Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. Here we show that silencing of the master genomic organizer Satb1 results in enhanced antigen-specific T Follicular Helper (TFH) differentiation. Increased TFH thereby promoted antigen-specific intra-tumoral CD19+B220+ B cell responses and spontaneous TLS assembly upon ovarian tumor challenge. Mechanistically, Satb1 deficiency drives increased TFH formation through de-repression of ICOS and PD-1. Accordingly, TGF-β1-driven downregulation of Satb1 licenses activated human CD4+ T-cells for enhanced antigen-specific T Follicular Helper (TFH) differentiation. Furthermore, Satb1 deficiency abrogates the generation of PD-1highCXCR5+Foxp3+ T Follicular Regulatory (TFR) cells during the TFH differentiation process. Importantly, functional TFH cell accumulation, in the absence of Satb1 specifically in CD4+ T cells, resulted in corresponding isotype-switched B cell responses and spontaneous formation of TLS, while B cell depletion accelerated malignant progression. Our results indicate that the formation of TLS in cancer depends on enhanced B cell responses driven by TFH cells generated through Satb1 down-regulation.

The relationship between stem-like, tissue resident memory (TRM) and exhausted T cells at tumor beds is incompletely understood. We found that more than 50% of the CD8+ T cells in human ovarian carcinomas are TRM cells. RNA seq of TRM and their re-circulating counterparts infiltrating multiple human ovarian carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector and exhaustion markers, along with increased clonality and marked proliferative and lipid metabolism signatures. Interestingly, both populations showed very little overlap in TCR repertoire. Single cell analysis of TRM population showed that this is a heterogeneous population composed of different clusters defined by gene expression and TCR repertoire. A distinctive cluster shows higher expression of cytotoxic mediators and exhaustion markers, along with higher clonality and higher proliferation rate, with a TCR repertoire shared by CD103+TCF7+ stem-like cells. Single cell ATACseq showed that TRM and their counterpart have different chromatin structure and, within both populations also appear variables epigenetic landscapes. In addition, the presence of TRM is correlated with better prognosis, while tumor antigen-specific TRM T cells transferred into syngeneic tumor-bearing mice were more effective at delaying tumor growth than their tumor antigen-specific re-circulating counterparts. Together, our data indicate that productive immune pressure against malignant progression depends on a subset of CD8+ stem-like T cells differentiating into a narrow cluster of TRM T cells of ~300 TCR clones, which represent true tumor antigen-specific effector lymphocytes.

T Follicular Helper cells (TFH) provide both co-stimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. However, is not clear how TFH differentiation is regulated. We found that deficiency of the chromatin organizer Satb1 results in increased TFH formation in CD4Cre+Satb1flx/flx mice through up-regulation of the canonical TFH markers ICOS and PD-1 and suppression of Foxp3+PD-1highCXCR5+ T follicular regulatory (TFR) cells as well. Accordingly, CD4Cre+Satb1flx/flx mice, or RAG1−/− mice transferred with Satb1-deficient CD4+ T cells showed a dramatic accumulation of CD4+CXCR5+PD-1high upon ovarian tumor challenge, compared to their Satb1+ counterparts, which was associated with reduced tumor growth. Importantly, intratumoral administration of Satb1-deficient CD4+ T cells re-directed to target ovarian cancer cells through chimeric receptors, but not their Satb1+ counterparts, induce the formation of Tertiary Lymphoid Structures in most tumors. Conclusion Satb1 controls three mechanisms relevant for TFH differentiation and, subsequently, antigen-specific humoral responses; namely, PD- 1 expression, ICOS de-repression and TFR formation. Our results suggest a novel role for Satb1 as a major regulator of TFH differentiation and TLS during tumor formation.

Cutaneous T cell lymphoma (CTCL) is a clinically unmet need. Using conditional knockout mice, we found that ablation of the genomic organizer Special AT rich sequence binding protein 1 (Satb1) induces a progressively fatal lymphoma characterized by mature, skin homing, Notch activated CD4 and CD8 T cells. Mechanistically, Satb1 restrains Stat5 phosphorylation and the expression of skin homing chemokine receptors in mature T cells. Notably, SUV39H1 and 2 methyltransferase dependent epigenetic repression of SATB1 is universally found in human Sezary Syndrome, but not other peripheral T cell malignancies. Accordingly, H3K27 and H3K9 trimethylation occlude the SATB1 promoter in Sezary cells. Inhibition of SUV39H1 and 2 methyltransferases with novel drugs, unlike EZH2 inhibition, restores SATB1 expression, selectively abrogating the growth of primary Sezary cells more effectively than Romidepsin. Therefore, SATB1 acts as a tumor suppressor in mature T cells upon NOTCH1 deregulation, and inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides and Sezary syndrome.