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The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Supplementary Figures and Tables.

The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Purpose: Chimeric antigen receptor (CAR) T cells have shown promise against solid tumors, but their efficacy has been limited, due in part, to immunosuppression by CD4+FoxP3+ regulatory T cells (Tregs). Although lymphodepletion is commonly used to deplete Tregs, these regimens are nonspecific, toxic, and provide only a narrow window before Tregs repopulate hosts. Importantly, CARs have also been shown to inadvertently potentiate Tregs by providing a source of IL2 for Treg consumption. We explored whether disruption of the IL2 axis would confer efficacy against solid tumors without the need for lymphodepletion. Experimental Design: We developed second- (CD28z) and third- (CD28-4-1BBz) generation CARs targeting EGFRvIII. To eliminate secretion of IL2, 2 amino acid substitutions were introduced in the PYAP Lck–binding motif of the CD28 domain (ΔCD28). We evaluated CARs against B16 melanomas expressing EGFRvIII. Results: CD28z CARs failed to engraft in vivo. Although 4-1BB addition improved expansion, CD28-4-1BBz CARs required lymphodepletion to treat solid tumors. CARs deficient in Lck signaling, however, significantly retarded tumor growth without a need for lymphodepletion and this was dependent on inclusion of 4-1BB. To evaluate CAR vulnerability to Tregs, we lymphodepleted mice and transferred CARs alone or with purified Tregs. Cotransfer with Tregs abrogated the efficacy of CD28-4-1BBz CARs, whereas the efficacy of ΔCD28-4-1BBz CARs remained unperturbed. Conclusions: In the absence of lymphodepletion, CARs targeting solid tumors are hindered by Treg immunosuppression and poor persistence. Here, CARs were modified to circumvent Treg suppression and to simultaneously improve in vivo engraftment. Modified CARs treated solid tumors without a need for lymphodepletion.