Low-dose total body irradiation facilitates antitumoral Th1 immune responses

Abstract

CD4⁺ T helper cells are capable of mediating long-term antitumoral immune responses. We developed a combined immunotherapy (COMBO) using tumor antigen-specific T helper 1 cells (Tag-Th1), dual PD-L1/LAG-3 immune checkpoint blockade, and a low-dose total body irradiation (TBI) of 2 Gy, that was highly efficient in controlling the tumor burden of non-immunogenic RIP1-Tag2 mice with late-stage endogenous pancreatic islet carcinomas. In this study, we aimed to explore the impact of 2 Gy TBI on the treatment efficacy and the underlying mechanisms to boost CD4⁺ T cell-based immunotherapies. Methods: Heavily progressed RIP1-Tag2 mice underwent COMBO treatment and their survival was compared to a cohort without 2 Gy TBI. Positron emission tomography/computed tomography (PET/CT) with radiolabeled anti-CD3 monoclonal antibodies and flow cytometry were applied to investigate 2 Gy TBI-induced alterations in the biodistribution of endogenous T cells of healthy C3H mice. Migration and homing properties of Cy5-labeled adoptive Tag-Th1 cells were monitored by optical imaging and flow cytometric analyses in C3H and tumor-bearing RIP1-Tag2 mice. Splenectomy or sham-surgery of late-stage RIP1-Tag2 mice was performed before onset of COMBO treatment to elucidate the impact of the spleen on the therapy response. Results: First, we determined a significant longer survival of RIP1-Tag2 mice and an increased CD4⁺ T cell tumor infiltrate when 2 Gy TBI was applied in addition to Tag-Th1 cell PD-L1/LAG-3 treatment. In non-tumor-bearing C3H mice, TBI induced a moderate host lymphodepletion and a tumor antigen-independent accumulation of Tag-Th1 cells in lymphoid and non-lymphoid organs. In RIP1-Tag2, we found increased numbers of effector memory-like Tag-Th1 and endogenous CD4⁺ T cells in the pancreatic tumor tissue after TBI, accompanied by a tumor-specific Th1-driven immune response. Furthermore, the spleen negatively regulated T cell effector function by upregulation PD-1/LAG-3/TIM-3 immune checkpoints, providing a further rationale for this combined treatment approach. Conclusion: Low-dose TBI represents a powerful tool to foster CD4⁺ T cell-based cancer immunotherapies by favoring Th1-driven antitumoral immunity. As TBI is a clinically approved and well-established technique it might be an ideal addition for adoptive cell therapy with CD4⁺ T cells in the clinical setting.

Keywords: RIP1-Tag2; T helper cells; Total body irradiation; cancer immunology; combined immunotherapy.

Figure 1

Combined immunotherapy of Tag-Th1 cells and dual immune checkpoint blockade (ICB) is crucially dependent on 2 Gy TBI. (A) RIP1-Tag2 mice at 10-11 weeks of age received weekly adoptive transfers of 10⁷ Tag-Th1 cells followed by PD-L1/LAG-3 blocking mAbs 24 h later. One day prior 1^st, 5^th, and 9^th cell application mice were 2 Gy TBI or sham irradiated. (B) Survival of RIP1-Tag2 mice treated with combined immunotherapy of Tag-Th1 cells, αPD-L1/αLAG-3 mAbs with (COMBO, red) or without 2 Gy TBI (blue) (n = 5-6 per group). (C) Blood glucose levels, representing a reliable blood marker for tumor burden of insulin-producing islet carcinoma cells, confirm rapid tumor progression in the non-irradiated treatment group (blue) compared to the COMBO group (red). (D) Another cohort of mice (n = 4 per group) was sacrified 10 days after first Tag-Th1 cell administration and immune cell infiltrates of the pancreatic tumor tissue were analyzed by flow cytometry. Irradiation impacted the fractions of CD3⁺ T cells (CD3) and CD3⁺CD4⁺ T cells (CD4) and to a lesser extend CD3⁺CD8⁺ T cells (CD8). (E) The ratio of Tag-Th1 cells to all CD4⁺ cells increased when 2 Gy TBI was added to the treatment.

