Faculty Bibliography

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterised by accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms hampering the quality of life and are entry sites for bacterial infection - a major cause of morbidity and mortality in advanced disease. What drives the pathological processes that compromise the skin barrier remains unknown. Here, we report on increased transepidermal water loss and compromised expression of 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 IL-13, IL-22 and Oncostatin M) that activate STAT3 signalling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, repression of filaggrins could be counteracted by a cocktail of antibodies targeting these cytokines/receptors, by siRNA-mediated knockdown of JAK1/STAT3, and by JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, Tofacitinib, increases filaggrin expression in lesional skin from mycosis fungoides patients. Taken together, these findings indicate that malignant T cells secrete cytokines, which induce skin barrier defects through 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 CTCL patients with advanced disease and a compromised skin barrier.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CL^pro (M^pro). The drug candidate PF-00835231 is the active compound of the first anti-3CL^pro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CL^pro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549^+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549^+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549^+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.

Background/Purpose: Patients with immune mediated inflammatory disorders (IMIDs) have an inherently heightened susceptibility to infection and may be considered high risk for developing COVID-19. While data regarding the COVID-19 vaccine's immunogenicity in an immunocompetent adult population is rapidly emerging, the ability of IMID patients to adequately respond to these vaccines is not known. Here, we investigate the humoral and cellular immune response to mRNA COVID-19 vaccines in patients with IMIDs on immunomodulatory treatment Methods: Patients with immune mediated inflammatory disorders (IMIDs) have an inherently heightened susceptibility to infection and may be considered high risk for developing COVID-19. While data regarding the COVID-19 vaccine's immunogenicity in an immunocompetent adult population is rapidly emerging, the ability of IMID patients to adequately respond to these vaccines is not known. Here, we investigate the humoral and cellular immune response to mRNA COVID-19 vaccines in patients with IMIDs on immunomodulatory treatment.
Result(s): The NY cohort baseline characteristics are found in Table 1. The Erlangen cohort consisted of 182 healthy subjects, 11 subjects with IMID receiving TNFi monotherapy, and 20 subjects with IMID on MTX monotherapy. In both cohorts, healthy individuals and those with IMID not on MTX were similar in age, while those IMID patients receiving MTX were generally older. In the NY cohort, of the healthy participants, 96.3% demonstrated adequate humoral immune response. Patients with IMID not on MTX achieved a similar rate of high antibody response rate (91.8%), while those on MTX had a lower rate of adequate humoral response (75.0%) (Figure 1A). This remains true even after the exclusion of patients who had evidence of prior COVID-19 infection (P= 0.014). Of note, 3 out of the 4 IMID patients receiving rituximab did not produce an adequate response. Similarly, in the Erlangen validation cohort, 98.3% of healthy controls, 90.9% of patients with IMID receiving TNFi monotherapy, and 50.0% receiving MTX monotherapy achieved adequate immunogenicity (Figure 1B). These differences remain significant when combining the cohorts, using a stricter definition of adequate response, and in a subgroup analysis by age. Cellular response was also analyzed in a subgroup of the NY cohort before and after second vaccination. Activated CD8+ T cells (CD8+ T cells expressing Ki67 and CD38) and the granzyme B-producing subset of these activated CD8+ T cells, were induced in immunocompetent adults and those with IMID not on MTX, but not induced in patients receiving MTX (Figure 2).
Conclusion(s): In two independent cohorts of IMID patients, MTX, a widely used immunomodulator for the treatment of several IMIDs, adversely affected humoral and cellular immune response to COVID-19 mRNA vaccines. Although precise cut offs for immunogenicity that correlate with vaccine efficacy are yet to be established, our findings suggest that different strategies may need to be explored in patients with IMID taking MTX to increase the chances of immunization efficacy against SARS-CoV-2, as has been demonstrated for other viral vaccines

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. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C ^stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C ^stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Formula presented] Disclosures: 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.
Copyright

[Figure: see text].

B lymphocytes play a central role in host immune defense. B cell receptor (BCR) signaling regulates survival, proliferation, and differentiation of B lymphocytes. Signaling through the BCR signalosome is a multi-component cascade that is tightly regulated and is important in the coordination of B cell differentiation and function. At different stages of development, B cells that have BCRs recognizing self are eliminated to prevent autoimmunity. microRNAs (miRNAs) are small single-stranded non-coding RNAs that contribute to post-transcriptional regulation of gene expression and have been shown to orchestrate cell fate decisions through the regulation of lineage-specific transcriptional profiles. Studies have identified miRNAs to be crucial for B cell development in the bone marrow and their subsequent population of the peripheral immune system. In this review, we focus on the role of miRNAs in the regulation of BCR signaling as it pertains to B lymphocyte development and function. In particular, we discuss the most recent studies describing the role of miRNAs in the regulation of both early B cell development and peripheral B cell responses and examine the ways by which miRNAs regulate signal downstream of B cell antigen receptor to prevent aberrant activation and autoimmunity.

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.