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. 2024 Feb 13;134(6):e171154.
doi: 10.1172/JCI171154.

PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice

Elselien Frijlink  1   2 , Douwe Mt Bosma  2 , Julia Busselaar  2 , Thomas W Battaglia  3 , Mo D Staal  2 , Inge Verbrugge  1 , Jannie Borst  2
Affiliations

PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice

Elselien Frijlink et al. J Clin Invest. .

Abstract

Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhanced spontaneous priming of thymus-derived (FOXP3+Helios+) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded tumor control by a simultaneous, RT-induced CD8+ cytotoxic T cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 (PD-L1+CD80+), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the control of such tumors by enabling a CTL response.

Keywords: Cancer immunotherapy; Costimulation; Immunology; Oncology; Radiation therapy.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Lymphocyte-depleted (C4/C5) human cancers have suboptimal responses to RT and are modeled by the murine TC-1 tumor.
(A) Kaplan-Meier OS curves obtained from TCGA for patients receiving RT (red) or not (gray) within the C1 “wound healing” (n = 2,136), C2 “IFN-γ dominant” (n = 2,296), C3 “inflammatory” (n = 1,903), C4 “lymphocyte-depleted” (n = 1,055), and C5 “immunologically quiet” (n = 354) cancer immune subtypes. P values (log-rank) were generated using a Cox proportional hazards model. (B) C3 “inflammatory” versus C4/C5 “lymphocyte-depleted” model predictions from transcriptome data on C57BL/6 syngeneic MC38 and TC-1 transplantable tumors. (C) Frequency of CD8+ T cells among CD45+ cells in MC38 (total n = 9) and TC-1 (total n = 7) tumors measured at the indicated tumor sizes (left) and representative flow cytometric plots (right) depicting the percentage of CD8+ T cells within TCRβ+ cells in 50 mm2 MC38 (gray) and TC-1 (black) tumors. (D) Tumor growth curves for mice bearing MC38 (n = 6/group, left) or TC-1 (n = 6/group, right) tumors that were treated with either 8 Gy over 3 days (3× 8 Gy) or a single dose of 20 Gy RT. Ratios indicate the number of mice among the total number of mice treated that showed full recovery upon RT. Error bars indicate the SD. *P < 0.05, by Mann-Whitney U test.
Figure 2
Figure 2. Myeloid cell– and Treg-rich TC-1 tumor shows a CD8+ T cell–dependent RT response.
(A) Frequency of the indicated immune cell populations among CD45+ cells measured by flow cytometry in 50 mm2 TC-1 tumors (n = 6). (B–D) cTregs and eTregs were defined as indicated in Supplemental Figure 2, B–D, and identified in the TdLN, non-TdLN, and tumor of 100 mm2 TC-1 tumor–bearing mice (n = 6) and age-matched naive (non-tumor-bearing) mice (n = 5). FlowSOM-guided clustering was performed on 5,000 randomly selected cells per sample within the CD3+ lymphocyte population. (B and C) Representative histograms depicting expression of the indicated markers on cTreg and eTreg populations in axillary LNs of naive and TC-1 tumor–bearing mice. (D) Frequency of Helios+ cells among cTregs and eTregs in axillary LNs of naive and TC-1 tumor–bearing mice. (E) Percentage of eTregs (left) and cTregs (right) among CD3+ T cells in the indicated tissues. (F–H) Monitoring by flow cytometry of the CD8+ T cell response to 20 Gy RT (n = 8) or control (0 Gy, n = 6) in TC-1 tumors. n-TdLN, non-TdLN. (F) Absolute number of total CD8+ T cells and (G) GZB-, IFN-γ–, or TNF-α–expressing CD8+ T cells per milligram of tumor tissue on after post-RT day 8. IFN-γ and TNF-α levels were measured after in vitro PMA/ionomycin stimulation. (H) OS of TC-1 tumor–bearing mice treated with 20 Gy RT on day 0 in combination with vehicle (PBS, n = 9) or depleting mAbs specific for CD8 (n = 5) or CD4 (n = 9). αCD8, anti-CD8 mAb. ***P < 0.001 (Mantel-Cox analysis). Data are from 1 experiment and are representative of at least 2 experiments. Error bars indicate the SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by Kruskal-Wallis test with uncorrected Dunn’s post hoc analysis (E) and Mann-Whitney U test (D, F, and G).
Figure 3
Figure 3. RT induces concomitant CTL and Treg responses in the TC-1 tumor model.
