Phosphatidylinositol 3-kinase delta (PI3KCD) is a critical signaling enzyme for B cell development, activation, function and immune regulation. Gain-of-function mutations in PI3KCD result in the congenital immunodeficiency known as Activated PI3KCD Syndrome (APDS). APDS patients are prone to repeated infections and other serious clinical manifestations. Here, we determine how B cell-intrinsic expression of the APDS-associated PI3KCDE1021K mutation impacts immune responses to the protozoan parasite Trypanosoma congolense. PI3KCDE1021K/B mice exhibit a significant expansion of IL10-expressing B cells within the spleen and peritoneal cavity, which was associated with impaired control of T. congolense infection. Despite the generation of robust germinal center, plasma cell and antibody responses, PI3KCDE1021K/B mice show elevation in the first wave of parasitemia and increased mortality. We further characterize the phenotype of the expanded IL10-producing B cell population in PI3KCDE1021K/B mice, which show hallmarks of innate-like regulatory B cells (Breg) and expression of multiple inhibitory molecules. This Breg expansion is associated with reduced IFNγ/IL10 ratio, reduced TNFα production and impaired activation of myeloid cells, likely compromising the innate response to infection. These findings highlight the profound impact of dysregulated PI3KCD activity on regulatory B cells that can functionally impair innate immune responses controlling a systemic parasite protozoan disease.

Abstract Background Gut B cells maintain homeostasis by producing IgA antibodies that control the entry of commensal bacteria and pathogens. An increase in the number of B cells in the inflamed colon was detected in ulcerative colitis (UC) patients. Pro-inflammatory IgG antibodies binding to commensal bacteria are significantly elevated in UC, promoting intestinal inflammation. Aims To determine the effects of intestinal inflammation on the B cell compartment using a preclinical model of UC. Methods 32 CD-1 WT males were treated for 6 days with 2.5% (w/v) dextran sulfate sodium (DSS), followed by 8 days of recovery (regular water). Disease activity index was monitored daily. Distal colonic mRNA expression of cytokine (tumor necrosis [TNF-α], B-cell activating factor [Baff]) and factors associated with B cell attraction (chemokine (C-X-C motif) ligand [CXCL]13) were assessed by qRT-PCR. Localization using B cells phenotype (B220, cluster of differentiation (CD) 19, CD138, GL7, and IgD) and activation markers (CD80 & CD86) were assessed by flow cytometry and immunofluorescence (IF) in Peyers’ patches, distal colon, and mesenteric lymph nodes (MLN). Levels of IgA, M, G1, G2b, and G2c in colon tissue and serum were determined by ELISA. Results In colitic mice, TNF-α, Baff, and CXCL-13 mRNA expression was significantly upregulated, and immunostaining demonstrated several aggregated sites of B cells with the phenotype of B220+, IgD+, and CD19+in the lamina propria. Aggregated B cells were positive for co-expression of IgD and CD19 markers and negative for expression of IgA and IgG antibodies. Flow cytometry data showed a significant increase in the percentage of B220+ and CD19+ B cells within the CD45+ live cell population in colitic mice’s distal colon and MLNs. In addition, in colitic mice, the percentage of B cells expressing CD86 significantly increased in the distal colon, Peyers’ patches, and MLN. Furthermore, in colitic conditions, the percentage of B cells producing IgG1 was increased in the Peyers’ patches and distal part of the colon but only in Peyers’ patches for IgA-producing B cells. In colitic mice, IgA/IgG2c ratio in colon tissue decreased, and serum IgG2b, IgG2c, and IgG1 antibody levels increased, with no difference for IgA and IgM. Conclusions Inflammatory conditions lead to B cell activation and recruitment with aggregated naïve B cells in the lamina propria that don’t switch to IgG or IgA. In colitis, systemic IgG subtypes increase, while Peyer’s patches may actively produce B cells expressing IgA or IgG1 during intestinal inflammation. These findings may guide the development of new UC therapies. Funding Agencies CCC

