Jintao Xu

Inflammatory brain damage is an important factor contributing to mortality or lasting neurological sequelae in CNS infections, such as cryptococcal meningoencephalitis (CM), but little is known about natural immunoregulatory mechanisms in the infected brain. Here we report that regulatory T cells (Tregs) are a central immunoregulatory component in CM. Tregs are present within the CNS in both human CM patients and in the experimental murine CM. Treg-depletion exacerbates Th1-driven brain inflammation and neurological symptoms, accelerating mortality, despite enhanced fungal clearance in mouse CM. Aligned brain NanoString, scRNA-seq, and flow cytometry analyses revealed that Tregs reduce brain inflammation, especially T-cell recruitment activation and differentiation, shielding the brain from neurological damage. The major CNS-Treg recruitment appears to be chemokine receptor CCR8-mediated, supporting the importance of the CCR8/CCL1 axis in Treg recruitment into the brain. Tregs in CM are major producers of anti-inflammatory IL-10 and the growth factor Amphiregulin (Areg), which is implicated in neuronal repair. IL-10 deletion in murine CM phenocopies Treg depletion. Areg deletion showed no survival effect; however, IFN-γ production by effector T cells in the brain was reduced, supporting Aregs as a potential internal regulator of Treg immunosuppressive function. Finally, a Treg-enhancing immunotherapy using low dose IL-2-immune complex treatment substantially improves mouse survival and neurological outcomes. Together, we identified that Tregs are crucial for neuronal protection in CM, predominantly via IL-10 production, the CCR8-CCL1 axis is an important CNS-Treg recruitment mechanism and Treg enhancement is a potential therapeutic strategy to mitigate immunopathology during CM, and possibly other forms of meningitis.

Supplementary Figure S8: Flow cytometric analysis of the phenotype of tumor-infiltrating CD8+ T cells between tumors treated with or without histotripsy or Trametinib.

Supplementary Figure S6: Effect of Trametinib on HIF-1α expression after 4 hours of in vitro exposure to cultured B16F10.

Supplementary Figure S4: CD8+ T cell presence within TDLN and tumors.

Supplementary Figure S5: CD8+ T cell infiltration guided by CXCR3 is also observed in the YUMM1.7 melanoma cell line.

Supplementary Figure S3: Validation of VG1 Antibody by immunofluorescence.

Supplementary Figure S1: Histotripsy causes abrogation of tumor hypoxia and subversion of HIF signaling.

Supplementary Figure S2: Transient HIF-1α downregulation is also observed in the YUMM1.7 melanoma cell line.

Histotripsy-focused ultrasound treatment gives rise to systemic antitumor immune responses. We investigated whether histotripsy effects on immunosuppressive tumor hypoxia were a potential mechanism for these immunostimulatory effects. Immunocompetent or CD8-deficient C57BL/6 mice with flank B16F10 or YUMM1.7 melanoma tumors underwent sham or subtotal histotripsy. Tumor growth, immune cell infiltration, and intratumoral hypoxia responses were examined using flow cytometry and fluorescence microscopy. Chemokine receptor CXCR3 and hypoxia-inducible factor-1α (HIF1α) were intercepted with antibodies and inhibitors to assess their roles in immune responses after histotripsy. Histotripsy-treated tumors exhibited rapid loss of intratumoral hypoxia and suppression of HIF1α and downstream prosurvival proteins. Histotripsy was followed by intratumoral upregulation of the CXCR3 ligand CXCL10 and CXCR3+/CD8+ T-cell infiltration. Tumor growth inhibition by histotripsy was significantly diminished in CD8-deficient mice and mice receiving anti-CXCR3 mAb. Post-histotripsy inhibition of hypoxia and tumor growth eventually receded in parallel with cessation of CD8+ T-cell influx, and pharmacologic HIF1α suppression with the MEK inhibitor trametinib substantially augmented the therapeutic effects of histotripsy. Transient abrogation of intratumoral hypoxia and HIF1α-associated hypoxia responses is mechanistically linked with intratumoral infiltration of activated CXCR3+/CD8+ T cells via CXCL10–CXCR3 engagement. These findings suggest that the immune effects of histotripsy may be regulated by hypoxia abrogation and that pharmacologic hypoxia abrogation could potentiate the immunotherapeutic effects of histotripsy.

Supplementary Figure S7: Flow cytometric analysis of immune cell infiltration in tumors treated with or without histotripsy or Trametinib.