Figure 2

TBI-induced moderate lymphodepletion of the host. (A) Repetitive white blood cell (WBC) count (left) and corresponding flow cytometric analyses of lymphocyte populations (right) of 2 Gy TBI-treated (♦) and sham-treated (●) C3H mice over 17 days (n=5-8 per group). (B) Representative in vivo CD3-ImmunoPET/CT images (left) and ex vivo organ quantification by γ-counting (right) 48 h after i.v. administration of ⁶⁴Cu-DOTA-CD3 mAb (72 h post-TBI) revealed higher irradiation-induced uptake in the lung (n = 3 per group). Mean percent injected dose per gram, %ID/g. (C) Weight- and decay-adjusted organ biodistribution (%ID/g, above) of the lymphatic organs were similar between the experimental groups. Organ weight reduction after TBI (middle) provoked significant decreases in the absolute uptake per organ (%ID, below) in TBI-treated mice. ALN = axillary lymph nodes, BLN = brachial lymph nodes, ILN = inguinal lymph nodes, MLN = mesenteric lymph nodes.

Figure 3

Adoptively transferred Tag-Th1 cells preferentially migrate to liver, lung, spleen and lymph nodes after TBI. (A) Representative in vivo (top) and post-mortem (lower) optical images of TBI-treated and sham-treated C3H mice 4 days after application of 10⁷ DID fluorescently-labeled Tag-Th1 cells (n = 5 per group). 2 Gy TBI was performed 1 day prior cell administration. (B) Organ biodistribution quantified by the organ-to-background ratio revealed higher Tag-Th1 cell derived fluorescence signals in the liver and lung after 2 Gy TBI. (C) Post-mortem optical imaging of representative TBI-treated and untreated C3H mice 27 days after administration of 10⁷ DID fluorescently-labeled Tag-Th1 cells (28 days post-2 Gy TBI) exhibited long term accumulation in the peritoneum, primarily the omentum majus (OM) and lymph nodes (n = 5 per group). (D) Tag-Th1 fractions of the entire CD4⁺ T cell populations in the spleen and lymph nodes after TBI (red) compared to the control group (blue) as analyzed by flow cytometry. lu = lung, li = liver, int = intestinum, cervical (CLN), axillary (ALN), brachial (BLN), inguinal (ILN) lymph nodes.

Figure 4

Reconfiguration of the host immune cell composition by 2 Gy TBI results in increased Tag-Th1 cell fractions. Flow cytometric analyses (A) 5 and (B) 11 days post-2 Gy TBI of the main immune cell populations in the blood, spleen, extraperitoneal lymph nodes (LN) and thymus (n = 5 per group). The decrease of viable CD45⁺ cells after TBI is delineated by the area of the inner circle in comparison to the outer circle (non-irradiated control cohort). Within each circle the relative fraction of each cell population is color-coded. CD4 = CD3⁺CD4⁺ T cells, CD3 = CD3⁺CD8⁺ T cells, BC = CD19⁺ B cells, NK = CD49b⁺ natural killer cells, gran = CD11b⁺Gr-1^High granulocytes, mono = CD11b⁺Gr-1^low monocytes/macrophages, DC = CD11c⁺ dendritic cells. (C) Absolute cell numbers of TBI-treated (red) and untreated C3H mice (blue) 4 and 10 days after Tag-Th1 cell administration in blood (cells/γL), spleen, and extraperitoneal lymph nodes as analyzed by flow cytometry. Absolute cell numbers were calculated by WBC count of each organ x Tag-Th1 cell fraction of viable CD45⁺ cells. (D) Relative Tag-Th1 cell fractions of total CD4⁺ T cells after TBI in all analyzed organs. (E) Fractions of functionally active (CD25⁺FoxP3^-) and regulatory (CD25⁺FoxP3⁺) transferred Tag-Th1 cells and endogenous CD4⁺ T cells in the lymph nodes 4 days after cell application.

