It has been shown that treatment with activating anti-CD40 antibodies preserves the responsiveness of CD4+
T cells to vaccination and prevented peptide-induced CD4+
T-cell anergy (29
). Furthermore, in vivo
ligation of CD40 with agonistic anti-CD40 antibodies prevented the induction of tolerance in tumor (HA)–specific T cells in mice with established pulmonary metastases of a renal cell carcinoma expressing HA. Although antigen-presenting cells were presumed to play an important role in the CD40-mediated conversion of T-cell tolerance to T-cell activation, the identity of the antigen-presenting cells involved and the underlying mechanism of reversion of T-cell tolerance in anti-CD40-treated mice were not determined. Our findings indicate that CD40 is required for MDSC-mediated immune suppression. Importantly, the treatment with agonistic anti-CD40 antibody prevented MDSC-mediated expansion of Tregs in mice with large tumor burdens, leading to augmented anti-tumor responses.
CD40/CD40L interaction plays a role in the development and homeostasis of naturally occurring CD4+
). The CD4+
subpopulation, which regulates autoreactivity, has been shown to be markedly reduced in CD40 KO mice. In vitro
, CD40-deficient, but not WT, dendritic cells produced diminished amounts of IL-2 upon Treg encounter and showed impaired ability to expand Tregs (30
). The identity of the CD40+
cells in vivo
that can mediate Treg expansion is not clear, however. Under pathologic conditions Treg homeostasis can be disrupted efficiently. An increased number of Tregs has been observed in tumor-bearing mice and in patients with various carcinomas (5
). Tumor-associated expansion of Tregs is the result of conversion of CD4+
T cells into CD4+
). Interestingly, this process occurs independent of both the thymus and proliferation; however, the mechanisms underlying the activation and expansion of tumor-specific CD4+
Tregs in the tumor-bearing host remains to be elucidated.
In this report, we show that the expression of CD40 on MDSCs is critically important for MDSC-mediated Treg expansion. Interference with CD40/CD40L interactions, through the use of CD40-deficient MDSC or anti-CD40, significantly reduces Tregs and prevents the suppression of tumor-specific T-cell responses in tumor-bearing mice. Previously, we showed that upon IFN-γ stimulation, the expressions of IL-10 and TGF-β by MDSCs were significantly enhanced and that the activation of T cells was required for Treg induction (25
). Our current working model is that activated T cells secrete IFN-γ, which enhances the expression of CD40, IL-10, and TGF-β. The engagement of CD40/CD40L between MDSC and Treg in the presence of TGF-β and IL-10 leads to activation and expansion of Treg.
We used two complementary models to show that CD40 expression was required for Treg expansion and tumor promotion mediated by Gr-1+CD115+ monocytic MDSCs. In the HA-MCA26 tumor model, endogenous MDSCs were depleted by sublethal irradiation. Reconstitution of CD40 KO MDSCs in combination with treatment of agonistic anti-CD40 antibodies did not yield additive or synergistic effects on the restoration of ex vivo proliferation and the decrease of Foxp3 expression by tumor-specific T cells that were reisolated from recipient mice. The result suggests that agonistic anti-CD40 antibodies disrupted CD40/CD40L ligation between MDSCs and Tregs, leading to a decrease in Treg expansion.
In the OVA-B16 tumor model, CD115-specific depletion was induced in MaFIA tumor-bearing mice by the treatment of AP20187. Although other CD115-expressing cells were also depleted, reconstitution of WT, but not CD40-deficient, MDSCs alone was sufficient to reestablish a high number of OT-II Tregs in the tumor, the ex vivo hypoproliferative response of OT-II T cells, and tumor growth to the level similar to those of MaFIA mice without CD115 depletion. Taken together, the data obtained from these studies using two complementary approaches strongly suggest that CD40 expression on MDSCs plays an important role in the establishment of immune tolerance in tumor-bearing mice through the expansion of Tregs.
At first glance, our findings seem to be contradictory to the well-established paradigm that CD40 triggering induces and enhances adaptive immunity. However, the outcome of CD40 ligation may vary, depending on the cell type that expresses CD40. Ligation on professional antigen-presenting cells, such as dendritic cells, induces immune activation, whereas on MDSC, it may promote the expansion of Tregs and is required for the suppression of the tumor-specific T-cell immune response. Furthermore, engagement of CD40L on T cells in an environment where the local cytokine milieu is rich in suppressive cytokines, such as IL-10 and TGF-β (25
), may favor Treg expansion. The use of agonistic anti-CD40 in this study may exert dual functions: blockade of the CD40/CD40L interaction between MDSCs and T cells, thereby preventing the activation and expansion of Tregs, and induction of maturation of dendritic cells or other antigen-presenting cells, thereby preventing the establishment of tolerance. Furthermore, we have observed an additional benefit of anti-CD40 treatment, the differentiation of MDSCs into dendritic cells or macrophages.3
Immune-based therapy remains one of the most promising options for the treatment of advanced metastatic cancer. However, therapeutic efficacy is severely compromised by the immune tolerance and suppression associated with advanced malignancy. In this report, we show that CD40 expression by MDSCs is required for MDSC suppressive activity and Treg expansion in tumor-bearing mice. Our findings reveal a novel role for CD40 in tumor-bearing hosts. The use of agonistic anti-CD40 in conjunction with immune-based therapy may represent a novel approach to not only to block MDSC-mediated Treg activation and expansion but to also intervene in MDSC-mediated immune suppression in hosts with advanced malignancies.