Cells In Secondary Lymphoid Organs Research Articles

State of pneumococcal vaccine immunity.

Like the other invasive encapsulated bacteria, Streptococcus pneumoniae is also covered with a polysaccharide structure. Infants and elderly are most vulnerable to the invasive and noninvasive diseases caused by S. pneumoniae. Although antibodies against polysaccharide capsule are efficient in eliminating S. pneumoniae, the T cell independent nature of the immune response against polysaccharide vaccines renders them weakly antigenic. The introduction of protein conjugated capsular polysaccharide vaccines helped overcome the weak immunogenicity of pneumococcal polysaccharides and decreased the incidence of pneumococcal diseases, especially in pediatric population. Conjugate vaccines elicit T cell dependent response which involve the interaction of specialized CD4+ T cells, called follicular helper T cells (Tfh) with germinal center B cells in secondary lymphoid organs. Despite their improved immunogenicity, conjugate vaccines still need to be administered three to four times in infants during the first 15 month of their life because they mount poor Tfh response. Recent studies revealed fundamental differences in the generation of Tfh cells between neonates and adults. As the portfolio of pneumococcal conjugate vaccines continues to increase, better understanding of the mechanisms of antibody development in different age groups will help in the development of pneumococcal vaccines tailored for different ages.

Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The “multiple hits hypothesis” suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H−/−, EBI2−/−, or CYP7B1−/− genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H−/−, EBI2−/−, or CYP7B1−/−) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 – oxysterol axis to the intestinal microbiota was detectable in the current study.

Visualization of immune pathways that enhance the neutralizing antibody response to vaccines after primary immunization.

We examined the relationship between the association of a vaccine antigen with immune cells in secondary lymphoid organs shortly after immunization and the resulting neutralizing antibody response induced by that antigen using three antigenic forms of anthrax protective antigen (PA) that induce qualitatively different antibody responses. The three PA forms used were wild-type PA, which binds to anthrax toxin receptors and elicits a robust antibody response that includes both neutralizing and non-neutralizing antibodies; a receptor-binding-deficient (RBD) mutant form of PA, which does not bind cellular receptors and elicits only barely detectable antibody responses; and an engineered chimeric form of PA, which binds cholera toxin receptors and elicits a robust total antibody response but a poor neutralizing antibody response. We found that both wild-type PA and the PA chimera associated with immune cells in secondary lymphoid organs after immunization, but the RBD mutant PA exhibited minimal association, revealing a relationship between antigen binding to toxin receptors on immune cells after immunization and subsequent antibody responses. A portion of wild-type PA that bound to immune cells was cell surface-associated and maintained its native conformation. Much lower amounts of conformationally intact PA chimera were associated with immune cells after immunization, correlating with the lower neutralizing antibody response elicited by the PA chimera. Thus, binding of an antigen to receptors on immune cells in secondary lymphoid organs after immunization and maintenance of conformational integrity of the cell-associated antigen help dictate the magnitude of the resulting neutralizing antibody response, but not necessarily the total antibody response.IMPORTANCEMany vaccines protect by the induction of antibodies that neutralize the action of the pathogen. Here, we followed the fate of three antigenic forms of a vaccine antigen in secondary lymphoid organs after immunization to investigate events leading to a robust neutralizing antibody response. We found that the magnitude of the neutralizing antibody response, but not the total antibody response, correlates with the levels of conformationally intact antigen associated with immune cells in secondary lymphoid organs after primary immunization. We believe that these results provide important insights into the genesis of neutralizing antibody responses induced by vaccine antigens and may have implications for vaccine design.

Downregulation of TCF7 and LEF1 is a key determinant of tumor-infiltrating regulatory T-cell function.

