Peripheral Blood T-Cell Fitness Is Diminished in Patients With Pancreatic Carcinoma but Can Be Improved With Homeostatic Cytokines.

Pancreatic ductal adenocarcinoma (PDAC) shows remarkable resistance to immunotherapy. Although cancer cell–intrinsic mechanisms are known to support immune escape, T-cell “fitness” also has emerged as a key determinant of immunotherapy outcomes. In PDAC, adoptively transferred T cells show limited expansion after infusion. Moreover, more than 50% of PDAC patients fail to mount productive T-cell responses to tumor vaccines. T-cell hypofunction in PDAC, however, remains ill defined. Here, we show that chemotherapy-refractory PDAC patients harbor increased frequencies of terminally differentiated effector, rather than exhausted, peripheral blood T cells, that show an altered transcriptional profile with decreased functionality.

We examined the proliferative capacity of T cells isolated from the blood of chemotherapy-refractory PDAC patients compared with healthy volunteers (Supplementary Figure 1A and B). We studied this patient subset because they represent a major population evaluated in immunotherapy trials and, to date, responses have been exceptionally poor. We found that T-cell subset frequencies were similar between patients and volunteers (Supplementary Figure 1C). However, patient-derived T cells showed significantly decreased proliferative capacity (Figure 1A) that was independent of age (Supplementary Figure 1D). This dysfunction was a result of decreased proliferation by effector memory and effector CD8+ and CD4+ T cells (Supplementary Figure 1E). In contrast, proliferation by naive-like and central memory T-cell subsets were similar (Supplementary Figure 1E). The frequency of naive-like T cells was reduced in patients with a proportional increase in effector T cells (Supplementary Figure 1F). Patient-derived T cells also showed a decreased capacity to secrete interleukin (IL)6 and granulocyte-macrophage–colony-stimulating factor, but not other effector cytokines (eg, interferon-γ and tumor necrosis factor-α) (Supplementary Figure 2A). We did not examine for alterations in cytolytic function, which was a limitation of our analysis. Nonetheless, these data show diminished proliferative capacity by effector and effector memory peripheral blood T cells in chemotherapy-refractory PDAC patients.

Supplementary Figure 1

Study participant characteristics for ( (C) Shown are percentages of CD4+ and CD8+ cells among total live CD3+ T cells detected in peripheral blood mononuclear cells or elutriated lymphocytes collected from healthy volunteers (n = 13) and patients with PDAC (n = 15). (D) Relationship of age to fold expansion of T-cell subsets. The correlation coefficient is shown in the figure. (E) Fold expansion of T-cell subsets in IL2 for 10 days. (F) Frequency of T-cell subsets detected before and after expansion in vitro with anti-CD3/CD28 Dynabeads plus IL2 for 10 days. ECOG OS, Eastern Cooperative Oncology Group overall survival. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 1

( (B) Expression of molecules on unstimulated peripheral blood T cells. (C) Expression of immunoregulatory molecules on T cells after polyclonal stimulation. PT, n = 15; HV, n = 13.

Supplementary Figure 2

( (B) Gene set enrichment analysis of T-cell effector signaling genes in peripheral blood CD8+ T cells isolated from patients (n = 6) compared with healthy volunteers (HV) (n = 6). (C) Heat map of genes expressed by CD3+CD8+ T cells. (D) Messenger RNA levels of genes associated with differentiation, signaling, homing, and survival detected in sorted peripheral blood T cells as indicated. GM-CSF, granulocyte-macrophage–colony-stimulating factor. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

T-cell differentiation is associated with distinct transcriptional profiles. From RNA sequencing of purified peripheral blood CD4+ and CD8+ T cells, we identified 310 and 955 differentially expressed genes (DEGs), respectively, between patients and volunteers with 155 DEGs shared across CD4+ and CD8+ T cells. Gene ontology analysis showed that DEGs down-regulated in patient CD8+ T cells were associated with cell proliferation and in patient CD4+ T cells were associated with apoptosis. Up-regulated genes in CD4+ T cells were associated with cell-cycle processes, and in CD8+ T cells were associated with cellular response to hypoxia and tumor necrosis factor–mediated signaling. Gene set enrichment analysis on genes up-regulated in CD8+ T cells showed enrichment associated with effector T cells (Supplementary Figure 2B). Based on this finding, we investigated genes involved in cellular differentiation and exhaustion. Surprisingly, we detected no association with T-cell exhaustion (eg, Eomesodermin [EOMES], PDCD1, and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]). Rather, patient-derived CD8+ T cells showed a decrease in cell survival genes (ie, B-cell lymphoma 2 [BCL2], IL7 receptor [IL7R]) and alterations in genes associated with effector activity including down-regulation of C-C chemokine receptor type 7 (CCR7) and up-regulation of interferon-gamma (IFNG), granzyme B (GZMB), granzyme A (GZMA), and Natural Killer Cell Receptor 2B4 (CD244) (Supplementary Figure 2C). Transcriptional changes in CD8+ T cells were confirmed by quantitative reverse-transcription polymerase chain reaction (Supplementary Figure 2D). Specifically, we detected down-regulation in RNA transcripts related to cellular signaling (salt inducible kinase 1 [SIK1]), differentiation (nucelar receptor related 1 protein [NR4A2], bZip Maf transcription factor [MAFF]), lymph node (A-kinase anchor protein 9 (AKAP9)) and bone marrow homing (C-X-C chemokine receptor type 4 [CXCR4]), and survival (IL7R).