Abstract Background Gut B cells maintain homeostasis by producing IgA antibodies that control the entry of commensal bacteria and pathogens. An increase in the number of B cells in the inflamed colon was detected in ulcerative colitis (UC) patients. Pro-inflammatory IgG antibodies binding to commensal bacteria are significantly elevated in UC, promoting intestinal inflammation. Aims To determine the effects of intestinal inflammation on the B cell compartment using a preclinical model of UC. Methods 32 CD-1 WT males were treated for 6 days with 2.5% (w/v) dextran sulfate sodium (DSS), followed by 8 days of recovery (regular water). Disease activity index was monitored daily. Distal colonic mRNA expression of cytokine (tumor necrosis [TNF-α], B-cell activating factor [Baff]) and factors associated with B cell attraction (chemokine (C-X-C motif) ligand [CXCL]13) were assessed by qRT-PCR. Localization using B cells phenotype (B220, cluster of differentiation (CD) 19, CD138, GL7, and IgD) and activation markers (CD80 & CD86) were assessed by flow cytometry and immunofluorescence (IF) in Peyers’ patches, distal colon, and mesenteric lymph nodes (MLN). Levels of IgA, M, G1, G2b, and G2c in colon tissue and serum were determined by ELISA. Results In colitic mice, TNF-α, Baff, and CXCL-13 mRNA expression was significantly upregulated, and immunostaining demonstrated several aggregated sites of B cells with the phenotype of B220+, IgD+, and CD19+in the lamina propria. Aggregated B cells were positive for co-expression of IgD and CD19 markers and negative for expression of IgA and IgG antibodies. Flow cytometry data showed a significant increase in the percentage of B220+ and CD19+ B cells within the CD45+ live cell population in colitic mice’s distal colon and MLNs. In addition, in colitic mice, the percentage of B cells expressing CD86 significantly increased in the distal colon, Peyers’ patches, and MLN. Furthermore, in colitic conditions, the percentage of B cells producing IgG1 was increased in the Peyers’ patches and distal part of the colon but only in Peyers’ patches for IgA-producing B cells. In colitic mice, IgA/IgG2c ratio in colon tissue decreased, and serum IgG2b, IgG2c, and IgG1 antibody levels increased, with no difference for IgA and IgM. Conclusions Inflammatory conditions lead to B cell activation and recruitment with aggregated naïve B cells in the lamina propria that don’t switch to IgG or IgA. In colitis, systemic IgG subtypes increase, while Peyer’s patches may actively produce B cells expressing IgA or IgG1 during intestinal inflammation. These findings may guide the development of new UC therapies. Funding Agencies:

The Canadian Society for Immunology (CSI) established a formal Equity, Diversity, and Inclusion (EDI) Committee with the goal of providing EDI advocacy and leadership within the CSI, as well as in the broader scientific community. A first task of this committee was to review the publicly available historical data on gender representation within the CSI’s membership, leadership, award recipients, and conference chairs/presenters as a step in establishing a baseline reference point and monitoring the trajectory of future success in achieving true inclusion. We found that, except for overall membership and a specific subset of awards, all categories showed a historical bias toward men, particularly prior to 2010. Bias persists in various categories, evident even in recent years. However, we note an encouraging trend toward greater gender parity, particularly in the roles of President, symposium presenters, and workshop chairs, especially from 2017 onward. We present these findings as well as our recommendations to enhance inclusivity. These include a more comprehensive collection and secure storage of self-identification data, emphasis on EDI as an essential component of all annual meeting activities, and innovative measures of outreach, collaboration, and leadership with the aim of making the CSI a model for improving EDI in other professional research societies.

The PI3K signalling pathway is known to regulate B cell metabolic programming upon activation, but the mechanisms involved are not well understood. Here we find that the PI3K pathway controls reprogramming of hexokinases (HKs), the enzymes that convert glucose into glucose-6-phosphate as a key rate-limiting step of glycolysis and other metabolic pathways. In primary mouse B cells, PI3K pathway inhibition substantially impaired the activation-induced increase in extracellular acidification rates (ECAR), a measure of glycolysis. In contrast, B cells isolated from PI3Kdelta gain-of-function mutant mice exhibit elevated ECAR. We find that B cell activation substantially elevates protein levels of HK2 and HK3 isoforms, but not HK1, in a PI3K-dependant manner. PI3K or mTOR inhibition significantly reduced induction of HK2 and HK3 expression, whereas Akt inhibition did not affect HK isoform expression. In human B lymphoma cells, HK isoforms differ significantly in their degree of mitochondrial localization, with HK1 being mitochondrial, HK3 being cytoplasmic and HK2 present in both mitochondria and cytoplasm. To assess whether HK isoforms have unique non-redundant functions, HK2-deficient B lymphoma cells were generated and were found to exhibit decreased ECAR as well as significant changes in metabolomic profile, despite normal expression and localization of HK1 and HK3. Taken together, our study reveals that PI3K-dependant reprogramming of hexokinase isoforms can impact on B cell glycolysis and other metabolic pathways. Studies in progress are examining the functional importance of HK2 in antibody responses using mice with B cell-specific deletion of this enzyme.