Forkhead box P3 (Foxp3)-expressing regulatory T (Treg) cells play essential roles in immune homeostasis but also contribute to establish a favorable environment for tumor growth by suppressing anti-tumor immune responses. It is thus necessary to specifically target tumor-infiltrating Treg cells to minimize effects on immune homeostasis in cancer immunotherapy. However, molecular features that distinguish tumor-infiltrating Treg cells from those in secondary lymphoid organs remain unknown. Here we characterize distinct features of tumor-infiltrating Treg cells by global analyses of the transcriptome and chromatin landscape. They exhibited activated phenotypes with enhanced Foxp3-dependent transcriptional regulation, yet being distinct from activated Treg cells in secondary lymphoid organs. Such differences may be attributed to the extensive clonal expansion of tumor-infiltrating Treg cells. Moreover, we found that TCF7 and LEF1 were specifically downregulated in tumor-infiltrating Treg cells both in mice and humans. These factors and Foxp3 co-occupied Treg suppressive function-related gene loci in secondary lymphoid organ Treg cells, whereas the absence of TCF7 and LEF1 accompanied altered gene expression and chromatin status at these gene loci in tumor-infiltrating Treg cells. Functionally, overexpression of TCF7 and LEF1 in Treg cells inhibited the enhancement of Treg suppressive function upon activation. Our results thus show the downregulation of TCF7 and LEF1 as markers of highly suppressive Treg cells in tumors and suggest that their absence controls the augmentation of Treg suppressive function in tumors. These molecules may be potential targets for novel cancer immunotherapy with minimum effects on immune homeostasis.

Aberrant follicular regulatory T cells associate with immunoglobulin hyperproduction in nasal polyps with ectopic lymphoid tissues

Ectopic lymphoid tissues (eLTs) and associated follicular helper T (TFH) cells contribute to local immunoglobulin hyperproduction in nasal polyps (NPs). Follicular regulatory T (TFR) cells in secondary lymphoid organs counteract TFH cells and suppress immunoglobulin production; however, the presence and function of TFR cells in eLTs in peripheral diseased tissues remain poorly understood. We sought to investigate the presence, phenotype, and function of TFR cells in NPs. The presence, abundance, and phenotype of TFR cells in NPs were examined using single-cell RNA sequencing, immunofluorescence staining, and flow cytometry. Sorted polyp and circulating T-cell subsets were cocultured with autologous circulating naïve B cells, and cytokine and immunoglobulin production were measured by ELISA. TFR cells were primarily localized within eLTs in NPs. TFR cell frequency and TFR cell/TFH cell ratio were decreased in NPs with eLTs compared with NPs without eLTs and control inferior turbinate tissues. TFR cells displayed an overlapping phenotype with TFH cells and FOXP3+ regulatory T cells in NPs. Polyp TFR cells had reduced CTLA-4 expression and decreased capacity to inhibit TFH cell-induced immunoglobulin production compared with their counterpart in blood and tonsils. Blocking CTLA-4 abolished the suppressive effect of TFR cells. Lower vitamin D receptor expression was observed on polyp TFR cells compared with TFR cells in blood and tonsils. Vitamin D treatment upregulated CTLA-4 expression on polyp TFR cells and restored their suppressive function invitro. Polyp TFR cells in eLTs have decreased CLTA-4 and vitamin D receptor expression and impaired capacity to suppress TFH cell-induced immunoglobulin production, which can be reversed by vitamin D treatment invitro.

