Tissue-specific interferon-gamma levels drive regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer

Regulatory T cells (Tregs) are proposed to restrain anti-tumor T cell responses either directly or by suppression of antigen-presenting cells and can act in a tissue-specific manner. Treg abundance is typically reported as a measure of suppression, without consideration of Treg functional quality. Here, we find that Tregs suppress type 1 conventional dendritic cells (DC1) and thereby induce dysfunctional CD8+ T cell responses against lung cancer. This immunoregulatory effect is spatially coordinated within tissue-specific lymph node microniches and requires antigen-specific contact between DC1 and Tregs. Suppressive clonally expanded TH1-like effector Tregs were differentially induced in the lung lymph node in response to tissue-specific levels of interferon-gamma. In cancer patients, TH1-like Tregs but not CD8+/Treg ratios correlate with poor responses to checkpoint blockade immunotherapy. Thus, Tregs that adopt the interferon-gamma-dependent TH1-like effector state have increased potential to restrain tumor-reactive T cell responses and represent a critical barrier to productive anti-tumor immunity. For scRNA-seq of ex vivo-primed T cells, CD8+ OT-I T cells and ZsG+ DC1 sorted from tumor-draining mLN were cultured alone or in the presence of Tregs sorted from tumor-draining mLN (added at the 1:8 Treg:OT-I ratio) as described above. After the 3 day co-culture, cells were collected for sequencing. For scRNA-seq of Tregs, LN were mashed through a 70 μm filter into RPMI (GIBCO), then incubated with αCD16/CD32 (clone 93, BioLegend) in FACS buffer (PBS (GIBCO) with 1% FBS (Atlanta Biologicals) and 2 mM EDTA (Invitrogen)) for 15 min at 4°C, washed with FACS buffer and stained with a mix of αCD45-APC (1:200, clone 30-F11, Biolegend) and Total-seq A mouse hashtag antibody (1:100, clone 30-F11, Biolegend) in FACS buffer to label individual mice (hashtag labeling was only performed for tdLN, not naïve LN). Cells were then washed twice in FACS buffer and tdLN cells from multiple mice were pooled together for subsequent CD4+ T cell enrichment sing the mouse CD4+ T cell isolation kit (Miltenyi Biotec), following manufacturer’s instructions. Next, cells were stained for FACS and Tregs were sorted as previously described. Cells from ex vivo co-cultures and sorted Tregs were processed for scRNA-seq using the Seq-Well platform with second strand chemistry, as previously described (Gierahn et al., 2017; Hughes et al., 2020). Whole transcriptome libraries were barcoded and amplified using the Nextera XT kit (Illumina) and were sequenced on a Novaseq 6000 (Illumina). Hashtag oligo libraries were amplified as described previously and were sequenced on a Nextseq 550 (Stoeckius et al., 2018).