Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile in pancreatic cancer

Clinical MedicineImmunologyOncology Open Access | 10.1172/jci.insight.152761

Max Heiduk,1,2 Ioana Plesca,3 Jessica Glück,1 Luise Müller,3 David Digomann,1 Charlotte Reiche,1 Janusz von Renesse,1 Rahel Decker,1 Christoph Kahlert,1,2,4 Ulrich Sommer,5 Daniela E. Aust,5,6 Marc Schmitz,2,3,4 Jürgen Weitz,1,2,4 Lena Seifert,1,2,4 and Adrian M. Seifert1,2,4

1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

Find articles by Aust, D. in: JCI | PubMed | Google Scholar

1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

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1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

Find articles by Weitz, J. in: JCI | PubMed | Google Scholar

1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

Find articles by Seifert, L. in: JCI | PubMed | Google Scholar

1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

2National Center for Tumor Diseases, Dresden, Germany; German Cancer Research Center, Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

3Institute of Immunology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

4German Cancer Consortium, Partner Site Dresden, German Cancer Research Center, Heidelberg, Germany.

5Institute of Pathology, Faculty of Medicine Carl Gustav Carus, and

6National Center for Tumor Diseases, Biobank Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address correspondence to: Adrian M. Seifert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus TU Dresden, 01307 Dresden, Germany. Phone: 49.351.4582190; Email: adrian.seifert@ukdd.de.

Authorship note: LS and AMS are co–senior authors.

Find articles by Seifert, A. in: JCI | PubMed | Google Scholar |

Authorship note: LS and AMS are co–senior authors.

Published November 22, 2022 - More info

Published in Volume 7, Issue 22 on November 22, 2022
JCI Insight. 2022;7(22):e152761. https://doi.org/10.1172/jci.insight.152761.
© 2022 Heiduk et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published November 22, 2022 - Version history
Received: June 28, 2021; Accepted: October 12, 2022 View PDF Abstract

BACKGROUND. Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. At diagnosis, only 20% of patients with PDAC are eligible for primary resection. Neoadjuvant chemotherapy can enable surgical resection in 30%–40% of patients with locally advanced and borderline resectable PDAC. The effects of neoadjuvant chemotherapy on the cytokine production of tumor-infiltrating T cells are unknown in PDAC.

METHODS. We performed multiplex immunofluorescence to investigate T cell infiltration in 91 patients with PDAC. Using flow cytometry, we analyzed tumor and matched blood samples from 71 patients with PDAC and determined the frequencies of T cell subsets and their cytokine profiles. Both cohorts included patients who underwent primary resection and patients who received neoadjuvant chemotherapy followed by surgical resection.

RESULTS. In human PDAC, T cells were particularly enriched within the tumor stroma. Neoadjuvant chemotherapy markedly enhanced T cell density within the ductal area of the tumor. Whereas infiltration of cytotoxic CD8+ T cells was unaffected by neoadjuvant chemotherapy, the frequency of conventional CD4+ T cells was increased, and the proportion of Tregs was reduced in the pancreatic tumor microenvironment after neoadjuvant treatment. Moreover, neoadjuvant chemotherapy increased the production of proinflammatory cytokines by tumor-infiltrating T cells, with enhanced TNF-α and IL-2 and reduced IL-4 and IL-10 expression.

CONCLUSION. Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile. Combinational treatment strategies incorporating immunotherapy in neoadjuvant regimens may unleash more effective antitumor responses and improve prognosis of pancreatic cancer.

FUNDING. This work was supported by the Jung Foundation for Science and Research, the Monika Kutzner Foundation, the German Research Foundation (SE2980/5-1), the German Cancer Consortium, and the Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden.

