GITR Ligation Improves Anti-PD1-Mediated Restoration of Human MMR-Proficient Colorectal Carcinoma Tumor-Derived T Cells

Abstract

Background & aims: In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on ex vivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM).

Methods: Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the in vitro T-cell agonistic activity of recombinant GITR ligation.

Results: Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4⁺ TIL, GITR expression was primarily expressed by CD45RA^- FoxP3^hi activated regulatory T cells. Within CD8⁺ TIL, GITR was predominantly expressed on functionally exhausted and putative tumor-reactive CD103⁺ CD39⁺ TIL. Strikingly, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4⁺ and CD8⁺ TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8⁺ TIL expansion compared with GITRL monotherapy. Moreover, GITRL/anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC.

Conclusions: GITR is overexpressed on CD4⁺ and CD8⁺ TIL from pMMR CRC and CRLM. Agonistic targeting of GITR enhances ex vivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined immunotherapies in primary pMRR CRC and CRLM.

Keywords: Colorectal carcinoma; Immune Checkpoint Stimulation; Liver metastasis; Microsatellite Stable; TNF Receptor Superfamily; Tumor-Infiltrating Lymphocytes.

Figure 1

TNFRSF members are predominantly expressed on intratumoral CD4⁺activated Th and Treg cells. (A) Frequencies of CD3⁺CD4⁺ among living CD45⁺ cells in tumor, adjacent tissues, and PBMC. CRC are depicted in blue (n = 43), and CRLM are depicted in red (n = 26). (B) Gating strategy and frequencies of Th, aTh, rTreg, and aTreg among CD3⁺CD4⁺ cells in tumor. CRC are depicted in blue (n = 42), and CRLM are depicted in red (n = 23). (C) Frequencies of Th, aTh, rTreg, and aTreg among CD3⁺CD4⁺ cells in tumor (T), adjacent tissues (A), and PBMC (P). CRC are depicted in blue (n = 42), and CRLM are depicted in red (n = 24). (D and E, respectively) Frequencies of any TNFRSF member (GITR, 4-1BB, or OX40) negative, single positive (SP), double positive (DP), or triple positive (TP) on aTreg, aTh, and Th in tumor (T), adjacent tissues (A), and PBMC (P). CRC are depicted in blue (n = 18), and CRLM are depicted in red (n = 9). Friedman (B) or Kruskal-Wallis test (A, C, D, and E) was applied to analyze differences between more than 2 different groups. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. A, adjacent tissues; aTh, activated T helper; aTreg, activated regulatory T cell; CRC, primary colorectal cancer; CRLM, liver metastasis; DP, double positive; MFI, median fluorescence intensity; P or PBMC, peripheral blood mononuclear cell; rTreg, resting regulatory T cell; SP, single positive; T, tumor; Th, T helper; TIL, tumor-infiltrating lymphocyte; TP, triple positive.

Figure 2

GITR is predominantly expressed on intratumoral CD4⁺activated Th and Treg cells. (A and B) Frequencies of GITR, 4-1BB, and OX40 on aTreg, aTh, and Th in tumor. CRC are depicted in blue (A) (n = 18), and CRLM are depicted in red (B) (n = 9). (C and D) Frequencies and MFI of GITR on aTreg, aTh, and Th in tumor. CRC are depicted in blue (C) (n = 42), and CRLM are depicted in red (D) (n = 23). (E) Histogram and frequencies of GITR-positive cells among aTreg, aTh, and Th in tumor (T), adjacent tissues (A), and PBMC (P). CRC are depicted in blue (n = 40), and CRLM are depicted in red (n = 22). Friedman (A–D) or Kruskal-Wallis test (E) was applied to analyze differences between more than 2 different groups. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. A, adjacent tissues; CRC, primary colorectal cancer; CRLM, liver metastasis; MFI, median fluorescence intensity; P or PBMC, peripheral blood mononuclear cell; T, tumor; TIL, tumor-infiltrating lymphocyte.