Modulation of immune responses to liposomal vaccines by intrastructural help

The encapsulation of HIV-unrelated T helper peptides into liposomal vaccines presenting trimers of the HIV-1 envelope glycoprotein (Env) on the surface (T helper liposomes) may recruit heterologous T cells to provide help for Env-specific B cells. This mechanism called intrastructural help can modulate the HIV-specific humoral immune response. In this study, we used cationic T helper liposomes to induce intrastructural help effects in a small animal model. The liposomes were functionalized with Env trimers by a tag-free approach designed to enable a simplified GMP production. The pre-fusion conformation of the conjugated Env trimers was verified by immunogold electron microscopy (EM) imaging and flow cytometry. The liposomes induced strong activation of Env-specific B cells in vitro. In comparison to previously established anionic liposomes, cationic T helper liposomes were superior in CD4+ T cell activation after uptake by dendritic cells. Moreover, the T helper liposomes were able to target Env-specific B cells in secondary lymphoid organs after intramuscular injection. We also observed efficient T helper cell activation and proliferation in co-cultures with Env-specific B cells in the presence of cationic T helper liposomes. Mouse immunization experiments with cationic T helper liposomes further revealed a modulation of the Env-specific IgG subtype distribution and enhancement of the longevity of antibody responses by ovalbumin- and Hepatitis B (HBV)-specific T cell help. Thus, clinical evaluation of the concept of intrastructural help seems warranted.

CCL21-Ser expression in melanoma cells recruits CCR7+ naïve T cells to tumor tissues and promotes tumor growth.

CCL21-Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21-Ser-expressing melanoma cells and the Ccl21a-deficient mice, we demonstrated the functional role of cancer cell-derived CCL21-Ser in melanoma growth in vivo. The B16-F10 tumor growth was significantly decreased in Ccl21a-deficient mice compared with that in wild-type mice, indicating that host-derived CCL21-Ser contributes to melanoma proliferation in vivo. In Ccl21a-deficient mice, tumor growth of melanoma cells expressing CCL21-Ser was significantly enhanced, suggesting that CCL21-Ser from melanoma cells promotes tumor growth in the absence of host-derived CCL21-Ser. The increase in tumor growth was associated with an increase in the CCR7+ CD62L+ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell-derived CCL21-Ser expression. These results suggest that CCL21-Ser from melanoma cells promotes the infiltration of CCR7+ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.

Comparative Pharmacology of Ofatumumab vs Ocrelizumab in Humanized CD20 Transgenic Mice (S9.007)

<h3>Objective:</h3> Benchmark the potency of subcutaneous ofatumumab (OMB-sc) and intravenous ocrelizumab (OCR-iv) at depleting B cells expressing human CD20 in transgenic mice. <h3>Background:</h3> Therapies that deplete CD20-positive B cells are effective in multiple sclerosis. Pilot work has suggested improved lymph node (LN) targeting for OMB-sc vs OCR-iv. <h3>Design/Methods:</h3> Humanized CD20 mice (C57BL/6-Ms4a1tm2[hCD20]Smoc) were treated once with OMB-sc or OCR-iv at various doses. Drug levels and B-cell counts were assessed via IgG-binding ELISA and flow cytometry, respectively, in blood and lymphoid organs (spleen, inguinal LNs, bone marrow [BM]). <h3>Results:</h3> Dose-proportional drug levels with B-cell depletion in all compartments was noted for both therapies. Three days post treatment, serum levels needed to achieve 50%/90% (EC50/EC90) efficacy were ~0.01/0.3 mg/mL, respectively, with OMB-sc and ~0.2/5.0 mg/mL, respectively, with OCR-iv, indicative of 20-fold higher B-cell–depleting potency for OMB-sc. OMB-sc reached its EC50 at the human-equivalent dose (huD) of 6 mg per mouse; OCR-iv was ~40-fold above its EC90 at the huD (200 mg). Drug level ratios (spleen and LN vs serum) were 5–20 for OMB-sc and ≤1 for OCR-iv, confirming lymphoid organ targeting for OMB-sc. Both treatments appeared equipotent at depleting noncirculating B cells (EC50s ~−0.2 mg/mL). Marginal zone and follicular B cells in secondary lymphoid organs were markedly depleted by OCR-iv but spared by OMB-sc at the huD. OMB-sc achieved drug levels 10-fold below the EC50 in BM at the huD, whereas OCR-iv reached its EC90, which suggests a lower impact on BM-resident CD20-expressing cells with OMB-sc <h3>Conclusions:</h3> OMB-sc demonstrated better efficiency in targeting lymphoid organs and greater potency for depleting circulating B cells vs OCR-iv. A sparing effect regarding marginal zone and follicular B cells was demonstrated with OMB-sc. OMB-sc may offer high efficacy and lower long-term safety risks with convenient dosing vs OCR-iv in humans. <b>Disclosure:</b> Marc Bigaud has received personal compensation for serving as an employee of Novartis Pharma. Dr. Anthony has received personal compensation for serving as an employee of University of Oxford. Dr. Anthony has received personal compensation for serving as an employee of Somerville College Oxford. Dr. Anthony has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Merck. Dr. Anthony has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Neuroplast. Dr. Anthony has received personal compensation in the range of $0-$499 for serving as an officer or member of the Board of Directors for Oxomics. The institution of Dr. Anthony has received research support from Numares. The institution of Dr. Anthony has received research support from MRC UK. Mrs. Zipfel has nothing to disclose. Tatjana Uffelmann has nothing to disclose. Mrs. Vostiarova has nothing to disclose. Mr. Engelhardt has nothing to disclose. Dr. Nuesslein-Hildesheim has received personal compensation for serving as an employee of Novartis Pharma AG. Dr. Nuesslein-Hildesheim has stock in Novartis Pharma AG. Bernd C. Kieseier, MD has nothing to disclose.