Graphical Abstractgraphical abstract Introduction

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, with only 10% of patients surviving 5 years after diagnosis (1). Surgical resection is the only potentially curative treatment; however, patients are often diagnosed at an advanced disease stage. At diagnosis only 20% of patients with PDAC are eligible for primary resection (PR) (2). Neoadjuvant chemotherapy (NEO) can enable surgical resection in 30%–40% of patients with locally advanced and borderline resectable PDAC (36). Even after complete tumor resection, 80% of patients develop tumor recurrence and die within 2 years (7, 8). Overall, patient outcomes have not improved significantly with current therapies over the past years (9). Accumulating evidence indicates that the immune system makes a crucial contribution to the antitumor effects of chemotherapy (1012). Beyond tumor cell–specific factors that determine cytotoxic and immune responses, the functional state of the host immune system has a relevant effect on patient prognosis. PDAC is characterized by a heterogeneous and mostly immunosuppressive immune infiltrate. T cells are the most prevalent immune cell type, with intermediate to high levels of T cell infiltration in PDAC (13, 14). The tumor-infiltrating lymphocyte composition and spatial distribution defined distinct immunological PDAC subtypes that correlated with patient prognosis (15, 16). The presence of intratumoral CD8+ T cells as well as the polarization of conventional CD4+ T (Tconv) cells toward a Th1 phenotype were both associated with prolonged survival in human PDAC, whereas Th2 cells promoted tumor progression in murine pancreatic cancer (15, 1719). Furthermore, high levels of immunosuppressive Tregs in the peripheral blood and tumor stroma were associated with poor clinical outcomes in human PDAC (20, 21). These data were almost entirely derived from limited immunohistochemical analyses, while functional studies are lacking. Analysis of rare long-term survivors of PDAC revealed persistence of T cell clones specific to tumor antigens (22). Patients with PDAC with high T cell infiltration and neoantigen qualities promoting T cell responses had improved survival (22, 23). The effect of NEO on the cytokine profile of PDAC-infiltrating T cells is unknown. In this study, we analyzed freshly isolated T cells from blood and matched tumor specimens from patients with PDAC who were either primarily resected or treated with NEO prior to surgery.

Results

Neoadjuvant chemotherapy increases the frequency of PDAC-infiltrating CD4+ Tconv cells and reduces the proportion of Tregs. To evaluate the effects of NEO on T cell infiltration in PDAC, we performed multiplex immunofluorescence for DAPI, PanCK, and CD3 on tumor specimens from 62 patients who were primary resected (PR) and 29 patients with PDAC (Supplemental Tables 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.152761DS1) who received neoadjuvant chemotherapy (NEO) prior to resection (Figure 1A). The intratumoral density of T cells was highly variable across tumors but unaffected by neoadjuvant treatment (Figure 1B). However, there was a significant difference in T cell distribution between the PR and NEO cohort (Figure 1B). Whereas PDAC-infiltrating T cells generally tended to reside in the stromal area, NEO increased T cell density in the ductal area. Patients with a moderate (tumor regression grade 2 [TRG2]) or major (TRG3) response showed increased T cell distribution compared with patients with a minor (TRG1) response to NEO (Figure 1C). FOLFOXIRI or FOLFIRINOX treatment increased T cell distribution compared with chemotherapy with gemcitabine and/or nab-paclitaxel (Supplemental Figure 1).

Neoadjuvant chemotherapy increases the frequency of tumor-infiltrating CD4+Figure 1

Neoadjuvant chemotherapy increases the frequency of tumor-infiltrating CD4+ Tconv cells and reduces the proportion of Tregs. (A) Paraffin-embedded human pancreatic ductal adenocarcinoma (PDAC) specimens from patients who were primary resected (PR) or received neoadjuvant chemotherapy (NEO) prior to surgery were stained for DAPI (blue), PanCK (red), and CD3 (green). Representative tissue segmentation and multiplex immunofluorescence images are shown. Scale bar: 100 μm. (B) Quantification of T cell density in whole PDAC specimens and distribution (density duct/density stroma) of patients who were PR (n = 62) or received NEO (n = 29). (C) T cell density and distribution according to tumor regression grade (TRG) of patients who received NEO with minor response (TRG1, n = 19) and moderate or major response (TRG ≥ 2, n = 10). (D) Flow cytometric analysis of circulating (blue) and matched PDAC-infiltrating leucocytes (red) from patients with PDAC who were PR and who received NEO. Quantification of CD8+ (n = 71; left) and CD4+ (n = 71; right) among all CD3+ T cells and (E) conventional CD4+ T (Tconv; CD4+FOXP3–; n = 46; left) cells and Tregs (CD4+FOXP3+; n = 46; right) among all CD4+ T cells. (F) Ratio of CD8+ T cells to Tregs (n = 46; left) and CD4+ Tconv cells to Tregs (n = 46; right). Each point represents data from 1 patient. Medians are shown as horizontal lines. Unpaired 2-tailed t test. *P < 0.05.