7051 Background: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib (ibr) has revolutionized the treatment of CLL, showing efficacy in the majority of patients. Nevertheless, resistance occurs in a subset of patients, often with dismal clinical outcomes. Ibr resistance occurs through several mechanisms, including mutations affecting BTK or PLCG2, or upregulation of alternative survival pathways. We investigated at the single-cell level the molecular mechanisms that underlie ibr resistance in CLL cells and their interactions with non-malignant tumor microenvironment (TME) cell populations. Methods: We retrospectively identified 7 CLL patients with ibr resistance (as second line therapy). Samples were obtained before ibr treatment and at progression. Chromium Single Cell Multiome ATAC + Gene Expression (GEX) (10x Genomics) was applied to a mixture of CD19+CD5+ and CD19- cells to jointly analyze DNA accessibility and gene expression in the same single nuclei of CLL B and TME cells. An average of 4,800 cells (range 2,600-15,000) per sample was retained after QC filtering. Principal component analysis (PCA) was performed on GEX counts. ATAC peaks were called with MACS2, and latent semantic indexing was used for dimensionality reduction. We then computed a weighted nearest neighbour graph, identified clusters using the SLM algorithm and performed cell type assignment with established marker genes. Results: We identified 28 distinct cell clusters, representing CLL B-cells or cell types that constitute the TME, including CD4+ and CD8+ T cells, monocytes, dendritic and NK cells. CLL B-cell clusters were patient and timepoint-specific, while TME clusters were occupied by cells from multiple patients. Five patients showed multiple distinct CLL B-cell clusters pre-treatment or at progression, reflecting cellular heterogeneity within the tumor compartment. Interestingly, some clusters that were dominated by post-treatment CLL B-cells also included cells from pre-treatment samples, suggesting pre-existence of the clone giving rise to the relapse. For each patient, we performed pairwise differential expression between CLL B-cell clusters. Gene ontology analysis revealed increased activation of IRF4 and MHC-related pathways in CLL B-cells at progression compared to pre-treatment in 6 and 4 out of 7 patients, respectively. The elevated IRF4 pathway activity was underpinned by increased chromatin accessibility in IRF4 signature genes. Conclusions: Differential blockade of B-cell receptor signaling by ibr has been reported to downregulate activity of IRF4, a key transcription factor in B-cell activation. Our results suggest that CLL B-cells that are resistant to ibrutinib have re-activated IRF4 activity beyond pre-treatment levels, implying a potential role for IRF4 in ibrutinib resistance.