T-cell-B-cell collaboration in the lung.

Collaboration between T and B cells in secondary lymphoid organs is a crucial component of adaptive immunity, but lymphocytes also persist in other tissues. Recent studies have examined T-cell-B-cell interactions in nonlymphoid tissues such as the lung. CD4+ T- resident helper cells (TRH) remain in the lung after influenza infection and support both resident CD8 T cells and B cells. Multiple lung-resident B-cell subsets (B-resident memory (BRM)) that exhibit spatial and phenotypic diversity have also been described. Though not generated by all types of infection, inducible bronchus-associated lymphoid tissue offers a logical place for T and B cells to interact. Perturbations to BRM and TRH cells elicit effects specific to Immunoglobulin A (IgA) production, an antibody isotype with privileged access to mucosa. Understanding the interplay of lymphocytes in mucosal tissues, which can be insulated from systemic immune responses, may improve the design of future vaccines and therapies.

Apelin expression is downregulated in T cells in a murine model of chronic colitis

Apelin (APL), an endogenous ligand for APJ, has been reported to be upregulated in a murine model of acute colitis induced by sodium dextran sulfate, as well as inflammatory bowel diseases (IBD) in humans. However, the mechanisms and functions of APL/APJ axis in the pathogenesis of IBD are unclear. We herein analyzed CD4+ T cells to determine the functions of APL in a murine model of chronic colitis induced in Rag deficient mice (Rag−/−). In colonic tissues of wild-type mice (WT), we found that APL was expressed especially in the lamina propria lymphocytes, where CD4+ T cells are dominant, rather than the epithelial cells. Unexpectedly, the APL expression was rather downregulated in the colonic tissue of the chronic colitis group compared to the control groups (Rag−/− before colitis induction and WT). The APL expression was downregulated when naïve T cells were differentiated into effecter T cells. A lack of APL resulted in decreased naïve T cells and increased effecter T cells in secondary lymphoid organs. A synthetic APL peptide, [Pyr1]-APL-13, increased IL-10 and decreased IFN-γ productions by effecter T cells. Administration of [Pyr1]-APL-13 improved survival rate in association with lessened colitis severity and decreased pro-inflammatory cytokine production. This is the first report showing immunological function of APL specifically on T cells, and these results indicate that APL/APJ axis may be a novel therapeutic target for IBD.