To further characterize different T cell subpopulations, we performed flow cytometry on T cells from the peripheral blood and tumors of 71 patients with PDAC (Supplemental Figure 2 and Supplemental Tables 3 and 4). Neoadjuvant chemotherapy did not alter the frequency of CD8+ and CD4+ T cells among all T cells (Figure 1D). To further delineate the composition of the CD4+ T cell population, we stained for the transcription factor FOXP3 to differentiate between CD4+ Tconv cells and Tregs. We found PDAC to be highly infiltrated by Tregs, which account for approximately 20% of all tumor-infiltrating CD4+ T cells (Figure 1E). Neoadjuvant chemotherapy markedly reduced the proportion of Tregs among CD4+ T cells (Figure 1E), significantly increasing the ratio of CD4+ Tconv cells to Tregs (Figure 1F). T cell frequencies did not differ between FOLFOXIRI and FOLFIRINOX compared with gemcitabine and/or nab-paclitaxel treatment (Supplemental Figure 3).

PDAC-infiltrating CD4+ T cells have enhanced proinflammatory cytokine production after NEO. Given the effect of NEO on T cell frequencies, we investigated the T cell cytokine profile using intracellular cytokine staining (Supplemental Figure 4). We analyzed the production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-2 in 22 patients who were PR and 13 patients who received NEO. IFN-γ was mostly produced by CD8+ T cells with no detectable difference between blood and tumor CD8+ T cells and irrespective of neoadjuvant treatment (Figure 2A). PDAC-infiltrating CD4+ Tconv cells produced higher levels of IFN-γ (PR, 28.9% ± 4.7% and NEO, 34.1% ± 7.5%, respectively) compared with CD4+ Tconv cells from matched blood (PR, 13.3% ± 4.7% and NEO, 13.3% ± 4.4%, respectively; Figure 2A). After NEO, IFN-γ expression by CD4+ Tconv cells and Tregs remained unchanged (Figure 2A). Notably, tumor-infiltrating Tregs showed increased TNF-α production in the NEO cohort compared with the PR cohort (Figure 2B). PDAC-infiltrating CD8+ T cells in the NEO cohort produced more IL-2 than corresponding circulating CD8+ T cells and tumor-infiltrating T cells in the PR cohort. Both CD4+ Tconv cells and Tregs from the tumor expressed less IL-2 than corresponding circulating T cells in the PR cohort. Neoadjuvant chemotherapy increased IL-2 production by all tumor-infiltrating T cell subsets (Figure 2C). Notably, response to NEO was associated with the cytokine profile of tumor-infiltrating T cells. The 2 patients with a major response (TRG3) showed the highest expression of IFN-γ and TNF-α (Figure 2D).