Organization of cellular membranes at the nanoscale is a topic of central importance for cell biology. Most of us can fondly recall colorful artistic renderings of the plasma membrane (PM) encountered as students, depicting an organized lipid bilayer and attendant protein and carbohydrate components. These images and the fluidic mosaic concept of membrane dynamics easily captures the imagination, but the truth is that much remains speculative about the organization and dynamics of membrane components at the nanoscale. Nowhere is this black box more evident (and important) than in cellular signal transduction, where transmembrane receptor engagement triggers modifications of lipids in the inner leaflet of the PM that occur over tiny scales of time and distance. The organization of transiently-modified lipids and proteins in space and over time is likely a key component of the specificity in signaling cascades,where somanyof themajorplayers are ubiquitous andmulti-functional. The hypothesis article in this issue by Cabral-Diaz and Antonescu[1] addresses such questions with a focus on the phosphatidylinositol 3-kinase signaling pathway. Consisting of multiple kinases and phosphatases modifying PI lipid headgroups, as well as PI lipid-binding proteins, the PI3K pathway is a fascinating module of regulated protein–membrane interactions. The PI3K pathway is a prime example of a signaling system that is ubiquitous and multifunctional, where specificity may be dictated by spatiotemporal organization within cellular membranes. Decades of research have refined our understanding of the kinase and phosphatase enzymes that control the generation of D3-phosphorylated PI species and their many functions in cell biology. Similarly, our understanding of the host of intracellular proteins binding to these key PI second messengers, and the biochemical and structural constraints governing their specific interactions with PIs, have advanced tremendously. This hypothesis addresses how these pieces might be put together in a spatially organized fashion at the nanoscale, and how this organization could affect the properties of this signaling system. Applying the simplest concept of a fluid membrane bilayer, the two D3 phosphorylated PIs generated by activated class-1 PI3K enzymes, namely phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphate, would freely diffuse within the inner leaflet of the PM along with the attendant PI-binding proteins. This would generate a uniformly “activated” membrane state, or perhaps a gradient of activation emanating from the activating receptor until signal terminationbyPI-dephosphorylation. Cabral-Diaz and Antonescu propose an alternativemodel incorporating knowledge of the non-homogenous nature of the PM, and particularly membrane nanodomains which potentially concentrate various lipid and protein components. Focusing on a well-studied subset of kinases, phosphatases and PI-binding proteins as examples of the concept, the Hypothesis proposes that nanodomains such as clathrin-coated pits (CCP) serve as pre-organized structures orchestrating PI3K signaling. The authors argue that nanoscale organization of signal activators, terminators and effectors can serve to limit PI lipid diffusion and restrict signaling to localized areas of themembrane, imparting tighter control and a higher degree of specificity to the downstream signaling processes. Expanding upon the CCP hypothesis the authors further propose that such organized PI3K signaling domains are remodeled and remain active in early endocytic compartments until final signal termination. Alternate models are also considered where activators and terminators are present in distinct nanodomains, and the differential implications of these are discussed. The authors outline evidence consistent with the hypothesis and touch upon some potential experimental approaches to directly test this. A number of technical hurdles need to be overcome to better resolve the nanoscale organization of multiple lipids and proteins within membranes. While biosensors based on PI-binding protein domains have been extremely useful, it is likely that biosensors disturb the normal mobility of PI lipids within membranes and their interactions with PI binding proteins hypothesized to be present in

Interleukin-16 (IL-16) is a novel biomarker that has been implicated in many cancers as well as inflammatory diseases. In this study, we examined plasma levels of 30 cytokines and chemokines in chronic lymphocytic leukemia (CLL) and monoclonal B cell lymphocytosis (MBL) patients, and examined their association with disease stage, CLL biomarkers and T cell subsets. Interleukin 16 (IL-16) was identified as a relatively uncharacterized cytokine significantly elevated in CLL patients compared to healthy controls and MBL patients. Plasma levels of IL-16 were significantly elevated by Rai stage 0, increased by Rai stage 3-4, correlated strongly with lymphocyte count and were decreased after Ibrutinib treatment. CLL cells expressed IL-16 mRNA and spontaneously secreted IL-16 in vitro. CLL cells express IL-16 mRNA at significantly higher levels in lymphoid tissues than blood, and we observed that IL-16 release was increased in co-cultures of CLL and autologous CD4 + T cells. Elevated plasma IL-16 levels were associated with abnormalities in the immune microenvironment including multiple inflammatory cytokines and chemokines and expansion of type 1 follicular helper T cells. Taken together, our results identify IL-16 as a novel biomarker in CLL with potential functional roles in cellular interactions between CLL cells and T cells.

FISH cytogenetics, TP53 sequencing, and IGHV mutational status are increasingly used as prognostic and predictive markers in chronic lymphocytic leukemia (CLL), particularly as components of the CLL International Prognostic Index (CLL-IPI) and in directing therapy with novel agents. However, testing outside of clinical trials is not routinely available in Canada. As a centralized CLL clinic at CancerCare Manitoba, we are the first Canadian province to evaluate clinical outcomes and survivorship over a long period of time, incorporating the impact of molecular testing and the CLL-IPI score. We performed a retrospective analysis on 1315 patients diagnosed between 1960 and 2018, followed over a 12-year period, where 411 patients had molecular testing and 233 patients had a known CLL-IPI score at the time of treatment. Overall, 40.3% (n = 530) of patients received treatment, and 47.5% (n = 252) of patients received multiple lines of therapy. High-risk FISH and CLL-IPI (4-10) were associated with higher mortality (HR 2.03, p = 0.001; HR 2.64, p = 0.002), consistent with other studies. Over time, there was an increase in the use of targeted agents in treated patients. The use of Bruton's tyrosine kinase inhibitors improved survival in patients with unmutated IGHV and/or TP53 aberrations (HR 2.20, p = 0.001). The major cause of death in patients who received treatment was treatment/disease-related (32%, n = 42) and secondary malignancies (57%, n = 53) in those who were treatment-naïve. Our data demonstrate the importance of molecular testing in determining survivorship in CLL and underpinning the likely immune differences in outcomes for those treated for CLL.