(A–D) TC-1 tumor–bearing mice were treated with 20 Gy RT (n = 10) or control (0 Gy, n = 4–6) when tumors reached approximately 20 mm2 in size (day 0). FTY720 or vehicle (NaCl) was administered orally on days –1, 3, and 5. On day 8, the CD8+ T cell response was analyzed by flow cytometry in the TdLN (A and B) and tumor (C and D). (A and C) Representative concatenated flow cytometric plots showing IFN-γ+ cells among CD8+ T cells in the TdLN (A) and tumor (C). (B and D) Frequency of CD44+TCF-1–, GZB+, and IFN-γ+ cells among CD8+ T cells in the TdLN (B) and tumor (D). IFN-γ was measured after in vitro PMA/Ionomycin stimulation. (E and F) Monitoring of the (FOXP3+CD25+) Treg response to 20 Gy RT (n = 6–8) or control (0 Gy, n = 6) in TC-1 tumor–bearing mice on day 8 after treatment. (E) Treg frequency among CD4+ T cells in the non-TdLN and TdLN, or among CD45+ cells within the tumor. (F) Percentage of Tregs among live cells in blood at the indicated time points (n = 6/group). (G) Frequency of Tregs in the indicated tissues on day 8 following 20 Gy RT (n = 10) or control (0 Gy, n = 4–6) with or without FTY720 treatment. (H) CD8+ T cell/Treg ratio in the TdLN and tumor after RT. (I) OS of TC-1 tumor–bearing mice treated with 0 Gy (n = 5) or 20 Gy (n = 11–14/group) RT in combination with a CD25-depleting mAb or vehicle (PBS) administered i.p. on day –1 and on day 5 after RT. *P < 0.05 (Mantel-Cox analysis). Data are from 1 experiment and are representative of at least 2 experiments. Error bars indicate the SD. *P < 0.05 and **P < 0.01, by 2-way ANOVA with Bonferroni’s post hoc test (B, D, F and G) and Mann-Whitney U test (E and H).
Figure 4
Figure 4. CTLA-4 blockade exacerbates RT-induced eTreg expansion.
Mice bearing 20 mm2 TC-1 tumors received RT (20 Gy, n = 9) or control (0 Gy, n = 6) on day 0. Treatment included vehicle (PBS) or a CTLA-4–blocking mAb on days 0, 3, 6, and 9, with longitudinal monitoring (A and B) and flow cytometric analysis of the non-TdLN, TdLN, and tumor on post-treatment day 8 (C–G). (A) Individual tumor growth curves and (B) OS for the treatment groups. Ratios indicate the number of mice that showed full recovery upon treatment compared with the total. (C) Percentage of total Tregs among CD3+ lymphocytes in the indicated tissues on day 8. (D–F) UMAP display of 2,500 randomly selected CD3+ T cells per sample in non-TdLN, TdLN, and tumor on day 8 for all treatment groups combined, with FlowSOM-guided clustering (see also Supplemental Figure 2B) (D) and marker visualization (E) used to highlight the eTreg response. (F) UMAP visualization of the response of the CD3+ T cell subpopulations in the TdLN and tumor to the indicated treatments. Red circles highlight the eTreg population. (G) Frequencies of eTregs and cTregs identified in D among CD3+ T cells found in the indicated tissues on post-treatment day 8. (H) TC-1 tumor–bearing mice received 20 Gy (n = 10/group) or control (0 Gy, n = 4–6), with CTLA-4 mAb blockade or vehicle on days 0, 3, and 6, with or without FTY720. Treg frequencies were measured in the TdLN and tumor on post-RT day 8 (same experiment as in Figure 3G). (I) Visual representation of how Tregs benefit from CTLA-4 blockade. Data are from 1 experiment and are representative of 2 experiments. Error bars indicate the SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Kruskal-Wallis with Dunn’s post hoc test (C and G) and 2-way ANOVA with Tukey’s multiple-comparison test (H).
Figure 5
Figure 5. CD86, but not CD80, drives the RT-induced eTreg response.
Mice bearing 20 mm2 TC-1 tumors received control treatment (0 Gy, n = 5) or 20 Gy RT on day 0 in combination with either vehicle (PBS, n = 8) or a blocking mAb against CD80 (n = 11) or CD86 (n = 11) on days 0, 3 and 6. The CD3+ lymphocyte response was monitored by flow cytometry in the non-TdLN, TdLN, and tumor on day 8. (A–C) UMAP visualization of 2,500 randomly selected CD3+ cells per sample found in the non-TdLN, TdLN, and tumors on day 8 of all treatment groups combined. FlowSOM-guided clustering (A) identifying the same cell populations as found in the previous figures and (B) representative heatmaps of the markers included to determine the CD3+ T cell subpopulations. (C) Visualization of the response of the CD3+ T cell subpopulations in the TdLN and tumor to the indicated treatments. Red circles highlight the eTreg population. (D) Frequencies of eTregs and cTregs identified in B among CD3+ cells found in the indicated tissues on post-treatment day 8. (E) Graphic visualization of how CD86, but not CD80, binds CD28 to support Treg expansion. Data are from 1 experiment and are representative of 2 experiments. Error bars indicate the SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ordinary 1-way ANOVA with Dunnett’s post hoc test (D).
