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Cutaneous T-cell lymphoma (CTCL) involves a clonal expansion of malignant cells accumulating in the skin, a primary barrier site. CTCL has long been hypothesized to be caused or perpetuated by chronic antigen stimulation due to unknown exposures. These antigenic triggers, defined as any element that may cause activation of malignant T cells through TCR signaling, have been hypothesized to range from chemicals to microbes. This review covers current evidence supporting chemical and microbial stimuli that may act as antigenic triggers of CTCL and summarizes novel areas of investigation, in which the potential antigenicity of the exposure is still unknown.

The relative and synergistic contributions of genetics and environment to interindividual immune response variation remain unclear, despite implications in evolutionary biology and medicine. Here we quantify interactive effects of genotype and environment on immune traits by investigating C57BL/6, 129S1 and PWK/PhJ inbred mice, rewilded in an outdoor enclosure and infected with the parasite Trichuris muris. Whereas cellular composition was shaped by interactions between genotype and environment, cytokine response heterogeneity including IFNγ concentrations was primarily driven by genotype with consequence on worm burden. In addition, we show that other traits, such as expression of CD44, were explained mostly by genetics on T cells, whereas expression of CD44 on B cells was explained more by environment across all strains. Notably, genetic differences under laboratory conditions were decreased following rewilding. These results indicate that nonheritable influences interact with genetic factors to shape immune variation and parasite burden.

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.

Antibiotics, among the most used medications in children, affect gut microbiome communities and metabolic functions. These changes in microbiota structure can impact host immunity. We hypothesized that early-life microbiome alterations would lead to increased susceptibility to allergy and asthma. To test this, mouse pups between postnatal days 5–9 were orally exposed to water (control) or to therapeutic doses of azithromycin or amoxicillin. Later in life, these mice were sensitized and challenged with a model allergen, house dust mite (HDM), or saline. Mice with early-life azithromycin exposure that were challenged with HDM had increased IgE and IL-13 production by CD4+T cells compared to unexposed mice; early-life amoxicillin exposure led to fewer abnormalities. To test that the microbiota contained the immunological cues to alter IgE and cytokine production after HDM challenge, germ-free mice were gavaged with fecal samples of the antibiotic-perturbed microbiota. Gavage of adult germ-free mice did not result in altered HDM responses, however, their offspring, which acquired the antibiotic-perturbed microbiota at birth showed elevated IgE levels and CD4+cytokines in response to HDM, and altered airway reactivity. These studies indicate that early-life microbiota composition can heighten allergen-driven Th2/Th17 immune pathways and airway responses in an age-dependent manner.

Antibiotics, among the most used medications in children, affect gut microbiome communities and metabolic functions. These changes in microbiota structure can impact host immunity. We hypothesized that early-life microbiome alterations would lead to increased susceptibility to allergy and asthma. To test this, mouse pups between postnatal days 5–9 were orally exposed to water (control) or to therapeutic doses of azithromycin or amoxicillin. Later in life, these mice were sensitized and challenged with a model allergen, house dust mite (HDM), or saline. Mice with early-life azithromycin exposure that were challenged with HDM had increased IgE and IL-13 production by CD4+T cells compared to unexposed mice; early-life amoxicillin exposure led to fewer abnormalities. To test that the microbiota contained the immunological cues to alter IgE and cytokine production after HDM challenge, germ-free mice were gavaged with fecal samples of the antibiotic-perturbed microbiota. Gavage of adult germ-free mice did not result in altered HDM responses, however, their offspring, which acquired the antibiotic-perturbed microbiota at birth showed elevated IgE levels and CD4+cytokines in response to HDM, and altered airway reactivity. These studies indicate that early-life microbiota composition can heighten allergen-driven Th2/Th17 immune pathways and airway responses in an age-dependent manner.

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 (MF ), 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 (SS), 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 patients with leukemic MF and SS, we combine T-cell receptor clonotyping with quantification of gene expression and cell surface markers at the single cell level. Our data reveal clonal evolution at a transcriptional and genetic level within the malignant populations of individual patients. We highlight highly consistent transcriptional signatures delineating skin- and blood-derived malignant T cells. Analysis of these 2 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 subclones.

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.

ObjectiveCancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood.DesignWe quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI–) on STAT3 activation (IL-6 vs IL-11).ResultsThe analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts.ConclusionAltogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.

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.