Figure 3

Intratumoral GITR-expressing Th, aTh, and aTreg feature an activated phenotype. (A and B) MFI of proliferation/activation markers Ki67 (A) (n = 16) and HLA-DR (B) (n = 6) on GITR^- and GITR⁺ cells among Th, aTh, and aTreg in tumor. CRC are depicted in blue, and CRLM are depicted in red. Wilcoxon matched test was used to analyze differences between 2 paired groups. ∗P ≤ .05, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; MFI, median fluorescence intensity; TIL, tumor-infiltrating lymphocyte.

Figure 4

Intratumoral CD8 cells are enriched for GITR. (A) Frequencies of CD3⁺CD8⁺ among living CD45⁺ cells in tumor, adjacent tissues, and PBMC. CRC are depicted in blue (n = 43), and CRLM are depicted in red (n = 26). (B and C) Frequencies of any TNFRSF member (GITR, 4-1BB, or OX40) negative, single positive (SP), double positive (DP), or triple positive (TP) on CD8⁺ T cells in tumor. CRC are depicted in blue (B) (n = 18), and CRLM are depicted in red (C) (n = 9). Mann-Whitney test (A) was used to analyze differences between 2 unpaired groups. Kruskal-Wallis test (B and C) was applied to analyze differences between more than 2 different groups. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. A, adjacent tissues; CRC, primary colorectal cancer; CRLM, liver metastasis; DP, double positive; P or PBMC, peripheral blood mononuclear cell; SP, single positive; T, tumor.

Figure 5

GITR delineates activated CD103⁺CD39⁺CD8⁺TIL. (A and B) Frequencies of GITR, 4-1BB, and OX40 on CD8⁺ T cells in tumor. CRC are depicted in blue (A) (n = 19), and CRLM are depicted in red (B) (n = 10). (C and D) Frequencies and MFI of GITR on CD8⁺ T cells in tumor (T), adjacent tissues (A), and PBMC (P). CRC are depicted in blue (C) (n = 42), and CRLM are depicted in red (D) (n = 23). (E) MFI of proliferation marker Ki67 on GITR^- and GITR⁺ cells among CD8+ TIL (n = 19). CRC are depicted in blue, and CRLM are depicted in red. (F) Frequencies of CD103⁺CD39⁺, CD103^-CD39⁺, CD103⁺CD39^-, and CD103^-CD39^- among CD8⁺ T cells in tumor. Blue and red bars represent CRC- and CRLM-derived TIL, respectively (n = 9). tSNE plot demonstrating CD103⁺ and CD39⁺ cells among GITR⁺ CD8⁺ T cells in tumor. Frequencies of GITR-positive cells among DN (CD103^-CD39^-), SP (CD103⁺CD39^-), and DP (CD103⁺CD39⁺) CD8⁺ T cells in tumor (n = 10). Wilcoxon matched test (E) was used to analyze differences between 2 paired groups. Friedman (A, B, and E) or Kruskal-Wallis test (C and D) was applied to analyze differences between more than 2 different groups. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. A, adjacent tissues; CRC, primary colorectal cancer; CRLM, liver metastasis; DP, double positive; MFI, median fluorescence intensity; P, peripheral blood mononuclear cell; SP, single positive; T, tumor; TIL, tumor-infiltrating lymphocyte.