We next profiled unstimulated peripheral blood T cells and found no significant differences in the expression of immunoregulatory molecules associated with T-cell exhaustion, including programmed cell death protein 1 (PD-1, or PDCD1), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), and lymphocyte-activation gene 3 (LAG3) (Supplementary Figure 3A). We found no significant differences in natural or induced regulatory T-cell frequencies (Supplementary Figure 3B). In contrast, the frequency of HLA-DR, but not CD25, expressing CD4+ and CD8+ T cells was increased in patients (Supplementary Figure 3C). Patient-derived T cells also showed increased CD57 expression and loss of CD27 (Figure 1B), which is seen with T-cell senescence and terminal differentiation.6, 7, 8 In addition, patient-derived CD4+ and CD8+ T cells showed an increased propensity to express immunoregulatory molecules (including PD-1 and LAG3) after in vitro stimulation (Figure 1C, Supplementary Figure 3D–F).

Supplementary Figure 3

(T-cell subsets from healthy volunteers (HV; (B) Percentages of induced (left) and natural (right) regulatory T-cell subsets among total CD4+ and CD8+ T cells (HV, n = 10; PT, n = 9). (C) Expression of molecules and (D and E) immunoregulatory molecules on CD3+ T-cell subsets after polyclonal stimulation with anti-CD3/CD28 Dynabeads in the presence of IL2 for 10 days (HV and PT, n = 11). (F) Flow cytometry gating strategy and fluorescence minus 1 controls. SSC-A, size scatter area. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Because T cells from patients showed a transcriptional profile consistent with decreased survival and lower levels of IL7R, we hypothesized that homeostatic growth factors may improve patient-derived T-cell function. We compared T cells activated in the presence of IL2 vs IL7/IL15, which support memory T-cell survival, proliferation, and recall responses.9, 10 We found a 2-fold increase in T-cell expansion ex vivo, with stimulation involving IL7/IL15 (Figure 2A). No difference in the expression of immunoregulatory molecules (PD1, LAG3, TIM3, and CD25) was observed after stimulation with IL2 compared with IL7/IL15 (Supplementary Figure 4A). However, CD4+ effector memory T cells, but not CD4+ or CD8+ effector T cells, showed increased expansion with IL7/IL15, implying that other factors regulate the decreased proliferative capacity of effector T cells (Figure 2B, Supplementary Figure 4B–D).

Figure 2

( (B) Fold expansion of CD4+ T-cell subsets. (C) Division index at day 5 and (D) cytokine release by mesothelin (Meso)-specific CAR T cells from patients (n = 9). TNF, tumor necrosis factor.

Supplementary Figure 4

(and CD8T cells (patients [PT], (B) Fold expansion of total and Teff CD8+ T-cell subsets in the presence of IL2 or IL7/IL15. Data for CD4+ T cells is shown in Figure 2. (C) Fold expansion of T naive-like, T central memory (Tcm), and T effector (Teff) CD3+ T-cell subsets in the presence of IL2 and IL7/IL15. (D) Flow cytometry gating strategy and fluorescence minus 1 controls. (E) Cytokine release by mesothelin (Meso)-specific CAR T cells from patients. Data for TNF-α is shown in Figure 2. Ctrl, control; GM-CSF, granulocyte-macrophage–colony-stimulating factor; SSC-A, size scatter area. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

We tested the capacity of IL7/IL15 compared with IL2 to improve the functionality of patient-derived T cells responding to restimulation with a specific tumor antigen by introducing a mesothelin-specific chimeric antigen receptor (CAR) into expanded T cells. The transfection efficiency of the mesothelin CAR was 90%–98% after 24 hours. CAR T cells derived in the presence of IL2 contracted when restimulated with antigen and showed less cytokine production compared with IL7/IL15, which improved both the cytokine release capacity and expansion of CAR T cells (Figure 2C and D, Supplementary Figure 4E).

Together, our study shows that peripheral blood T cells in patients with chemotherapy-refractory PDAC harbor intrinsic alterations that limit their functionality, which may influence their potential to be harnessed for antitumor activity. This study offers insights into the defects in T-cell immunosurveillance associated with PDAC and suggests that strategies to reverse T-cell dysfunction may be necessary for advancing immunotherapy.