Figure 5

Low-dose TBI provokes enhanced tumor-infiltration of Tag-Th1 and endogenous CD4⁺ T cells in RIP1-Tag2 mice. (A) 10⁷ tumor antigen-specific Tag-Th1 cells were injected i.p. into 10- to 11-week-old RIP1-Tag2 tumor-bearing mice with progressed tumors 1 day after 2 Gy TBI or sham-treatment. Pancreas, spleen, and pancreas draining lymph nodes were harvested for ex vivo analyses 10 days after cell administration (11 days after 2 Gy TBI) (n = 4 per group). (B) Immune cell infiltrates of pancreatic tumor tissue of nonirradiated (blue) and 2 Gy-irradiated (red) mice were analyzed by multicolor flow cytometry. Cell subsets were classified as CD3⁺ T cells, CD19⁺ B cells, NKp46⁺ NK cells (NK), CD11c⁺ dendritic cells (DC), CD11b⁺Ly6G^- macrophages/monocytes (mono), and CD11b⁺Ly6G⁺ granulocytes. (C) Flow cytometric analyses revealed higher infiltrates of adoptive Tag-Th1 cells, endogenous CD3⁺CD4⁺ T cells (CD4 endo), but not endogenous CD3⁺CD8⁺ T cells (CD8 endo) cells. (D) Adoptively transferred and host T cells isolated from the spleen were analyzed for activation status (CD69⁺), phenotypic differentiation (CD44^-CD62L⁺ naïve (T_N), CD44⁺CD62L⁺ central memory (T_CM), CD44L⁺CD62L^- effector memory (T_EM) T cells) and expression of checkpoint molecules. (E) Blood levels of Th1-associated cytokines (mean±SEM) of healthy C3H and tumor-bearing RIP1-Tag2 mice 4 days after Tag-Th1 cell administration and 5 days after 2 Gy TBI (or sham-irradation) (n = 4-5 per group). The values of each individual mouse were derived from the arithmetic mean of duplicates.

Figure 6

Th1-based immunotherapy is negatively regulated by the spleen. (A) Immune cell infiltrates of the spleen of nonirradiated (blue) and 2 Gy TBI (red) mice analyzed 10 days after Tag-Th1 cell administration (11 days post-2 Gy TBI) by multicolor flow cytometry (n = 4 per group). Cell subsets were classified as CD3⁺ T cells, CD19⁺ B cells, NKp46⁺ NK cells (NK), CD11c⁺ dendritic cells (DC), CD11b⁺Ly6G^- macrophages/monocytes (mono), and CD11b⁺Ly6G⁺ granulocytes. (B) Splenic T cell fractions of Tag-Th1 cells, endogenous CD3⁺CD4⁺ (CD4 endo) and CD3⁺CD8⁺ T cells (CD8 endo). (C) Adoptively transferred and host T cells isolated from the spleen were analyzed for activation status (CD69⁺), phenotypic differentiation (CD44^-CD62L⁺ naïve (T_N), CD44⁺CD62L⁺ central memory (T_CM), CD44L⁺CD62L^- effector memory (T_EM) T cells) and expression of checkpoint molecules. (D) RIP1-Tag2 mice at 10-11 weeks of age received weekly adoptive transfers of 10⁷ Tag-Th1 cells followed by PD-L1/LAG-3 blocking mAbs 24 h later. One day prior, 1^st and 5^th cell application mice were 2 Gy TBI or sham treated. Spleens of RIP1-Tag2 mice were surgically removed in one experimental group one week prior to therapy initiation (COMBO + spleen ex). The other groups underwent either sham surgery and COMBO treatment (COMBO) or both sham surgery and sham treatment (sham) (Treatment groups: n = 10, sham group: n = 5).