Deficient expression of SATB1 hampers thymocyte development and results in inept T-cell lineages. Recent data implicate dysregulated SATB1 expression in the pathogenesis of mycosis fungoides, the most frequent variant of cutaneous T-cell lymphoma. Here, we report on a disease stage-associated decrease of SATB1 expression and an inverse expression of STAT5 and SATB1 in situ. STAT5 inhibited SATB1 expression through induction of microRNA-155. Decreased SATB1 expression triggered enhanced expression of IL-5 and IL-9 (but not IL-6 and IL-32), whereas increased SATB1 expression had the opposite effect, indicating that the microRNA-155 target SATB1 is a repressor of IL-5 and IL-9 in malignant T cells. In accordance, inhibition of STAT5 and its upstream activator JAK3 triggered increased SATB1 expression and a concomitant suppression of IL-5 and IL-9 expression in malignant T cells. In conclusion, we provide a mechanistic link between the proto-oncogenic JAK3/STAT5/microRNA-155 pathway, SATB1, and cytokines linked to CTCL severity and progression, indicating that SATB1 dysregulation is involved in cutaneous T-cell lymphoma pathogenesis.

Cutaneous T-cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic, and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome showed a highly heterogeneous landscape of genetic perturbations, and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway previously implicated in cutaneous T-cell lymphoma pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of cutaneous T-cell lymphoma. Using this mouse model, we show that T-cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient–derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk–dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient–derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk–dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

Diffuse large B cell lymphoma (DLBCL) is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-κB activity, a role for oncogenic lesions that activate the alternative NF-κB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-κB pathway, occurs in ∼15% of DLBCLs and that it often coexists with BCL6translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-κB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-κB pathway in DLBCL development.

Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus (SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.

Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus(SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.

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.

Antigen receptor editing—a process of secondary rearrangements of antigen receptor genes in autoreactive lymphocytes—is a well-established tolerance mechanism in B cells, whereas its role in T cells remains controversial. Here, we investigated this issue using a novel Tcra knock-in locus, which ensured appropriate timing of TCRα expression and allowed secondary rearrangements. Under these conditions the only response to self-antigen that could be unambiguously identified was negative selection of CD4/CD8 double positive thymocytes. No evidence could be obtained for antigen-induced TCR editing, whereas replacement of the transgenic TCRα chain by ongoing gene rearrangement occurred in some cells irrespective of the presence or absence of self-antigen.

Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.

Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.

The use of coronavirus disease 2019 (COVID-19) vaccines will play the major role in helping to end the pandemic that has killed millions worldwide. COVID-19 vaccines have resulted in robust humoral responses and protective efficacy in human trials, but efficacy trials excluded individuals with a prior diagnosis of COVID-19. As a result, little is known about how immune responses induced by mRNA vaccines differ in individuals who recovered from COVID-19. Here, we evaluated longitudinal immune responses to two-dose BNT162b2 mRNA vaccination in 15 adults who had experienced COVID-19, compared to 21 adults who did not have prior COVID-19. Consistent with prior studies of mRNA vaccines, we observed robust cytotoxic CD8+T cell responses in both cohorts after the second dose. Furthermore, SARS-CoV-2–naive individuals had progressive increases in humoral and antigen-specific antibody-secreting cell (ASC) responses after each dose of vaccine, whereas SARS-CoV-2–experienced individuals demonstrated strong humoral and antigen-specific ASC responses to the first dose but these responses were not further enhanced after the second dose of the vaccine at the time points studied. Together, these data highlight the relevance of immunological history for understanding vaccine immune responses and may have implications for personalizing mRNA vaccination regimens used to prevent COVID-19, including for the deployment of booster shots.

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders.

Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that VH to JH joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the DH-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular VH gene family or JH element in VHJH junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.

The complement system enhances antibody responses to T-dependent antigens, but paradoxically, deficiencies in C1 and C4 are strongly linked to autoantibody production in humans. In mice, disruption of the C1qa gene also results in spontaneous autoimmunity. Moreover, deficiencies in C4 or complement receptors 1 and 2 (CR1/CR2) lead to reduced selection against autoreactive B cells and impaired humoral responses. These observations suggest that C1 and C4 act through CR1/CR2 to enhance humoral immunity and somehow suppress autoimmunity. Here we report high titers of spontaneous antinuclear antibody (ANA) in C4−/− mice. This systemic lupus erythematosus–like autoimmunity is highly penetrant; by 10 mo of age, all C4−/− females and most males produced ANA. In contrast, titers and frequencies of ANA in Cr2−/− mice, which are deficient in CR1 and CR2, never rose significantly above those in normal controls. Glomerular deposition of immune complexes (ICs), glomerulonephritis, and splenomegaly were observed in C4−/− but not Cr2−/− mice. C4−/−, but not Cr2−/−, mice accumulate activated T and B cells. Clearance of circulating ICs is impaired in preautoimmune C4−/−, but not Cr2−/−, mice. C4 deficiency causes spontaneous, lupus-like autoimmunity through a mechanism that is independent of CR1/CR2.

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.

