Faculty Bibliography

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.

Staphylococcus aureus enterotoxins (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. aureus and its enterotoxins induce enhanced expression of oncogenic mir-155 providing mechanistic insight into the role of S. aureus in CTCL. Our findings support that environmental stimuli such as bacteria can fuel disease progression in CTCL.

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

The advent of COVID-19 vaccines will play a major role in helping to end the pandemic that has killed millions worldwide. Vaccine candidates have demonstrated robust humoral responses and have protected against infection. However, efficacy trials were focused on individuals with no prior exposure to SARS-CoV-2, and, as a result, little is known about immune responses induced by these mRNA vaccines in individuals who recovered from COVID-19. Here, we evaluated immune responses in 32 subjects who received two-dose BNT162b2 mRNA vaccination. In individuals naive to SARS-CoV-2, we observed robust increases in humoral and antigen-specific antibody-secreting cell (ASC) responses following each dose of vaccine, whereas individuals with prior exposure to SARS-CoV-2 demonstrated strong humoral and antigen-specific ASC responses to the first dose but muted responses to the second dose of the vaccine for the time points studied. These data highlight an important gap in our knowledge and may have major implications for how these vaccines should be used to prevent COVID-19.

RATIONALE/BACKGROUND:Microbiome studies of the lower airway based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS:Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. MEASUREMENTS/METHODS:We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole genome (WGS) and RNA metatranscriptome sequencing. Short chain fatty acids (SCFA) were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. MAIN RESULTS/RESULTS:Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFAs levels were compared with WGS and metatranscriptome. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS:Functional characterisation of the lower airway microbiota through metatranscriptome identify metabolically active organisms capable of producing metabolites with immunomodulatory capacity such as SCFAs.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.

Objective/UNASSIGNED:To investigate the humoral and cellular immune response to mRNA COVID-19 vaccines in patients with immune-mediated inflammatory diseases (IMIDs) on immunomodulatory treatment. Methods/UNASSIGNED:Established patients at NYU Langone Health with IMID (n=51) receiving the BNT162b2 mRNA vaccination were assessed at baseline and after second immunization. Healthy subjects served as controls (n=26). IgG antibody responses to the spike protein were analyzed for humoral response. Cellular immune response to SARS-CoV-2 was further analyzed using high-parameter spectral flow cytometry. A second independent, validation cohort of controls (n=182) and patients with IMID (n=31) from Erlangen, Germany were also analyzed for humoral immune response. Results/UNASSIGNED:Although healthy subjects (n=208) and IMID patients on biologic treatments (mostly on TNF blockers, n=37) demonstrate robust antibody responses (over 90%), those patients with IMID on background methotrexate (n=45) achieve an adequate response in only 62.2% of cases. Similarly, IMID patients do not demonstrate an increase in CD8+ T cell activation after vaccination. Conclusions/UNASSIGNED:In two independent cohorts of IMID patients, methotrexate, 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 methotrexate to increase the chances of immunization efficacy against SARS-CoV-2 as has been demonstrated for augmenting immunogenicity to other viral vaccines. KEY MESSAGES/UNASSIGNED:These results suggest that patients on methotrexate may need alternate vaccination strategies such as additional doses of vaccine, dose modification of methotrexate, or even a temporary discontinuation of this drug. Further studies will be required to explore the effect of these approaches on mRNA vaccine immunogenicity.