Graft-Versus-Host Disease Induces Immune Dysregulation through Irreversible Damage to Specialized Immune-Interacting Fibroblastic Stromal Cells in Secondary Lymphoid Organs

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially life-saving treatment for patients with leukemia and other hematological disorders. However, its success is limited by graft-versus-host disease (GVHD), a life-threatening syndrome caused by alloreactive donor T cells that induce damage in normal host tissues. Many patients who survive transplantation develop chronic GVHD which can be severe and has few effective therapeutic options. Chronic GVHD is characterized both by immune hyperreactivity, reminiscent of autoimmunity, and defective immune responses, including poor responses to vaccination and infectious challenges. The mechanisms underlying the complex immune dysregulation of chronic GVHD remain incompletely understood. Previous work indicates that pathogenic alloreactive T cells can damage lymph node fibroblastic stromal cells, a group of specialized cells increasingly recognized to regulate multiple aspects of immune homeostasis. Using mouse models of allo-HCT, we sought to identify the populations of specialized fibroblastic stromal cells and other stromal elements that were most sensitive to GVHD-mediated injury and to assess the functional impact of this damage during chronic GVHD. We used a sublethal allo-HCT model with a low T cell dose that allowed for long-term survival over 100 days after transplantation (B6 into lethally irradiated B6xBALB/c F1 mice, 2x 5.5 Gy). Unlike allo-HCT recipients of T cell-depleted bone marrow (no GVHD control), recipients of T cell-depleted bone marrow plus allo-T cells (1-5x106 splenocytes) developed low-grade systemic GVHD with mild reversible weight loss. At day 30-60 after transplantation, allo-T cell recipients showed modestly decreased thymopoiesis, consistent with the thymus being a sensitive target organ of GVHD, as previously reported. In contrast to this mild injury, peripheral lymph nodes (pLN) showed profound long-lasting depletion of CD31-/podoplanin+ fibroblastic stromal cells as assessed by flow cytometry. Among these cells, MAdCAM-hi marginal reticular cells (MRCs) and CD157-hi fibroblastic reticular cells were most severely affected, including most cells lineage-traced by a Ccl19-Cre transgene. Total blood endothelial cells and lymphatic endothelial cells (LECs) were only modestly decreased by GVHD at day 30, but the LEC subset characterized by high MAdCAM expression consistent with a floor LEC phenotype virtually disappeared. Thus, MRCs and floor LECs lining the marginal sinus were severely affected by GVHD, suggesting that antigen uptake and processing could be defective in these pLN. Single cell RNA-seq revealed a near complete loss of floor LECs, MRCs and Ccl19/Ccl21+ T zone FRCs in GVHD-affected pLNs. Instead, residual fibroblastic stromal cells showed accumulation of collagen-expressing Cxcl14+ fibroblasts with loss of immune-interacting properties. In terms of functional consequences, GVHD pLNs had a markedly decreased abundance of naïve B and T cells, the latter despite relatively preserved naïve T cell production in the thymus. Compared to pLNs, the spleen was less profoundly affected and remained more receptive than pLNs to homing of adoptively transferred T cells. Immune responses to SARS-CoV2 mRNA used as a potent and clinically relevant T-dependent antigen were profoundly impaired in GVHD pLNs. In summary, our data suggest that specialized fibroblastic stromal cells and LECs in secondary lymphoid organs are even more sensitive to GVHD than thymic epithelial cells, with little evidence of functional recovery. We speculate that subclinical damage to pLN stromal compartments may underlie many aspects of immune dysregulation in chronic GVHD, including persistently impaired responses to vaccination and loss of peripheral tolerance.

Lymphoid Lineage γδ T Cells Were Successfully Generated from Human Pluripotent Stem Cells via Hemogenic Endothelium.

