Disruption of CD8+ Treg activity results in expansion of T follicular helper cells and enhanced antitumor immunity

Abstract

Tumor growth is associated with the inhibition of host antitumor immune responses that can impose serious obstacles to cancer immunotherapy. To define the potential contribution of Qa-1-restricted CD8 regulatory T cells (Treg) to the development of tumor immunity, we studied B6.Qa-1 D227K mice that harbor a point mutation in the MHC class Ib molecule Qa-1 that impairs CD8 Treg suppressive activity. Here, we report that the growth of B16 melanoma is substantially delayed in these Qa-1-mutant mice after therapeutic immunization with B16 melanoma cells engineered to express granulocyte macrophage colony-stimulating factor compared with Qa-1 B6-WT controls. Reduced tumor growth is associated with enhanced expansion of follicular T helper cells, germinal center B cells, and high titers of antitumor autoantibodies, which provoke robust antitumor immune responses in concert with tumor-specific cytolytic T cells. Analysis of tumor-infiltrating T cells revealed that the Qa-1 DK mutation was associated with an increase in the ratio of CD8(+) T effectors compared with CD8 Tregs. These data suggest that the CD8(+) T effector-Treg ratio may provide a useful prognostic index for cancer development and raise the possibility that depletion or inactivation of CD8 Tregs represents a potentially effective strategy to enhance antitumor immunity.

Figure 1. GVAX immunization combined with disruption of CD8 Treg activity in B6-DK mice significantly potentiates antitumor immunity

A) B6-WT mice were challenged with 2×10⁵ B16 cells s.c. and immunized with irradiated 1×10⁶ GVAX (grey box) or not (black box) on days 5, 8 and 11. Qa-1 expression was determined on gated subsets of splenocytes and TILs 20–24h after the last immunization. Data are representative of 2 independent experiments and presented as ratio of mean fluorescence intensity (MFI) of specific Qa-1 staining and MFI of background (bg) staining with streptavidin only. B) CD8 Treg efficiently infiltrate into tumors. Mice were challenged with 5×10⁵ B16 cells. The lymphoid fraction of collagenase/dispase digested tumors was collected by gradient centrifugation on days 10, 20, and 30 after challenge. The proportions of CD8 Treg (CD122⁺Ly49⁺) were determined by FACS as percent (%) of CD8⁺CD3⁺ cells. A representative FACS plot from day 20 after challenge is shown on the left. Tumor growth is shown on the right. C) B6-WT (■) or B6-DK (□) mice were challenged with 2×10⁵ B16 and immunized on the same day with irradiated 1×10⁵ GVAX followed by immunization with irradiated 5×10⁵ B16 and 5×10⁵ GVAX s.c. on day 7 and 14, n=15 mice per group. Data are presented as cumulative survival (left panel) or tumor growth (right panel) of B16 challenged mice from two independent experiments. D) B6-WT (■) or B6-DK (grey □) mice were treated as in B). Percent (%; left panel) or absolute number (right panel) of CD4 T_FH cells (CD200⁺ICOS⁺PD-1^hi gated from CD4⁺CD3⁺) from either spleens or tumors (Tm) of treated animals was determined 3 days after the last immunization. ICOS expression by tumor-infiltrating CD3⁺CD4⁺ lymphocytes was determined by FACS 3 days after the last immunization. Data are representative of at least 3 independent experiments. E) Cellular analysis of tumor-bearing B6-WT (■) or B6-DK (grey □) mice immunized with GVAX. The proportion and absolute number of CD8, Ly49⁺ CD8, NK (NK1.1⁺CD3⁻) or CD4 Treg (CD25⁺FoxP3⁺ gated from CD3⁺CD4⁺) lymphocytes from the spleens, tumor draining lymph nodes (dLN) or tumors (Tm) was determined by FACS 3 days after the last immunization. Data are representative of at least 2 independent experiments.

Figure 2. GVAX immunization induces robust antibody response to tumor-associated antigens in B6-DK mice

A) B6-WT (■) or B6-DK (grey □) mice were vaccinated with irradiated 1×10⁶ B16-OVA and 5×10⁵ GVAX on day 7 and 14. Sera were collected 14, 21, and 28 days after vaccination and analyzed for anti-OVA IgG1 antibodies by ELISA, n=3 mice per group. Data are representative of two independent experiments. B) Histology of spleens from A) on day 28. Green, GL7; red, B220. Germinal center (GC) area, pixel² per field of view (FOV) and GC numbers per FOV were quantified with ImageJ. C) Mice were challenged with 5×10⁵ B16-OVA and immunized with irradiated 1×10⁵ GVAX followed by immunization with irradiated 1×10⁶ B16-OVA and 5×10⁵ GVAX s.c. on day 7 and 14, n=5 mice per group. Sera were collected on day 25 after challenge and analyzed for anti-OVA IgG1 antibodies by ELISA. Data are representative of 2 independent experiments.

