Ctla-4 blockade plus adoptive T-cell transfer promotes optimal melanoma immunity in mice

Abstract

Immunotherapeutic approaches to the treatment of advanced melanoma have relied on strategies that augment the responsiveness of endogenous tumor-specific T-cell populations [eg, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade-mediated checkpoint inhibition] or introduce exogenously prepared tumor-specific T-cell populations [eg, adoptive cell transfer (ACT)]. Although both approaches have shown considerable promise, response rates to these therapies remain suboptimal. We hypothesized that a combinatorial approach to immunotherapy using both CTLA-4 blockade and nonlymphodepletional ACT could offer additive therapeutic benefit. C57BL/6 mice were inoculated with syngeneic B16F10 melanoma tumors transfected to express low levels of the lymphocytic choriomeningitis virus peptide GP33 (B16GP33), and treated with no immunotherapy, CTLA-4 blockade, ACT, or combination immunotherapy of CTLA-4 blockade with ACT. Combination immunotherapy resulted in optimal control of B16GP33 melanoma tumors. Combination immunotherapy promoted a stronger local immune response reflected by enhanced tumor-infiltrating lymphocyte populations, and a stronger systemic immune responses reflected by more potent tumor antigen-specific T-cell activity in splenocytes. In addition, whereas both CTLA-4 blockade and combination immunotherapy were able to promote long-term immunity against B16GP33 tumors, only combination immunotherapy was capable of promoting immunity against parental B16F10 tumors as well. Our findings suggest that a combinatorial approach using CTLA-4 blockade with nonlymphodepletional ACT may promote additive endogenous and exogenous T-cell activities that enable greater therapeutic efficacy in the treatment of melanoma.

Figure 1. Experimental schema

Ly5.2+/C57BL/6 mice were inoculated subcutaneously with 10⁶ B16GP33 melanoma cells on day 0. Mice received no immunotherapy (control), CTLA-4 blockade, adoptive cell transfer (ACT), or combination immunotherapy of CTLA-4 blockade plus ACT. CTLA-4 blockade was administered by three intraperitoneal injections of 200 mg anti-CTLA-4 mAb on days 2, 5, and 8. GP33-specific T cells were harvested from Ly5.1+/B6.SJL mice 8 days after LCMV injection. ACT was administered by one intravenous injection of 10⁴ Ly5.1+/CD8+/GP33-specific T cells on day 1. On days 9 or 14 after B16GP33 tumor inoculation, mice were euthanized for TIL and/or splenocyte analysis. Tumors were measured every three days.

Figure 2. Combination immunotherapy promotes optimal control of melanoma tumors

A) Exponential B16GP33 tumor growth was observed in control mice. CTLA-4 blockade resulted in no initial inhibition of B16GP33 tumor growth followed by durable arrest of tumors beginning on day 12 after tumor inoculation. ACT resulted in early inhibition of B16GP33 tumor growth followed by exponential tumor growth beginning on day 12. Combination immunotherapy resulted in durable and optimal inhibition of B16GP33 tumor growth. (B) Kaplan-Meier survival curves of mice in the four treatment groups indicate that whereas control mice and mice treated with ACT alone met criteria for euthanasia by day 18, half of mice treated with CTLA-4 and the majority of mice treated with CTLA-4 and ACT experienced long-term survival for up to 8 weeks. This experiment (n=4 mice per group) was performed four times with similar results. (* p<0.05 compared with control group; † p<0.05 compared with CTLA-4 blockade group; ‡ p < 0.05 compared with ACT group.)

Figure 3. Combination immunotherapy promotes optimal tumor infiltration of CD8+ T cells

Tumor infiltrating lymphocytes populations were evaluated by flow cytometry on days 9 and 14 after B16GP33 tumor inoculation. When total CD8+ T cells were evaluated (representative individual data shown in A, group data shown in B), control mice showed an initial small infiltration on day 9 that largely dissipated by day 14. CTLA-4 blockade resulted in an early infiltration on day 9 that persisted through day 14. ACT resulted in a strong early infiltration on day 9 that largely dissipated by day 14. Combination immunotherapy resulted in a strong early infiltration on day 9 that was stronger than that seen in control mice and after CTLA-4 blockade; this infiltration persisted on day 14 to levels that were stronger than those seen in control, CTLA-4 blockade, and ACT mice. (C) When only Ly5.1+/CD8+ T cells (derived from Ly5.1+ donor mice during ACT) were evaluated, no infiltration was seen in mice that did not received ACT (control and CTLA-4 blockade). In mice that did receive ACT, a brisk infiltration of exogenously-derived Ly5.1+/CD8+ T cells seen on day 9 was largely lost by day 14. No differences in the levels of exogenously-derived Ly5.1+/CD8+ T cells were observed between mice treated with CTLA-4 blockade alone and combination immunotherapy. (D) When only Ly5.2+/CD8+ T cells (derived from the tumor-bearing mouse and not from the adoptively transferred cells) were evaluated, ACT resulted in a slight increase in intratumoral T cell infiltration on day 9 compared with controls, but this effect was short-lived. In contrast, CTLA-4 blockade resulted in a gradual increase in endogenously-derived T cell infiltration, and the magnitude of this infiltration was largely additive in mice treated with combination immunotherapy. This experiment (n=4 mice per group) was performed three times with similar results. (* p<0.05 compared with control group; † p<0.05 compared with CTLA-4 blockade group; ‡ p < 0.05 compared with ACT group.)

