Platelets subvert T cell immunity against cancer via GARP-TGFβ axis

Abstract

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as major platelet-derived soluble factors to obliterate CD4⁺ and CD8⁺ T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of the TGFβ-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFβ per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer.

Fig. 1. Targeting platelets genetically potently enhances adoptive T cell therapy of cancer

(A)Platelet counts from the peripheral blood of WT and Pf4-Cre-Hsp90b1^flox/flox (KO) mice (n=15 per group). (B) Bleeding time was measured in WT and KO mice by pricking the lateral tail vein (n=5 per group). (C) B16-F1 melanoma tumors were established in WT and Pf4-Cre-Hsp90b1^flox/flox mice, followed by adoptive transfer of activated Thy1.1⁺ Pmel cells on day 11 plus IL-2-anti IL-2 antibody complexes on day 11, 13, 15, and 17. Shown are average tumor growth curves (n=7–9 per group). (D) Same as in (C) except mice did not receive T cells (n=5–6 per group). (E–F) Pmel cells from the tumor-draining LNs of the adoptively transferred mice in (D) were stimulated with hgp100 peptide for 4 hours followed by intracellular staining for IFNγ (E) and TNFα (F). MFI: mean fluorescence intensity. Repeated measures two-way ANOVA was used to compare the tumor growth curves. Two-tailed, independent Student’s t-test was used in A, B, E and F. Data are represented as mean ± standard error of the mean (SEM).

Fig. 2. Platelet-derived soluble factors suppress T cell function

(A) Naïve splenic CD8⁺ T lymphocytes were activated polyclonally with or without mouse platelet releasate (PR) for 3 days followed by measuring forward scatter profile (FSC) and multiple intracellular molecules. Data from multiple experiments (n>5 times) were summarized at the bottom of the panel. (B) Human peripheral blood CD4⁺ or CD8⁺ cells were activated polyclonally for 7 days with or without human platelet releasate, followed by measuring the indicated markers (representative of 2 experiments). Correlation between % PR and cytokine quantity was established using Spearman correlation coefficient. (C–D) Naive CD8⁺ T cells were polyclonally activated in the presence or absence of platelet releasate for 3 days, followed by flow cytometry of indicated markers. Numbers represent percentage of cells in the corresponding quadrants (n=3–4 per group). Comparisons in A, C and D were performed using two-tailed, independent Student’s t-tests.

Fig. 3. Priming of antigen-specific T cells in the platelet releasate abrogates their anti-tumor immunity in vivo.

(A) TRP1 transgenic CD4⁺ T cells were primed under Th17-differentiating conditions, in control media or human platelet releasate (n=6 per group). They were then adoptively transferred on day +10 to B16-F10-bearing mice who also received sub-lethal dose of total body irradiation on day +9. (B) Percentages of TRP1 cells in the peripheral blood on day +37 (n=6–7 per group). Mann-Whitney test for non-normal distribution was used to compare the two groups. (C)Pmel T cells were primed with IL-12 and human gp100 peptide, in control media or human platelet releasate. They were then adoptively transferred on day +8 to B16-F10-bearing mice who also received cyclophosphamide on day +7 (n=4–8 per group). (D) Percentages of Pmel cells in the peripheral blood on day +33 (n=4–8 per group). Tumor growth curves were compared using repeated measures two-way ANOVA. Percentages of T cells were compared using two-tailed, independent Student’s t-tests. Data are represented as mean ± SEM.

Fig. 4. TGFβ and lactate contribute to platelet-mediated T cell suppression

(A) Human platelet releasate was fractionated by size-exclusion chromatography, followed by quantifying individual fractions for their suppressive activity. T cell suppression indices of all fractions are shown. Suppression index of media (percent of undivided CD8 T cells) was set as 1 and is indicated. (B) Fraction A was further resolved by DEAE column, and sub-fractions were assayed for T cell suppression. The most active fractions (boxed) were resolved by SDS-PAGE and coomassie blue stain. The protein identities of the major bands were determined by mass spectrometry and immunoblot. (C) CFSE-labeled naïve CD8⁺ T cells were stimulated with anti-CD3ε/CD28 antibodies and IL-2 for 3 days in media or Fraction A (Fr. A) with or without TGFβ blockade. Cells were assayed for CFSE dilution, GZMB and IFNγ production by CD8⁺ cells (n=3 per group). Representative histograms are shown. Corresponding quantifications are displayed below the flow cytometry data. (D) Suppressive activity of various sub-fractions B1-B8. (E) Upfield 600 MHz ¹H-excitation sculpting NMR spectra of fractions B2-5 (bottom to top). Solid and dashed boxes highlight varying concentrations of lactate methyl- and methine proton resonances, respectively. Spectra are normalized to the defined concentration of sodium 3-trimethylsilyl-2,2,3,3-d₄-propionate (TSP). (F) Purified CD8⁺ T cells were cultured for 3 days in the presence of agonistic CD3ε and CD28 antibodies and IL-2. Cell surface expression of CD25 and CD69, as well as blastogenesis (FSC) was assayed by flow cytometry. Correlation between lactic acid concentration and cytokine production was established using Spearman correlation coefficient. Difference between groups in (C) was tested based on a two-tailed, independent Student’s t-test.