γδ T cells are a rare and unique prototype of T cells that share properties with natural killer cells in secondary lymphoid organs. Although many studies have revealed the function and importance of adult-derived γδ T cells in cancer biology and regenerative medicine, the low numbers of these cells hamper their application as therapeutic cell sources in the clinic. To solve this problem, pluripotent stem cell-derived γδ T cells are considered alternative cell sources; however, few studies have reported the generation of human pluripotent stem cell-derived γδ T cells. In the present study, we investigated whether lymphoid lineage γδ T cells were successfully generated from human pluripotent stem cells via hemogenic endothelium under defined culture conditions. Our results revealed that pluripotent stem cells successfully generated γδ T cells with an overall increase in transcriptional activity of lymphoid lineage genes and cytolytic factors, indicating the importance of the optimization of culture conditions in generating lymphoid lineage γδ T cells. We uncovered an initial step in differentiating γδ T cells that could be applied to basic and translational investigations in the field of cancer biology. Based on our result, we will develop an appropriate method to purify γδ T cells with functionality and it helpful for the study of basic mechanism of γδ T cells in pathophysiologic condition as well as clinic application.

Classical and γδ T cells are each independently sufficient to establish protection against a classical strain of Klebsiella pneumoniae.

Infections with classical strains of the Gram-negative bacterium Klebsiella pneumoniae pose a significant clinical challenge due to rising antibiotic resistance. We previously established a lung inoculation plus challenge model using live, classical K. pneumoniae in order to study host protection. Here, we employ this model to dissect adaptive immune responses to this critical pathogen. First, we performed convalescent serum transfers from inoculated mice to naïve recipients and found that classical K. pneumoniae infection outcomes, unlike hypervirulent K. pneumoniae infection outcomes, were not improved. This suggests that circulating antibody responses alone are not sufficient to mediate protection against this classical strain. Hence, we evaluated the role of T cells in protection against classical K. pneumoniae reinfection and demonstrated that mice lacking T cells are unable to establish a protective response. However, mice individually deficient in either of the major T cell subsets, γδ or αβ (classical T cells), effectively mount a protective response, indicating either subset alone is sufficient to mediate protection. Sequestration of T cells in secondary lymphoid organs during the challenge infection did not ablate protection, indicating the circulating T cell pool is not required for the protective phenotype. Finally, we demonstrate that depletion of T cells during initial infection eliminates protection against challenge. Collectively, these experiments demonstrate the imperative contribution of T cells to protective immunity against classical K. pneumoniae and will guide further inquiries into host effector responses required to control this infection.

The dark side of Tregs during aging.

In the last century, we have seen a dramatic rise in the number of older persons globally, a trend known as the grey (or silver) tsunami. People live markedly longer than their predecessors worldwide, due to remarkable changes in their lifestyle and in progresses made by modern medicine. However, the older we become, the more susceptible we are to a series of age-related pathologies, including infections, cancers, autoimmune diseases, and multi-morbidities. Therefore, a key challenge for our modern societies is how to cope with this fragile portion of the population, so that everybody could have the opportunity to live a long and healthy life. From a holistic point of view, aging results from the progressive decline of various systems. Among them, the distinctive age-dependent changes in the immune system contribute to the enhanced frailty of the elderly. One of these affects a population of lymphocytes, known as regulatory T cells (Tregs), as accumulating evidence suggest that there is a significant increase in the frequency of these cells in secondary lymphoid organs (SLOs) of aged animals. Although there are still discrepancies in the literature about modifications to their functional properties during aging, mounting evidence suggests a detrimental role for Tregs in the elderly in the context of bacterial and viral infections by suppressing immune responses against non-self-antigens. Interestingly, Tregs seem to also contribute to the reduced effectiveness of immunizations against many pathogens by limiting the production of vaccine-induced protective antibodies. In this review, we will analyze the current state of understandings about the role of Tregs in acute and chronic infections as well as in vaccination response in both humans and mice. Lastly, we provide an overview of current strategies for Treg modulation with potential future applications to improve the effectiveness of vaccines in older individuals.

ORAI3 is dispensable for store-operated Ca2+ entry and immune responses by lymphocytes and macrophages.

Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3-/- mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3-/- mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3-/- mice. Moreover, Orai3-/- mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21

AbstractTumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease.