The phosphatidylinositol 3-kinase (PI3K) signaling cascade downstream of the B cell receptor (BCR) signalosome is essential for B cell maturation. Proper signaling strength is maintained through the PI3K negative regulator phosphatase and tensin homolog (PTEN). Although a role for microRNA (miRNA)-dependent control of the PTEN-PI3K axis has been described, the contribution of individual miRNAs to the regulation of this crucial signaling modality in mature B lymphocytes remains to be elucidated. Our analyses reveal that ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and dampening of the PI3K pathway in mature B cells. This dysregulation has a profound impact on the survival of B lymphocytes and results in increased class switch recombination and decreased plasma cell differentiation. Furthermore, we demonstrate that ablation of one copy of Ptenis sufficient to ameliorate the phenotypes associated with miR-29 loss. Our data suggest a critical role for the miR-29-PTEN-PI3K regulatory axis in mature B lymphocytes.

Staphylococcus aureus is responsible for various diseases in humans, and recurrent infections are commonly observed. S. aureus produces an array of bicomponent pore-forming toxins that target and kill leukocytes, known collectively as the leukocidins. The contribution of these leukocidins to impair the development of anti–S. aureus adaptive immunity and facilitate reinfection is unclear. Using a murine model of recurrent bacteremia, we demonstrate that infection with a leukocidin mutant results in increased levels of anti–S. aureus antibodies compared with mice infected with the WT parental strain, indicating that leukocidins negatively impact the generation of anti–S. aureus antibodies in vivo. We hypothesized that neutralizing leukocidin-mediated immune subversion by vaccination may shift this host-pathogen interaction in favor of the host. Leukocidin-immunized mice produce potent leukocidin-neutralizing antibodies and robust Th1 and Th17 responses, which collectively protect against bloodstream infections. Altogether, these results demonstrate that blocking leukocidin-mediated immune evasion can promote host protection against S. aureus bloodstream infection.

Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes—including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells—and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive.

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.

ABSTRACTStaphylococcus aureusand its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8+T cells play a crucial role in anti-cancer responses and high CD8+T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8+T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8+T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivoby inhibiting CD8+T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureusmay contribute to cancer immune evasion and disease progression in CTCL.

Hymes: Celgene: Consultancy. Odum:Micreos human Health B.V: Consultancy. Geskin:Merck: Other: Supported/Contracted Research; UpToDate: Patents & Royalties: Royalty, Receipt of Intellectual Property Rights / Patent Holder; Mallinckrodt: Consultancy, Honoraria, Other: Supported/Contracted Research; Helsinn: Consultancy, Honoraria, Other: Supported/Contracted Research; Stratpharma: Other: Supported/Contracted Research; Medivir: Consultancy, Honoraria; Medscape: Speakers Bureau; Actelion: Other: Supported/Contracted Research.

It has been proposed that bacteria play a direct role in progression of cutaneous T cell lymphoma (CTCL), although definitive evidence is missing, and the underlying mechanism of how microbes contribute to disease progression remains unknown. The skin of CTCL patients is frequently colonized with Staphylococcus aureus (S. aureus)strains and infections with hospital and community associated strains of S. aureusare a frequent cause of morbidity and mortality among patients with advanced CTCL. Here we provide a comprehensive analysis of the association between CTCL and S. aureuscolonization, and use our unique pre-clinical animal model of CTCL to determine the cause-effect relationship between skin-associated S. aureusand CTCL progression.

ABSTRACTStaphylococcus aureusis implicated in disease progression in cutaneous T-cell lymphoma (CTCL). Here, we demonstrate that malignant T cell lines derived from CTCL patients as well as primary malignant CD4+T cells from Sézary syndrome patients are considerably more resistant to alpha-toxin-induced cell death than their non-malignant counterparts. Thus, in a subset of Sézary syndrome patients the ratio between malignant and non-malignant CD4+T cells increases significantly following exposure to alpha-toxin. Whereas toxin-induced cell death is ADAM10 dependent in healthy CD4+T cells, resistance to alpha-toxin in malignant T cells involves both downregulation of ADAM10 as well as other resistance mechanisms. In conclusion, we provide first evidence that Staphylococcus aureusderived alpha-toxin can tilt the balance between malignant and non-malignant CD4+T cells in CTCL patients. Consequently, alpha-toxin may promote disease progression through positive selection of malignant CD4+T cells, identifying alpha-toxin as a putative drug target in CTCL.

The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Ighlocus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Ighlocus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Ighlocus.

B cell development is a highly regulated process that requires stepwise rearrangement of immunoglobulin genes to generate a functional B cell receptor (BCR). The polycomb group protein BMI1 is required for B cell development, but its function in developing B cells remains poorly defined. We demonstrate that BMI1 functions in a cell-autonomous manner at two stages during early B cell development. First, loss of BMI1 results in a differentiation block at the pro-B cell to pre-B cell transition due to the inability of BMI1-deficient cells to transcribe newly rearranged Ighgenes. Accordingly, introduction of a pre-rearranged Ighallele partially restored B cell development in Bmi1−/−mice. In addition, BMI1 is required to prevent premature p53 signaling, and as a consequence, Bmi1−/−large pre-B cells fail to properly proliferate. Altogether, our results clarify the role of BMI1 in early B cell development and uncover an unexpected function of BMI1 during VDJ recombination.