Phosphoinositide 3-kinase delta (PI3Kδ) plays key roles in normal B cell activation and is chronically activated in malignant B cells. Targeting of PI3Kδ using FDA-approved drugs Idelalisib or Umbralisib has shown efficacy in treatment of multiple B cell malignancies. Duvelisib, an inhibitor targeting both PI3Kδ and PI3Kγ (PI3Kδγi) has also been used for treatment of several leukemias and lymphomas and was suggested to offer potential additional benefits in supressing T cell and inflammatory responses. Transcriptomics analyses indicated that while most B cell subsets predominantly express PI3Kδ, plasma cells upregulate PI3Kγ. We thus assessed whether PI3Kδγi treatment can impact chronic B cell activation in the context of an autoantibody-mediated disease. Using the TAPP1R218LxTAPP2R211L (TAPP KI) mouse model of lupus-like disease driven by dysregulated PI3K pathway activity, we performed 4 week PI3Kδγi treatments and found significant reduction in CD86+ B cells, germinal center B cells, follicular helper T cells and plasma cells in multiple tissues. This treatment also significantly attenuated the abnormally elevated serum levels of IgG isotypes observed in this model. The profile of autoantibodies generated was markedly altered by PI3Kδγi treatment, with significant reductions in IgM and IgG targeting nuclear antigens, matrix proteins and other autoantigens. Kidney pathology was also impacted, with reduced IgG deposition and glomerulonephritis. These results indicate that dual inhibition of PI3Kδ and PI3Kγ can target autoreactive B cells and may have therapeutic benefits in autoantibody-mediated disease.

African trypanosomiasis, a neglected tropical disease, is caused by diverse species of the protozoan parasite belonging to the genus Trypanosoma. Although anti-trypanosomal medications exist, the increase in drug resistance and persistent antigenic variation has necessitated the development of newer and more efficacious therapeutic agents which are selectively toxic to the parasite. In this study, we assessed the trypanocidal efficacy of Crosspteryx fibrifuga leaf extract (C.f/L-extract) in vitro. Following treatment of T. congolense parasites with C.f/L-extract, we observed a significant decrease in parasite number and an elevation in the expression of the apoptotic markers, Annexin V and 7-Aminoactinomycin D (7AAD). Interestingly, at the same concentration (50 μg/mL), C.f/L-extract was not cytotoxic to murine whole splenocytes. We also observed a significant increase in pro-inflammatory cytokines and nitric oxide secretion by bone marrow derived macrophages following treatment with C.f/L-extract (10 μg/mL and 50 μg/mL) compared to PBS treated controls, suggesting that the extract possesses an immune regulatory effect. Treatment of T. congolense infected mice with C.f/L-extract led to significant decrease in parasite numbers and a modest increase in mouse survival compared to PBS treated controls. In addition, there was a significant increase in CD4+IFN-γ+ T cells and a decrease in CD4+IL-10+ T cells in the spleens of T. congolense infected mice treated with C.f/L-extract. Interestingly, C.f/L-extract treatment decreased the activity of superoxide dismutase (an enzyme that protects unicellular organisms from oxidative stress) in T. congolense parasites but not in splenocytes. Collectively, our study has identified C.f/L-extract as a potential anti-trypanosomal agent that warrant further investigation and possibly explored as a treatment option for T. congolense infection.

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutation in the TAFAZZIN gene. Tafazzin (Taz) deficiency in BTHS patients results in an increased risk of infections. Mesenchymal stem cells (MSCs) are well known for their immune-inhibitory function. We examined how Taz-deficiency in murine MSCs impact their ability to modulate the function of lipopolysaccharide (LPS)-activated wild type (WT) B lymphocytes. MSCs from tafazzin knockdown (TazKD) mice exhibited a reduction in mitochondrial cardiolipin compared to wild type (WT) MSCs. However, mitochondrial bioenergetics and membrane potential were unaltered. In contrast, TazKD MSCs exhibited increased reactive oxygen species generation and increased glycolysis. The increased glycolysis was associated with an elevated proliferation, phosphatidylinositol-3-kinase expression and expression of the immunosuppressive markers indoleamine-2,3-dioxygenase, cytotoxic T-lymphocyte-associated protein 4, interleukin-10, and cluster of differentiation 59 compared to controls. Inhibition of glycolysis with 2-deoxyglucose attenuated the TazKD-mediated increased expression of cytotoxic T-lymphocyte-associated protein 4 and interleukin-10. When co-cultured with LPS-activated WT B cells, TazKD MSCs inhibited B cell proliferation and growth rate and reduced B cell secretion of immunoglobulin M compared to controls. In addition, co-culture of LPS-activated WT B cells with TazKD MSCs promoted B cell differentiation toward interleukin-10 secreting plasma cells and B regulatory cells compared to controls. The results indicate that Taz deficiency in MSCs promote reprogramming of activated B lymphocytes toward immunosuppressive phenotypes.