Figure 6
Figure 6. CD86 blockade in the context of RT improves cDC costimulatory status and CTL priming.
(A–D) Mice bearing 20 mm2 TC-1 tumors received 0 Gy (n = 6) or 20 Gy RT on day 0 in combination with either vehicle (PBS, n = 8) or a blocking mAb against CD80 (n = 7) or CD86 (n = 8) on days 0, 3, and 6. The cDC response was monitored by flow cytometry in the TdLN on day 8. (A) Absolute counts of migratory cDCs1 and cDC2s. (B) Representative concatenated (n = 6–8) flow cytometric plots depicting the percentage of CD80+ and/or PD-L1+ T cells among migratory (Mig.) cDC1s and cDC2s in the TdLN per treatment group. The numbers in the boxes indicate percentages. (C and D) Quantification of the cell populations represented in B among migratory cDC1s (C) and migratory cDC2s (D) from the TdLN. (E–H) The CD8+ T cell response was monitored by flow cytometry in the same experiment described in Figure 5. (E and F) Opt-SNE visualization of 1,000 randomly selected CD44+CD62L– cells among CD8+ T cells per sample found in TdLNs on day 8, concatenated per treatment group. (E) Representative heatmap of TCF-1 expression and (F) visualization of the TCF-1– subpopulation in the TdLN (encircled) in different treatment groups. (G) Frequency of CD44+TCF-1– cells among CD8+ T cells found in the TdLN and among CD45+ cells in the tumor on post-treatment day 8. (H) Concatenated (n = 11) contour plots depicting expression of the indicated markers on CD44+TCF-1– cells and CD44+TCF-1+ cells within CD8+ T cells in the TdLN. Numbers indicate percentages. Data are from 1 experiment and are representative of 2 experiments. Error bars indicate the SD. *P < 0.05, **P < 0.01, and ****P < 0.0001, by ordinary 1-way ANOVA with Dunnett’s post hoc test (A and C–E).
Figure 7
Figure 7. CD86-mediated CD28 costimulation is required for PD-1–dependent eTreg expansion.
(A) PD-1 expression on Ki67+CD8+ T cells (green) and eTregs (red) in the tumor as identified in Figure 5A, presented as a heatmap and a representative histogram across all experimental conditions. (B–D) TC-1 tumor–bearing mice received 0 Gy (n = 4) or 20 Gy RT on day 0 with vehicle (PBS, n = 8) or blocking mAbs against PD-1 (n = 11), CD86 (n = 10), or their combination (n = 10) on days 0, 3, and 6. CD3+ lymphocyte responses were analyzed by flow cytometry in non-TdLNs, TdLNs, and tumor on day 8. (B) UMAP visualization of the treatment response of the CD3+ T cell subpopulations. The red circle indicates eTregs, and the green circle indicates Ki67+CD8+ T cells (see also Supplemental Figure 8, B and C). (C) Frequencies of eTregs and cTregs identified in Supplemental Figure 8B among CD3+ T cells in the indicated tissues. (D) Quantification of the Ki67+CD8+ T cell population among total CD3+ T cells in the TdLN and tumor. (E) Individual tumor growth curves and (F) OS of TC-1 tumor–bearing mice receiving RT on day 0 with vehicle (n = 27), blocking mAbs against PD-1 (n = 26), CD86 (n = 26), or a combination (n = 28) on days 0, 3, and 6. Proportion of mice that fully recovered is indicated. (G) Proposed effect of combined CD86 and PD-1 blockade on Tregs. (i) PD-L1/L2 on cDCs engages PD-1, which inhibits CD28 costimulation of Tregs. (ii) PD-1 blockade enables CD28 costimulation of Tregs. (iii) CD86 blockade inhibits CD28 costimulation of Tregs, which cannot be overruled by PD-1 blockade, impeding the Treg response. Data are from 1 experiment and are representative of 2 experiments. Error bars indicate the SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by ordinary 1-way ANOVA with Dunnett’s post hoc test (C), Brown-Forsythe ANOVA with Dunnett’s T3 post hoc analysis (D), and Mantel-Cox analysis (F).
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