Figure 3. Combination immunotherapy promotes optimal tumor infiltration of CD8+ T cells

Tumor infiltrating lymphocytes populations were evaluated by flow cytometry on days 9 and 14 after B16GP33 tumor inoculation. When total CD8+ T cells were evaluated (representative individual data shown in A, group data shown in B), control mice showed an initial small infiltration on day 9 that largely dissipated by day 14. CTLA-4 blockade resulted in an early infiltration on day 9 that persisted through day 14. ACT resulted in a strong early infiltration on day 9 that largely dissipated by day 14. Combination immunotherapy resulted in a strong early infiltration on day 9 that was stronger than that seen in control mice and after CTLA-4 blockade; this infiltration persisted on day 14 to levels that were stronger than those seen in control, CTLA-4 blockade, and ACT mice. (C) When only Ly5.1+/CD8+ T cells (derived from Ly5.1+ donor mice during ACT) were evaluated, no infiltration was seen in mice that did not received ACT (control and CTLA-4 blockade). In mice that did receive ACT, a brisk infiltration of exogenously-derived Ly5.1+/CD8+ T cells seen on day 9 was largely lost by day 14. No differences in the levels of exogenously-derived Ly5.1+/CD8+ T cells were observed between mice treated with CTLA-4 blockade alone and combination immunotherapy. (D) When only Ly5.2+/CD8+ T cells (derived from the tumor-bearing mouse and not from the adoptively transferred cells) were evaluated, ACT resulted in a slight increase in intratumoral T cell infiltration on day 9 compared with controls, but this effect was short-lived. In contrast, CTLA-4 blockade resulted in a gradual increase in endogenously-derived T cell infiltration, and the magnitude of this infiltration was largely additive in mice treated with combination immunotherapy. This experiment (n=4 mice per group) was performed three times with similar results. (* p<0.05 compared with control group; † p<0.05 compared with CTLA-4 blockade group; ‡ p < 0.05 compared with ACT group.)

Figure 4. Combination immunotherapy promotes systemic T cell responsiveness to melanoma antigen

Splenocytes were evaluated by flow cytometry on day 14 after B16GP33 tumor inoculation. Splenocytes were stimulated for 5 hours in vitro with the LCMV peptide GP33 (or no peptide) in the presence of IL-2 and brefeldin A, after which intracellular expression of IFNγ was measured. Although levels of GP33-induced IFNγ+ CD8+ T cells were higher after CTLA-4 blockade and ACT as compared with control mice, these levels were not significantly higher than background (no peptide stimulation) in these groups. In contrast, significantly higher levels of GP33-induced IFNγ expression were observed after combination immunotherapy, and these levels were significantly above background. (Representative individual data shown in A, group data shown in B.) This experiment was also performed on day 9 after B16GP33 tumor inoculation with similar results (data not shown). This experiment (n=3-4 mice per group) was performed two times with similar results. (* p<0.05 compared with control group; † p<0.05 compared with CTLA-4 blockade group; ‡ p < 0.05 compared with ACT group; x p<0.05 compared with background control.)

Figure 5. Combination immunotherapy improves the balance of CD8+ effector : CD4+ regulatory TILs

Tumor infiltrating lymphocytes were evaluated by flow cytometry on day 14 after B16GP33 tumor inoculation for levels of CD8+ and CD4+ T cells. CTLA-4 blockade and ACT resulted in the induction of higher levels of CD4+CD25+FoxP3+ T cells compared with control mice. (Representative individual data of CD4+CD25+ T cells shown in A, representative individual data of intracellular FoxP3 expression gated on CD4+CD25+ T cell populations shown in B.) (C) When represented as ratios of activated CD8+/CD44^high effector T cells to CD4+ regulatory T cells, a trend toward a favorable tumor infiltrating T cell profile was observed after combination immunotherapy (p=0.078 vs. control). This experiment (n=3-4 mice per group) was performed two times with similar results.

Figure 5. Combination immunotherapy improves the balance of CD8+ effector : CD4+ regulatory TILs

Tumor infiltrating lymphocytes were evaluated by flow cytometry on day 14 after B16GP33 tumor inoculation for levels of CD8+ and CD4+ T cells. CTLA-4 blockade and ACT resulted in the induction of higher levels of CD4+CD25+FoxP3+ T cells compared with control mice. (Representative individual data of CD4+CD25+ T cells shown in A, representative individual data of intracellular FoxP3 expression gated on CD4+CD25+ T cell populations shown in B.) (C) When represented as ratios of activated CD8+/CD44^high effector T cells to CD4+ regulatory T cells, a trend toward a favorable tumor infiltrating T cell profile was observed after combination immunotherapy (p=0.078 vs. control). This experiment (n=3-4 mice per group) was performed two times with similar results.

Figure 6. Combination immunotherapy promotes optimal immunity against recurrent melanoma tumors

Some of the mice treated with CTLA-4 blockade exhibited persistent B16GP33 tumors that showed no progression, and all of the mice treated with combination immunotherapy exhibited minimal to no visible B16GP33 tumors with no progression. (A) When these selected mice were challenged with contralateral injections of B16GP33 tumors > 30 days after initial B16GP33 inoculation, no tumor growth was observed in mice that had initially been treated with CTLA-4 blockade or combination immunotherapy, indicating successful induction of immunity to B16GP33. As a reference, B16GP33 tumor growth in naïve mice that had not received any previous tumor injections or immunotherapy is also shown. (B) When these selected mice were challenged with contralateral injections of B16F10 tumors (identical to B16GP3 tumors with the exception of absent GP33 expression), only mice that had initially been treated with combination immunotherapy exhibited immunity to B16F10. As a reference, B16F10 tumor growth in naïve mice that had not received any previous tumor injections or immunotherapy is also shown. This experiment (n=3 mice per group) was performed two times with similar results. († p<0.05 compared with CTLA-4 blockade group.)