The flipped classroom (FC) model of instruction has inherent barriers to implementation in medical education due to amount of content taught versus time allotted, the widespread adoption of interdisciplinary course structure causing a plethora of instructors responsible for content delivery, and trends to reduce the number of weeks to teach preclinical foundational science. Here we report on a FC model executed in an interdisciplinary endocrinology block in a time-saving manner, while preserving student preferences and satisfaction and improving written assessment performance. In this study, traditional lectures were 100% replaced with pre-session assignments (custom video modules) resulting in less time (- 9 h) spent on first pass learning. In-person, active-learning, case-based sessions were created (+ 8 h) to complete the FC model and achieve higher level understanding. Written assessment performance in the endocrinology block was compared between two cohorts: the FC model and traditional lecture model. The FC model cohort outperformed the traditional lecture cohort on written, multiple-choice assessments (both in-house and NBME assessments). Furthermore, a measured (survey data) student preference for the FC model was observed.

1 Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada 2 Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada 3 Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 4 Department of Immunology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada

BackgroundDespite immune cell dysregulation being an important event preceding the onset of rheumatoid arthritis (RA), the phenotype of T and B cells in preclinical RA is less understood. The aim of this study was to characterize T and B cell populations in RA patients and their autoantibody (aAb) negative and positive first-degree relatives (FDR). MethodsCryopreserved peripheral blood mononuclear cells (PBMCs) collected at scheduled visits from aAb-(n=25), and aAb+ FDR (n=10) and RA patients (n=13) were thawed and stained using optimized antibody cocktails as per a specific 13-color T or B cell panel. Immunophenotyping was performed using a Cytoflex LX (Beckman-Coulter) flow cytometer and FlowJo software was used for analyzing the frequency of immune cell populations. ResultsMulticolor flow cytometry experiments identified an increased TIGIT expression in circulating lymphocytes of aAb+ FDR and RA patients, relative to aAb- FDR (P<0.01). These TIGIT(+) T cells exhibited a memory phenotype and expressed high levels of PD-1, ICOS, HLA-DR, CXCR3 and CXCR5. Moreover, increased TIGIT(+) CD4 T cell frequency correlated with the frequency of PD-1(+) CD4 T cells (r = 0.4705: P = 0.0043) and circulating levels of ACPA and RF. We also identified a decreased frequency of CD27+IgD- switched memory B cells in RA patients (P < 0.01), while increased frequency of TIGIT+ CD4 T cells in FDR correlated with the frequency of PD1(+)PTEN(+) B cells (r = 0.6838, P = 0.0004) and autoantibody positivity (P = 0.01). ConclusionWe demonstrate TIGIT as a distinct CD4 T cell marker for differentiating aAb- FDR from aAb+FDR and might play a critical role in regulating T and B cell crosstalk in preclinical RA.

Barth syndrome (BTHS) is a rare X-linked genetic disease caused by mutations in TAFAZZIN. The tafazzin (Taz) protein is a cardiolipin remodeling enzyme required for maintaining mitochondrial function. Patients with BTHS exhibit impaired mitochondrial respiratory chain and metabolic function and are susceptible to serious infections. B lymphocytes (B cells) play a vital role in humoral immunity required to eradicate circulating antigens from pathogens. Intact mitochondrial respiration is required for proper B-cell function. We investigated whether Taz deficiency in mouse B cells altered their response to activation by anti-cluster of differentiation 40 (anti-CD40) + interleukin-4 (IL-4). B cells were isolated from 3–4-month-old wild type (WT) or tafazzin knockdown (TazKD) mice and were stimulated with anti-CD40 + IL-4 for 24 h and cellular bioenergetics, surface marker expression, proliferation, antibody production, and proteasome and immunoproteasome activities determined. TazKD B cells exhibited reduced mRNA expression of Taz, lowered levels of cardiolipin, and impairment in both oxidative phosphorylation and glycolysis compared to WT B cells. In addition, anti-CD40 + IL-4 stimulated TazKD B cells expressed lower levels of the immunogenic surface markers, cluster of differentiation 86 (CD86) and cluster of differentiation 69 (CD69), exhibited a lower proliferation rate, reduced production of immunoglobulin M and immunoglobulin G, and reduced proteasome and immunoproteasome proteolytic activities compared to WT B cells stimulated with anti-CD40 + IL-4. The results indicate that Taz is required to support T-cell-dependent signaling activation of mouse B cells.