Figure 3. Disrupted CD8 Treg activity in B6-DK mice is associated with decreased tumor formation and increased antibody response to tumor-associated antigens (TAA)

Rag2^−/− mice were adoptively transferred with OT-II WT or B6-DK polarized in vitro to T_FH cells, naïve B cells, and CD8 Treg. Mice were immunized with NP-OVA in CFA, boosted on day 10 with NP-OVA in IFA, and challenged with 5×10⁵ B16-OVA 4 days later. A) Tumor volume was recorded every 2–3 days B) Sera were collected on day 15–16 after challenge and analyzed for anti-OVA IgG1 or anti-NP IgG1 antibodies by ELISA. C) Absolute numbers of T_FH, GC B (B220⁺Fas⁺IgM⁻) or CD8 Treg (CD3⁺CD8⁺ CD122⁺Ly49⁺) cells from B6-WT (■) or B6-DK (grey □) mice were determined in the spleens, tumor draining lymph nodes (dLN) or tumors (Tm). D) IL-21 and IFNγ levels were quantified by ELISA in serum collected from B6-WT (■) or B6-DK (grey □) mice on day 15–16 after challenge. Data are representative of at least 3 independent experiments, n=5 mice per group.

Figure 4. High autoantibody titers induced after GVAX immunization in the absence of CD8 Treg activity

A) Mice were vaccinated with irradiated 5×10⁵ B16-OVA and 5×10⁵ GVAX on day 7 and 14. Sera were collected 4 weeks later and analyzed for anti-dsDNA IgG or anti-ANA IgG antibodies by ELISA B) Mice were challenged with 5×10⁵ B16-OVA and immunized with irradiated 1×10⁵ GVAX followed by immunization with irradiated 5×10⁵ B16-OVA and 5×10⁵ GVAX s.c. on day 7 and 14. Sera were analyzed as above. C) Mice were challenged and treated as in B). Sera were analyzed for anti-MIF or anti-Angiopoietin-2 (Ang-2) IgG antibodies by ELISA. Data are representative of at least two independent experiments. D) Remodeling of the tumor microenvironment with therapeutic antibodies results in decreased tumor vasculature. Histology of B16-OVA tumors that were collected on day 20 from either B6-WT or B6-DK mice. Mice were immunized with irradiated 5×10⁵ B16-OVA and 5×10⁵ GVAX on day 7 and 14 after challenge. Vessel numbers were counted manually per slide, and vessel diameter was measured on the longest axis by NIS Elements software. Data are representative of three independent experiments.

Figure 5. Antitumor antibodies produced in B6-DK mice enhance tumor rejection and remodel tumor microenvironment

A) Serum was prepared from either B6-WT (■) or B6-DK (grey □) mice serially vaccinated with irradiated 1×10⁶ B16-OVA and 5×10⁵ GVAX. Rag2^−/− hosts were challenged with 1×10⁶ B16-OVA and treated with concentrated serum i.p. on days 0, 8, and 15 after challenge. Mice also received naïve OT-I Tg CD8 T cells on day 2 and 1 µg IL15/IL15Rα complex on days 2, 7, and 14 after challenge (mice n=4). B) Histology of B16-OVA tumors that were collected on day 20 from either B6-WT (■) or B6-DK (grey □) mice. Mice were either untreated or immunized with irradiated 5×10⁵ B16-OVA and 5×10⁵ GVAX on day 7 and 14 after challenge. Green, anti-mouse IgG; red, complement membrane-activated complex (MAC), blue, DAPI. Data were quantified using ImageJ and are presented as fluorescent area, pixels² per FOV (right panel). Data are representative of at least two independent experiments. C) Histology of B16-OVA tumors that were collected on day 20 from either B6-WT (■) or B6-DK (grey □) mice. Mice were untreated or immunized with irradiated 5×10⁵ B16-OVA and 5×10⁵ GVAX on day 7 and 14 after challenge. Green, anti-mouse IgG; red, F4/80. Data were quantified using NIS Elements software and are presented as fluorescent area, pixels² per region of interest (ROI) or using ImageJ and presented as fluorescent area, pixels² per FOV (right panel). Data are representative of at least two independent experiments. P values were calculated to compare F4/80⁺ fluorescent area within IgG⁺ and IgG⁻ regions. D) Tissue sections obtained from above were analyzed for IgG deposition (green) in relation to stromal components by staining with anti-CD31 (red, 100×), anti-ERTR7 (red, 100×) and anti-desmin (red, 200×). Nuclei were stained with DAPI (blue). Data shown are representative of IgG deposit area within tumors from B6-DK mice containing 5–10 distinct foci.