IL-17–producing follicular Th cells enhance plasma cell differentiation in lupus-prone mice

Follicular Th (Tfh) cells are key CD4+ Th cells involved in regulating B cell differentiation in the germinal center. Mice with conditional KO (CKO) of B lymphocyte-induced maturation protein-1 (BLIMP-1) expression in CD11chi DCs (Prdm1 CKO) spontaneously develop a lupus-like disease. We found an increase in the number of Tfh cells in secondary lymphoid organs in lupus-prone Prdm1-CKO mice. B cells stimulated with Tfh cells become Ab-secreting cells (ASCs); there was an increase in ASC differentiation mediated by Tfh cells from Prdm1-CKO mice compared with Tfh cells from control mice. This was mainly due to the increased expression of molecules within the IL-23/IL-17 pathway in Tfh cells from Prdm1-CKO mice. There was an increased frequency of IL-17A+ Tfh cells in Prdm1-CKO mice. These Tfh cells secrete IL-17A and an increased amount of IL-21. Furthermore, neutralizing IL-17A and IL-21 reduced ASC differentiation induced by Tfh cells. Lastly, we found the expression of IL-1β and IL-6 was increased in BLIMP-1–deficient DCs, which are key for Th17 induction. Altogether, the lack of BLIMP-1 expression in DCs induces not only an increased number of Tfh cells, but also functionally distinct Tfh cells that lead to increased ASC differentiation.

Myosin 18A is a novel checkpoint regulator in B cell differentiation and antibody-mediated immunity

Abstract The B cell antibody response is tightly regulated to facilitate pathogen-specific immunity and prevent self-reactivity, with actin cytoskeleton dynamics playing an important role in regulating B cell activation. The newly discovered unconventional myosin family protein Myosin 18A (Myo18A) is most closely related to Myo2A, and regulates important cellular processes in non-lymphoid cells. Myo18A is expressed in both precursor and mature B cells, and interacts with ezrin, Myo2A and tyrosine phosphorylated proteins suggesting that it may regulate physiological functions of B cells. We investigated the function of Myo18A in antibody-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Basally, Myo18A deficiency led to expansion of both bone marrow progenitor B cells, and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia with greater levels of splenic IgM secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing antibodies against the major antigen hemagglutinin, associated with early expansion and persistent accumulation of antigen-specific germinal center B cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into antibody secreting cells, and induce Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, self-tolerance and humoral immunity. Supported by an NIH grant (R21 AI117350) to Neetu Gupta

Dendritic Cells and Their Immunotherapeutic Potential for Treating Type 1 Diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells through a process that is primarily mediated by T cells. Emerging evidence suggests that dendritic cells (DCs) play a crucial role in initiating and developing this debilitating disease. DCs are professional antigen-presenting cells with the ability to integrate signals arising from tissue infection or injury that present processed antigens from these sites to naïve T cells in secondary lymphoid organs, thereby triggering naïve T cells to differentiate and modulate adaptive immune responses. Recent advancements in our knowledge of the various subsets of DCs and their cellular structures and methods of orchestration over time have resulted in a better understanding of how the T cell response is shaped. DCs employ various arsenal to maintain their tolerance, including the induction of effector T cell deletion or unresponsiveness and the generation and expansion of regulatory T cell populations. Therapies that suppress the immunogenic effects of dendritic cells by blocking T cell costimulatory pathways and proinflammatory cytokine production are currently being sought. Moreover, new strategies are being developed that can regulate DC differentiation and development and harness the tolerogenic capacity of these cells. Here, in this report, we focus on recent advances in the field of DC immunology and evaluate the prospects of DC-based therapeutic strategies to treat T1D.

Late Allograft Rejection: A Case Report

Recognition of donor antigens by recipient T cells in secondary lymphoid organs initiates the adaptive inflammatory immune response leading to the rejection of allogeneic transplants. We present a unique case of a 38-year-old male with late allograft rejection due to immunosuppressive cytotoxic therapy. There is limited literature available as well as management. This case highlights the need for further investigative research of this entity and its pathogenesis.