PDAC-infiltrating CD4+ T cells have enhanced proinflammatory cytokine produFigure 2

PDAC-infiltrating CD4+ T cells have enhanced proinflammatory cytokine production after neoadjuvant chemotherapy. Intracellular cytokine production of circulating and matched PDAC-infiltrating T cells from patients with PDAC. Percentages of (A) IFN-γ, (B) TNF-α, and (C) IL-2 production by CD8+ T cells (left), CD4+ Tconv cells (middle), and Tregs (right). IFN-γ+ and TNF-α+ cells of CD8+ T cells, n = 22 PR, n = 13 NEO; of Tconv cells, n = 20 PR, n = 11 NEO; of Tregs, n = 19 PR, n = 10 NEO. IL-2+ cells of CD8+ T cells, n = 20 PR, n = 12 NEO; of Tconv cells, n = 20 PR; n = 11 NEO; of Tregs, n = 19 PR, n = 10 NEO. (D) Heatmap depicting the percentage of IFN-γ–, TNF-α–, and IL-2–expressing CD8+ T cells, CD4+ Tconv cells, and Tregs standardized to z score ordered by tumor regression grade (TRG). Missing values are shown in gray. Each point represents data from 1 patient. Medians are shown as horizontal lines. Unpaired 2-tailed t test. *P < 0.05; **P < 0.01.

PDAC-infiltrating T cells have reduced antiinflammatory cytokine production after NEO. To assess the production of cytokines that are associated with an antiinflammatory response, we stained for IL-17a, IL-4, and IL-10 (Supplemental Figure 4). All T cell subsets showed minimal IL-17a production (Figure 3A). All T cell subsets had markedly lower IL-4 production in the NEO cohort than in the PR cohort (Figure 3B). The production of IL-10 by CD8+ and CD4+ Tconv cells was generally low (Figure 3C). Tregs in the blood and tumor had reduced IL-10 production after NEO, suggestive of a lower suppressive capacity (Figure 3C). Notably, the highest expression of IL-4 was found in patients with a minor response (TRG1) to NEO (Figure 3D).

PDAC-infiltrating T cells have reduced antiinflammatory cytokine productionFigure 3

PDAC-infiltrating T cells have reduced antiinflammatory cytokine production after neoadjuvant chemotherapy. Intracellular cytokine production of circulating and matched PDAC-infiltrating T cells from patients with PDAC. Percentages of (A) IL-17a, (B) IL-4, and (C) IL-10 production by CD8+ T cells (left), CD4+ Tconv cells (middle), and Tregs (right). IL-17a+ and IL-4+ cells of CD8+ T cells, n = 22 PR, n = 13 NEO; of Tconv cells, n = 20 PR, n = 11 NEO; of Tregs, n = 19 PR, n = 10 NEO. IL-10+ cells of CD8+ T cells, n = 20 PR, n = 10 NEO; of Tconv cells, n = 19 PR, n = 9 NEO; of Tregs, n = 18 PR, n = 9 NEO. (D) Heatmap depicting the percentage of IL-17a–, IL-4–, and IL-10–expressing CD8+ T cells, CD4+ Tconv cells, and Tregs standardized to z score ordered by tumor regression grade (TRG). Missing values are shown in gray. Each point represents data from 1 patient. Medians are shown as horizontal lines. Unpaired 2-tailed t test. *P < 0.05.

Neoadjuvant chemotherapy decreases the proportion of functionally exhausted CD8+ T cells in PDAC. Next, we applied a t-SNE analysis on CD8+ T cells from patients with PR and those who received NEO (Figure 4A). Whereas the proinflammatory cytokines IFN-γ, TNF-α, and IL-2 were produced by many CD8+ T cells mostly simultaneously, expression of the cytokines IL-17a, IL-4, and IL-10 was rare (Figure 4B). To define populations based on the expression pattern of the different cytokines, we used FlowSOM clustering (Figure 4, C and D). A highly proinflammatory CD8+ T cell population (P1, as denoted in FlowSOM analysis and shown in Figure 4D), defined by the coexpression of IFN-γ, TNF-α, and IL-2, was one of the most prevalent populations (Figure 4D) and modestly higher after NEO (Figure 4E). Moreover, we found a trend toward less functionally exhausted CD8+ T cells that lack cytokine production in the NEO cohort (Figure 4E).

