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Cutaneous T-cell lymphoma (CTCL) is characterized by malignant T-cells proliferating in a unique tumor microenvironment (TME) dominated by keratinocytes. Skin colonization and infection by Staphylococcus aureus(S. aureus) is a common cause of morbidity and suspected of fueling disease activity. Here we show that expression of HLA-DR, high-affinity receptors for Staphylococcal enterotoxins (SE), by keratinocytes correlates with IFN-γ expression in the TME. Importantly, IFN-γ induces HLA-DR, SE-binding, and SE-presentation by keratinocytes to malignant T-cells from Sézary syndrome (SS) patients, and malignant and non-malignant T-cell lines derived from SS and Mycosis fungoides patients. Likewise, preincubation of keratinocytes with supernatant from patient-derived SE-producing S. aureustriggers proliferation in malignant T-cells and cytokine release (including IL-2), when cultured with non-malignant T-cells. This is inhibited by pre-treatment with engineered bacteriophage S. aureus-specific endolysins. Furthermore, mutations in the HLA-DR binding sites of SE type-A, and siRNA-mediated knockdown of Janus Kinase-3 (JAK3) and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that, upon exposure to patient-derived S. aureusand SE, keratinocytes stimulate IL-2Rγ/JAK3-dependent proliferation of malignant and non-malignant T-cells in an environment with non-malignant T-cells. These findings suggest that keratinocytes in the TME play a key role in S. aureusmediated disease activity in CTCL.

•Enterotoxins from S aureusbacteria induce drug resistance in primary malignant T cells in SS.•Targeting bacteria, their toxins, and downstream signaling pathways in malignant T cells abrogate the induction of drug resistance.

Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.

Staphylococcus aureusis suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureusskin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureusisolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureusbacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureusis profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureusinduction of IFNγand the IFNγ-inducible chemokine CXCL10in healthy skin. Whereas patient-derived S. aureusstimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureuson activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureusand blocks their potential tumor-promoting effects on malignant T cells.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA–mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

Staphylococcus aureusenterotoxins (SE) are believed to fuel disease activity in cutaneous T-cell lymphoma (CTCL). Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sezary syndrome, the most common forms of CTCL. Yet, it remains incompletely characterized how SE fuel disease activity. Here, we show that SE induce expression of the oncogenic microRNA mir-155 in primary malignant T cells. Thus, SE and S. aureus-isolates from lesional patient skin induce mir-155 expression, at least partly, through the IL-2Rg/JAK/STAT5 pathway, and the effect is augmented by the presence of non-malignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus,express pY-STAT5, and display enhanced mir-155 expression, when compared with non-lesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased pY-STAT5 and mir-155 expression in lesional skin in two patients with Sezary syndrome. In conclusion, we demonstrate that S. aureusand its enterotoxins induce enhanced expression of oncogenic mir-155 providing mechanistic insight into the role of S. aureusin CTCL. Our findings support that environmental stimuli such as bacteria can fuel disease progression in CTCL.

Cutaneous T cell lymphoma (CTCL) is a heterogeneous group of mature T cell neoplasms characterized by the accumulation of clonal malignant CD4+ T cells in the skin. The most common variant of CTCL, Mycosis Fungoides, is confined to the skin in early stages but can be accompanied by extracutaneous dissemination of malignant T cells to the blood and lymph nodes in advanced stages of disease. Sézary Syndrome, a leukemic form of disease is characterized by significant blood involvement. Little is known about the transcriptional and genomic relationship between skin and blood residing malignant T cells in CTCL. To identify and interrogate malignant clones in matched skin and blood from leukemic MF and SS patients, we combine T cell receptor clonotyping, with quantification of gene expression and cell surface markers at the single cell level. Our data reveals clonal evolution at a transcriptional and genetic level within the malignant populations of individual patients. We highlight highly consistent transcriptional signatures delineating skin-derived and blood-derived malignant T cells. Analysis of these two populations suggests that environmental cues, along with genetic aberrations, contribute to transcriptional profiles of malignant T cells. Our findings indicate that the skin microenvironment in CTCL promotes a transcriptional response supporting rapid malignant expansion, as opposed to the quiescent state observed in the blood, potentially influencing efficacy of therapies. These results provide insight into tissue-specific characteristics of cancerous cells and underscore the need to address the patients’ individual malignant profiles at the time of therapy to eliminate all sub-clones.