Introduction: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Despite significant improvement in the treatment of CLL, drug resistance is emerging when using the single agents ibrutinib or venetoclax. To achieve greater depth of response, combination treatments are being used to eradicate disease. Altered mitochondrial metabolism is a key factor in CLL survival. In order to gain insights into the underlying biology of a promising drug combination treatment, we investigated the combination of venetoclax and ibrutinib on mitochondrial function as well as the B-cell receptor (BCR), apoptotic and adenosine monophosphate activated protein kinase /silent information regulator 1 / peroxisome proliferator-activated receptor-coactivator-1α (AMPK/SIRT1/PGC-1α) signaling pathways in CLL cells. We also evaluated a proposed mechanism of resistance using interleukin-4 (IL-4) to demonstrate the role of a nicotinamide phosphoribosyltransferase (NAMPT) specific inhibitor, FK866, in order to overcome resistance in vitro.

PI3Kδ is critical in generating humoral and regulatory immune responses. In this study, we determined the impact of PI3Kδ in immunity to Trypanosoma congolense, an African trypanosome that can manipulate and evade Ab responses critical for protection. Upon infection with T. congolense, PI3KδD910A mice lacking PI3Kδ activity paradoxically show a transient enhancement in early control of parasitemia, associated with impaired production of regulatory IL-10 by B cells in the peritoneum. C57BL/6 wild-type (WT) mice treated with the PI3Kδ inhibitor (PI3Kδi) Idelalisib showed a similar transient decrease in parasitemia associated with reduced IL-10. Strikingly, however, we find that PI3KδD910A mice were ultimately unable to control this infection, resulting in uncontrolled parasitemia and death within 2 wk. Assessment of humoral responses revealed delayed B cell activation, impaired germinal center responses, and compromised Ab responses to differing degrees in PI3KδD910A and PI3Kδi-treated mice. To test the role of Abs, we administered serum from WT mice to PI3KδD910A mice and found that lethality was prevented by postinfection serum. Interestingly, serum from naive WT mice provided partial protection to PI3KδD910A mutants, indicating an additional role for natural Abs. Together our findings suggest that although PI3Kδ drives immune regulatory responses that antagonize early control of parasite growth in the peritoneum, it is also required for generation of Abs that are critical for protection from systemic trypanosome infection. The essential role of PI3Kδ for host survival of African trypanosome infection contrasts with findings for other pathogens such as Leishmania, underlining the critical importance of PI3Kδ-dependent humoral immunity in this disease.

Follicular helper T cells (T FH ) have specialized properties in promoting normal B cell activation but their role in chronic lymphocytic leukemia (CLL) is unknown. We find that T FH cells are elevated in CLL patients and are phenotypically abnormal, expressing higher levels of PD-1, TIGIT, CD40L, IFNγ and IL-21, and exhibiting abnormal composition of T FH 1, T FH 2 and T FH 17 subsets. Frequencies of CD4-positive T cells expressing T FH 1 markers and IL-21 were positively correlated with patient lymphocyte counts and RAI stage, suggesting that accumulation of abnormal T FH cells is concomitant with expansion of the leukemic B cell clone. Treatment with ibrutinib led to normalization of T FH frequencies and phenotype. T FH cells identified in CLL bone marrow display elevated expression of several functional markers compared to blood T FH cells. CLL T cell-B cell co-culture experiments revealed a correlation of patient T FH frequencies with functional ability of their CD4-positive T cells to promote CLL proliferation. Conversely, CLL cells can preferentially activate the T FH cell subset in co-culture. Together our results indicate that CLL development is associated with expansion of abnormal T FH populations that produce elevated levels of cytokines and costimulatory molecules which may help support CLL proliferation.