Figure 6

GITR is up-regulated on PD1^hiCD8⁺TIL featuring a (pre-)exhausted phenotype. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. Ex vivo function and phenotype of TIL were analyzed using flow cytometry with or without in vitro stimulation. (A) Frequencies of IFN-$\gamma$ and TNF-$\alpha$ positive cells in GITR^- and GITR⁺ among CD8⁺ T cells in tumor after 5 hours of PMA/ionomycin stimulation (n = 8). CRC are depicted in blue, and CRLM are depicted in red. (B) Frequencies of PD1^-, PD1^int, or PD1^hi and LAG3^- or LAG3⁺ among CD8⁺ T cells in tumor. Blue and red bars represent CRC-and CRLM-derived TIL, respectively. (C) Gating strategy and frequencies of GITR, PD1, and LAG3 among CD8⁺ T cells in tumor. Frequencies of GITR among PD1^-, PD1^int, or PD1^hi and LAG3^- or LAG3⁺ CD8⁺ T cells in tumor. CRC are depicted in blue (n = 26), and CRLM are depicted in red (n = 13). (D) Correlation of frequencies of GITR-positive cells to frequencies of PD1^hi or LAG3⁺ cells among CD8⁺ T cells in tumor. Blue and red dots represent individual CRC (n = 26 and n = 14, respectively) and CRLM (n = 13 and n = 8, respectively) patients. (E) Histogram and MFI of transcription factor TOX on PD1^-, PD1^int, GITR^- PD1^hi, or GITR⁺ PD1^hi CD8⁺ T cells in tumor (n = 7). (F) Histogram and MFI of TCF1 on GITR^- PD1^hi or GITR⁺ PD1^hi CD8⁺ T cells in tumor (n = 8). Histogram and MFI of proliferation marker Ki67 on GITR^- PD1^hi or GITR⁺ PD1^hi CD8⁺ T cells in tumor (n = 14). CRC are depicted in blue, and CRLM are depicted in red. Wilcoxon matched test (A and F) was used to analyze differences between 2 paired groups. Friedman test (C and D) was applied to analyze differences between more than 2 different groups. Correlation analysis was performed according to Spearman. ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; IFN-$\gamma$, interferon gamma; MFI, median fluorescence intensity; TIL, tumor-infiltrating lymphocyte; TNF-$\alpha$, tumor necrosis factor alpha.

Figure 7

GITR ligation enhances CD4⁺and CD8⁺TIL expansion. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. TIL were stimulated using CD3/CD28 stimulation beads in the absence (ctrl) or presence of a hexameric GITR ligand (GITLR 1 μg/mL + anti-HA 2.5 μg/mL). After 8 days, TIL expansion was measured and defined as fold increase over control conditions (proliferation index). (A) Flow cytometric gating strategy used for ex vivo phenotyping after in vitro polyclonal assay. Cell numbers were normalized using counting beads (red box). (B) pMMR CRC-derived CD4⁺ and CD8⁺ TIL expansion upon GITR ligation (n = 19). (C) CD4⁺ and CD8⁺ TIL expansion upon various dosages of GITR ligation (0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1.0 μg/mL, 10.0 μg/mL, and 100.0 μg/mL, respectively) (n = 8). (D) pMMR CRLM-derived CD4⁺ and CD8⁺ TIL expansion upon GITR ligation (n = 6). (E) Gating strategy and frequencies of Ki67⁺ cells among CD4⁺ and CD8⁺ TIL upon GITR ligation, respectively. CRC are depicted in blue, and CRLM are depicted in red. Wilcoxon matched test (B, D, and E) was used to analyze differences between 2 paired culture conditions. Friedman test (C) was applied to analyze differences between more than 2 different culture conditions. ∗P ≤ .05, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; Ctrl, control; TIL, tumor-infiltrating lymphocyte.

Figure 8

CD4⁺Th TIL respond to GITR ligation in the absence of CD4⁺Treg. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. TIL were stimulated using CD3/CD28 stimulation beads in the absence (ctrl) or presence of hexameric GITR ligand (GITRL 1 μg/mL + anti-HA 2.5 μg/mL) with or without magnetic CD25-depletion. TIL expansion was measured and defined as fold increase over control conditions (proliferation index). (A) pMMR CRC- and CRLM-derived CD4⁺ FoxP3^- and CD4⁺ FoxP3⁺ TIL expansion upon GITR ligation (n = 18). (B) MFI of FoxP3 in pMMR CRC- and CRLM-derived CD4⁺ FoxP3⁺ TIL in absence (ctrl) or presence of hexameric GITR ligand (n = 18). (C) pMMR CRC-derived CD4⁺ and CD8⁺ TIL expansion upon GITR ligation with or without CD25-depletion. (n = 8). Wilcoxon matched test was used to analyze differences between 2 paired culture conditions. ∗P ≤ .05, ∗∗P ≤ .01. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; MFI, median fluorescence intensity; TIL, tumor-infiltrating lymphocyte.