Fig. 5. Platelet-intrinsic GARP plays critical roles in generating active TGFβ

(A) Baseline serum was collected from wild type C57BL/6J mice followed by a single dose of anti-mouse thrombocyte sera (n=7). Serum was collected 24, 48, and 72 hours post injection. (B) Representative flow cytometry plots. Platelet-specific marker CD41⁺ population was gated on and analyzed for the expression of cell surface GARP and latent TGFβ. Numbers represent percentages of the gated population over all CD41⁺ events. (C–D) Graphical representation of flow cytometry data from (B) (n=4–9 per group). (E) Serum and plasma levels of active TGFβ from indicated mice (n=5 per group). (F) Serum and plasma levels of total TGFβ from indicated mice (n=5 per group). Comparison was performed using two-tailed, independent Student’s t-test. Data are represented as mean ± SEM.

Fig. 6. Platelet-derived GARP-TGFβ complex blunts adoptive T cell therapy of melanoma

(A–C) B16-F1-bearing WT and Plt-GARP^KO mice (n=7–8 per group) expressing congenic marker Thy1.2 were given Cy on day 9, followed by adoptive transfer of activated Thy1.1⁺ Pmel cells on day 10. (A) Tumor growth curves. (B) The frequency of Pmel cells in mice was enumerated 3 weeks post-adoptive transfer of T cells by flow cytometry in the peripheral blood (CD8⁺Thy1.1⁺/total CD8⁺). (C) IFNγ-producing ability of antigen-specific donor T cells (Pmel) from indicated mice 3 weeks after T cell transfer. (D)) B16-F1 melanoma tumors were established in WT and Plt-Tgfβ KO mice, followed by adoptive transfer of activated Thy1.1⁺ Pmel cells on day 11 plus IL-2-anti IL-2 antibody complexes on day 11, 13, 15, and 17. Shown are average tumor growth curves (n=4–9 per group). Repeated measures ANOVA was used in panel A and D. Two-tailed, independent Student’s t-test was used in panels B and C. Data are represented as mean ± SEM.

Fig. 7. Targeting platelet-derived GARP-TGFβ complex results in reduction of TGFβ activity in the tumor microenvironment and protection against colon cancer

(A) WT or Plt-GARP^KO mice (n=5 per group) were injected in the right flank with 1×10⁶ MC38 colon cancer cells. Tumor size was measured every 3 days with digital vernier caliper. (B) Kaplan-Mayer survival curve in MC38-bearing mice (n=5 per group). (C) In a separate experiment, 6 weeks after MC38 injection, mice were sacrificed and the primary tumors were resected and weighed. The inset shows the photographs of primary tumors resected from mice 6 weeks after injection. (D) Serum was obtained from mice 6 weeks after MC38 injection and active TGFβ1 was measured by ELISA. (E) IHC for p-SMAD-2/3 in MC38 tumors from indicated mice; representative images are shown. Scale bar: 12.5 μm. (F) Independent histopathological quantification of p-SMAD-2/3 staining intensity from panel (E) (n=4 per group). (G) Flow cytometric analysis of peripheral blood myeloid derived suppressor cells. (H) Percentage of regulatory T cells (CD25+ Foxp3+) in the CD4⁺ tumor-infiltrating lymphocytes (TIL) from the indicated mice. Repeated measures two-way ANOVA was used in panel A; Kaplan-Meier curves and log rank tests were used in panel B. Two-tailed, independent Student’s t-test was used in panels C, D, F, G and H. Data are represented as mean ± SEM.

Fig. 8. Pharmacological inhibition of platelets enhances adoptive T cell therapy of cancer

(A) C57BL/6J mice were inoculated with B16-F1 subcutaneously on day 0, given Cy on Day 7, followed by adoptive therapy with activated Pmel cells on day 8 (Cy + T, n=7–8 per group) or not (Cy, n=4–5 per group). Each of the above groups of mice received concurrent aspirin and clopidogrel (AP: anti-platelets) or water. Left: Average tumor growth curves. Right: Recurrence-free survival. (B) Pmel cells in the peripheral blood (day +62 post tumor inoculation), inguinal lymph nodes (ILN) and spleens (upon sacrifice) of mice in different treatment groups were enumerated by flow cytometry. WBC: white blood cells. (C) C57BL/6J mice were inoculated with 2.5 B16-F1 subcutaneously on day 0. Mice were lymphodepleted with Cy on day 9 followed by adoptive transfer of either activated Pmel wild type cells or IFNγ −/− Pmel cells on day 10. AP was given as described in A. (T^WT: wild type Pmel cells; T^KO: IFNγ KO Pmel cells, n=6–10 per group). (D) C57BL/6J mice were inoculated with 2.5 B16-F1 subcutaneously on day 0. Mice were lymphodepleted with Cy on day 9 followed by adoptive transfer of activated Pmel cells on day 10. IFNγ neutralizing antibody (clone XMG1.2, BioXCell) was delivered i.p. at 100 μg/mouse every other day starting on day 11 until sacrifice. AP was given as described in A (n=4–10 per group). Repeated measures two-way ANOVA was used to compare the tumor growth curves in A, C and D. Kaplan-Meier curves and log rank tests were used for relapse free survival analysis. Two-tailed, independent Student’s t-test was used in B. Data are represented as mean ± SEM.