Neoadjuvant chemotherapy decreases the proportion of functionally exhaustedFigure 4

Neoadjuvant chemotherapy decreases the proportion of functionally exhausted CD8+ T cells in PDAC. t-SNE analysis based on intracellular cytokine expression of tumor-infiltrating CD8+ T cells from patients who were primary resected (PR) (n = 8) and patients who received NEO (n = 8). (A) t-SNE analysis of CD8+ T cells merged (left) and separated distribution from patients who were PR and patients who received NEO (right). (B) t-SNE expression of indicated cytokines. (C) FlowSOM clustering into 10 clusters (P1–P10). (D) Heatmap depicting mean fluorescence intensity for cytokine expression of each cluster (left), and bar graph showing the distribution of each cluster within PR and NEO CD8+ T cells (right). (E) Proportion of CD8+ T cells within indicated clusters (PR vs. NEO). Each point represents data from 1 patient. Data are shown as the mean ± SEM. Unpaired 2-tailed t test. *P < 0.05.

Neoadjuvant chemotherapy increases the proportion CD4+ Tconv cells and Tregs with a proinflammatory profile in PDAC. Furthermore, we performed t-SNE analysis and FlowSOM clustering on CD4+ Tconv cells (Figure 5, A–D) and Tregs (Figure 6, A–D). PDAC-infiltrating CD4+ Tconv cells consisted mostly of a population with little cytokine production (P8, as denoted in FlowSOM analysis and shown in Figure 5D). Notably, NEO markedly increased a TNF-α– and IL-2–producing population (P4, as denoted in FlowSOM analysis) and reduced mostly IL-4–producing (P9, as denoted in FlowSOM analysis) CD4+ Tconv cells (Figure 5, D and E). PDAC-infiltrating Tregs expressed low amounts of cytokines (Figure 6B), but 2 populations coexpressing IL-2 and TNF-α (P3, as denoted in FlowSOM analysis and shown in Figure 6D) and producing mostly TNF-α (P4, as denoted in FlowSOM analysis) were increased after NEO. The most frequent population of Tregs was characterized by little cytokine production (P8, as denoted in FlowSOM analysis), which was significantly reduced in the NEO cohort (Figure 6, D and E).

Neoadjuvant chemotherapy decreases the proportion of functionally exhaustedFigure 5

Neoadjuvant chemotherapy decreases the proportion of functionally exhausted CD4+ Tconv cells in PDAC. t-SNE analysis based on intracellular cytokine expression of tumor-infiltrating CD4+ Tconv cells from patients who were primary resected (PR) (n = 8) and patients who received NEO (n = 8). (A) t-SNE analysis of CD4+ Tconv cells merged (left) and separated distribution from patients who were PR and patients who received NEO (right). (B) t-SNE expression of indicated cytokines. (C) FlowSOM clustering into 10 clusters (P1–P10). (D) Heatmap depicting mean fluorescence intensity for cytokine expression of each cluster (left), and bar graph showing distribution of each cluster within PR and NEO CD4+ Tconv cells (right). (E) Proportion of CD4+ Tconv cells within indicated clusters (PR vs. NEO). Each point represents data from 1 patient. Data are shown as the mean ± SEM. Unpaired 2-tailed t test. *P < 0.05; **P < 0.01.

Neoadjuvant chemotherapy increases cytokine production of PDAC-infiltratingFigure 6

Neoadjuvant chemotherapy increases cytokine production of PDAC-infiltrating Tregs. t-SNE analysis based on intracellular cytokine expression of tumor-infiltrating Tregs from patients who were primary resected (PR) (n = 8) and patients who received NEO (n = 8). (A) t-SNE analysis of Tregs merged (left) and separated distribution from patients who were PR and patients who received NEO (right). (B) t-SNE expression of indicated cytokines. (C) FlowSOM clustering into 10 clusters (P1–P10). (D) Heatmap depicting mean fluorescence intensity for cytokine expression of each cluster (left), and bar graph showing the distribution of each cluster within PR and NEO Tregs (right). (E) Proportion of Tregs within indicated cluster (PR vs. NEO). Each point represents data from 1 patient. Data are shown as the mean ± SEM. Unpaired 2-tailed t test. *P < 0.05.

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