two or more experiments.

Figure 3

Role of direct presentation in Trm formation. (a) Rag^−/− mice were reconstituted with a 1:1 mix of splenocytes from wt and Kaede mice, or a 50:50 mice of splenocytes from wt and Kaede CCR7^−/− mice. Following reconstitution, mice were implanted sc. with tumors and tumor-infiltrating CD8⁺CD103⁺ T cells were analyzed for Kaede expression after 14 days. (b) Representative gating on CD103⁺ T cells, and Kaede expression by these cells. (c) Summary of percent of CD103⁺ cells that are Kaede⁺ in mice given Kaede or Kaede CCR7^−/− cells. Key: ** = p < 0.01. Results are representative of two or more experiments.

Figure 4

CD4 depletion minimally impacts CD8 T cell responses to tumor implantation. (a) PK5L1940 pancreatic adenocarcinoma cells were injected sc. into C57BL/6 mice, C57BL/6 mice treated with anti-CD40L on d0, 1, and 2, or C57BL/6 mice treated with anti-CD4 on d-1. ii) Representative flow cytometry of tumor infiltrating T cells 14d following implantation. Plots show gated CD90.2⁺ T cells, and subgates for CD8⁺ T cells, and SIY-pentamer⁺ CD8 T cells. (b) Summary of T cell infiltration of PK5L1948, PK5L1941, PK5L1932, and PK5L1940 tumors derived from Pdx-Cre+/− Kras(G12D)+/− Trp53(R172H)+/− Luciferase-SIY + mice implanted sc. into C57BL/6 mice or C57BL/6 mice treated with anti-CD4 on d-1. Graphs show tumor the proportion of CD4, CD8, CD8 SIY-specific T cells, and CD103 expression on CD8 SIY-specific T cells. Key: NS = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Results are representative of two or more experiments.

Figure 5

Decreased tumor-specific memory following anti-CD40L or anti-CD4 treatment at tumor implantation. (a) PK5L1940 pancreatic adenocarcinoma cells were injected sc. into C57BL/6 mice or C57BL/6 mice treated with anti-CD40L on d0, 1, and 2, or C57BL/6 mice treated with anti-CD4 on d-1. Control mice received no treatment. (b) Mice were treated with Lm-SIY on d21 and analyzed for SIY-specific T cells in the spleen on d28 by intracellular cytokine staining for IFNg following SIY peptide stimulation. (c) Quantitative analysis of SIY-specific cells in the spleen at d28.

Figure 6

CD8 T cell can provide CD40L for Trm formation. (a) Rag^−/− mice were adoptively transferred with splenocytes from wt or CD40L^−/− mice, then following reconstitution, implanted sc. with PK51940 tumors. d14 tumors were harvested and analyzed for tumor infiltrating CD8 T cell phenotypes. Flow plots show representative CD103 expression in tumor CD8 T cells from mice reconstituted with (i) wt or (ii) CD40L^−/− splenocytes. (iii) Summary of CD103 expression in tumor T cells. (b) Rag^−/− mice were reconstituted with CD8-depleted CD40L^−/− splenocytes, along with purified CD40L^−/− CD8⁺ splenocytes, or purified wt CD8⁺ splenocytes. Following reconstitution, mice were implanted sc. with PK51940 tumors. d14 tumors were harvested and analyzed for tumor infiltrating CD8 T cell phenotypes. Flow plots show representative CD103 expression in tumor CD8 T cells from mice reconstituted with (i) CD40L^−/− CD8 T cells, or (ii) wt CD8 T cells. (iii) Summary of CD103 expression in tumor T cells. . Key: ** = p < 0.01. Results are the combination of two experiments.

Figure 7

Concomitant tumor immunity is dependent on CD40L. (a)(i) Mice were implanted with a primary sc. PK5L1940 tumor on d0 and a secondary sc. tumor on the opposite flank at d0 or d7 (with and without CD8 depletion). Graphs show the size of the primary (1°) and secondary tumor (2°) at d14. (ii) Mice bearing a primary sc. PK5L1940 tumor were injected with a secondary tumor sc. on d7 or d21. The number of SIY-specific CD8 T cells were determined 0, 7 and 14d following secondary tumor implantation. (b) Mice were implanted with a primary sc. PK5L1940 tumor or no treatment at d0, and a secondary sc. tumor on d7. Groups of mice also received anti-CD40L on d0, 1, and 2, anti-CD4 on d-1, or anti-CD8 on d6. Additional groups received FTY-720 IP on days 6–11. Graphs show the size of the (i) primary (1°) and (ii) secondary tumor (2°) on d21. (iii) Proportion of mice secondary tumor free on d21. (c) Mice were implanted with a primary sc. PK5L1940 tumor or no treatment at d0 or were vaccinated with Lm-SIY or Lm-Ova IV on d0. Mice received a secondary sc. tumor on d7. Peripheral blood was analyzed for (i) SIY-specific T cells or (ii) ova-specific T cells on d7. (iii) Size of secondary tumors on d21. (iv) proportion of mice tumor-free on d21. Key: NS = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Results are the combination of 4–5 experiments.