Figure 9

GITR ligation increases CD3⁺TIL pro-inflammatory cytokine secretion. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. TIL were stimulated using CD3/CD28 stimulation beads in absence (ctrl) or presence of hexameric GITR ligand (GITRL 1 μg/mL + anti-HA 2.5 μg/mL). After 8 days, supernatants were analyzed for soluble factors, and TIL were phenotyped after PMA/ionomycin restimulation. (A) Flow cytometric gating strategy and relative frequencies of IFN-$\gamma$ and TNF-$\alpha$ producing pMMR CRC-derived CD3⁺ TIL upon PMA/ionomycin restimulation after GITR ligation compared with control conditions (n = 11). (B) Concentration of secreted IFN-$\gamma$ defined as fold increase over control conditions. CRC are depicted in blue (n = 18), and CRLM are depicted in red (n = 4). (C) Concentration of secreted cytokines defined as fold increase over control conditions. CRC are depicted in blue (n = 6). Wilcoxon matched test was used to analyze differences between 2 paired culture conditions. ∗P ≤ .05. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; Ctrl, control; IFN-$\gamma$, interferon gamma; TIL, tumor-infiltrating lymphocyte; TNF-$\alpha$, tumor necrosis factor alpha.

Figure 10

GITR ligation skews TIL to a pro-inflammatory chemokine profile. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. TIL were stimulated using CD3/CD28 stimulation beads in absence (ctrl) or presence of hexameric GITR ligand (GITRL 1 μg/mL + anti-HA 2.5 μg/mL). After 8 days, supernatants were analyzed for soluble factors. (A) Concentration of secreted cytokines and chemokines defined as fold increase over control conditions of CRC-derived TIL (n = 10). (B) Concentration of secreted cytokines and chemokines defined as fold increase over control conditions of CRLM-derived TIL (n = 5). Wilcoxon matched test was used to analyze differences between 2 paired culture conditions. ∗P ≤ .05, ∗∗P ≤ .01. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; CRLM, liver metastasis; sFAS, soluble FAS; TIL, tumor-infiltrating lymphocyte.

Figure 11

GITR ligation further enhances anti-PD-1-mediated reinvigoration of CD4⁺and CD8⁺TIL. TIL were isolated from tumor tissues using enzymatic digestion and subsequent Ficoll density gradient centrifugation. TIL were stimulated using CD3/CD28 stimulation beads in presence of isotype antibodies (iso IgG4), anti-PD1 blocking antibodies ($\propto$ PD1), anti-PD1 blocking antibodies in combination with GITRL ($\propto$ PD1 + GITRL), or GITRL (GITRL). After 8 days, TIL were phenotyped directly or after PMA/ionomycin restimulation. TIL expansion and cytokine secretion were measured and defined as fold increase over isotype control conditions (proliferation and stimulation index, respectively). (A) pMMR CRC-derived CD4⁺ TIL Ki67 expression and expansion upon iso IgG4, $\mathrm{\alpha }$PD1, $\propto$ PD1 + GITRL, and GITRL (n = 12). (B) pMMR CRC-derived CD8⁺ TIL Ki67 expression and expansion upon iso IgG4, $\propto$ PD1, $\propto$ PD1 + GITRL, and GITRL (n = 12). (C) Flow cytometric gating strategy and relative frequencies of IFN-$\gamma$ and TNF-$\propto$ producing pMMR CRC-derived CD4⁺ TIL upon PMA/ionomycin restimulation after $\propto$ PD1, $\propto$ PD1 + GITRL, and GITRL compared with isotype control conditions (n = 10). (D) Flow cytometric gating strategy and relative frequencies of IFN-$\gamma$ and TNF-$\propto$ producing pMMR CRC-derived CD8⁺ TIL upon PMA/ionomycin restimulation after $\propto$ PD1, $\propto$ PD1 + GITRL, and GITRL compared with isotype control conditions (n = 10). Friedman (A–D) was applied to analyze differences between more than 2 different groups. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Boxes and whiskers represent mean and 95% confidence interval. CRC, primary colorectal cancer; IFN-$\gamma$, interferon gamma; Iso IgG4, isotype control; PI, proliferation index; SI, stimulation index; TIL, tumor-infiltrating lymphocyte; TNF-$\propto$, tumor necrosis factor alpha.