Cell-deficient Recipients Research Articles

IL-21 from IFN-g +IL-21 +hybrid T cells promotes germinal center B cell proliferation

Abstract Clearance of infection with Plasmodium spp. requires both antibody and Th1-type responses. While Th1-like cells control the peak in P. chabaudi infection, however specific IgG is delayed. Both Tfh and GC-Tfh can promote specific antibodies and long-lived plasma cells to fully clear infection. GC-Tfh numbers remain stable throughout P. chabaudi infection, while Th1/Tfh hybrid (IFN-g +/IL-21 +CXCR5 int) cells expand. Testing if triple cytokine reporter mice (Il21-Kat Il4-GFP Ifng/Thy1.1 Knock-In) report Tfh functionality accurately, we confirmed that Tfh-like Il-21 +Ifng −cells were enriched for GC Tfh (CXCR5 hiPD-1 hiCXCR6 −) while the majority of Ifng +Il21 +cells were CXCR5 intCXCR6 +. GC-Tfh appear functional sooner than Ifng +Il21 +with expression of CCR7 intand Il4 +as early as day 5p.i. Transfer of cytokine-expressing subsets from triple reporter and Il21 −/−Ifng-Thy1.1 KI mice into T cell deficient recipients, which are then infected with P. chabaudi, also shows that Il21 +Ifng −Tfh-like cells from day 8 of P. chabaudi infection are functional. Il21 +Ifng −cell recipients generated more GC B cells, larger GCs and more parasite-specific IgG compared to other groups. Il21 +Ifng +hybrid T cells, also promoted GCs, but Th1-like Ifng +T cells from WT or Il21 −/−mice did not. As GCs were larger, but not more numerous, in Il21 +Ifng −recipient spleens, we hypothesized that B cell proliferation was increased. Indeed, there were more BrdU +dark zone (CXCR4 +CD86 −) GCB cells in recipients of Il21 +Ifng −cells than other groups, particularly recipients of Ifng +T cells from IL-21 KO. Combined, this data suggests that IL-21 from hybrid Th1/Tfh promotes GCB cells in vivo, though less than Il21 +Ifng −T cells. R01AI13506101

Marginal Zone B Cells Facilitate CD4 T Cell Proliferation in Response to Factor VIII in Mice with Hemophilia Α

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies represents a significant barrier to FVIII replacement therapy in patients with hemophilia A. Despite this, the immune factors that regulate anti-FVIII antibody formation remain incompletely understood. We have previously shown that shortly after injection, FVIII co-localizes with marginal zone (MZ) B cells in the marginal sinus of the spleen in mice with hemophilia A. Moreover, mice depleted of MZ B cells did not produce detectable anti-FVIII IgG antibodies. Given the ability of MZ B cells to trap and rapidly respond to blood-borne antigens, produce both IgM and IgG antibodies and activate CD4 T cells, while also trafficking antigen to B cell follicles, these results suggest that MZ B cells likely play a critical role in the initiation and subsequent orchestration of an immune response to FVIII. In contrast to other model antigens, anti-FVIII antibody formation occurs only following multiple FVIII exposures in both human and mouse models. As CD4 T cells facilitate long-lasting antibody formation, and have previously been shown to play an important role in anti-FVIII antibody development (Bray GL, et al. Am J Hematol 1993; Reding MT, et al. Thromb Haemost 2000), it is possible that each FVIII exposure event eventually propagates a CD4 T cell response to a threshold necessary to generate an optimal antibody response. Consistent with this, we have previously shown that FVIII-specific CD4 T cell proliferation requires more than 1 prior exposure to FVIII. An improved understanding of the overall timing and factors influencing CD4 T cell activation in the immune response to FVIII is necessary for the development of strategies to prevent antibody formation. OBJECTIVE: Define the key factors that regulate CD4T cell activation following FVIII exposure and the number of exposure events required to observe FVIII-specific CD4 T cell proliferation. METHODS: Hemophilia A mice +/- CD4T cell depletion or MZ B cell depletion or S1PR1-TSS mice were challenged with 1-4 weekly exposures to FVIII. Anti-FVIII IgM and IgG was examined in plasma by enzyme-linked immunoassay (ELISA). To assess FVIII-specific CD4T cell proliferation, we engineered FVIII-OVA, which contains the ovalbumin peptide recognized by OTII T cell receptor transgenic T cells. CFSE-labeled OTII splenocytes were adoptively transferred into mice with hemophilia A, mice lacking B cells and MZ B cell-depleted mice, followed by administration of FVIII-OVA. RESULTS: Anti-FVIII IgG formation is CD4T cell-dependent, however IgM is readily produced in the absence of CD4T cells. In contrast, MZ B cell depletion prevented the formation of both IgM and IgG antibodies against FVIII. FVIII is unable to cause proliferation of CD4T cells or produce IgG antibodies following initial exposure, but instead significantly increases following the 3rd exposure to FVIII. Mapping the kinetics of antibody formation has uncovered that anti-FVIII IgM formation peaks and anti-FVIII IgG formation begins after the 3rd FVIII exposure, at the time of CD4 T cell proliferation. We next examined the influence of B cells on CD4 T cell proliferation, and observed that FVIII-specific CD4T cell proliferation is significantly impaired in B cell-deficient recipients. Moreover, in mice who have undergone MZ B cell depletion, there is likewise an absence of anti-FVIII IgM and IgG and a lack of measurable CD4 T cell proliferation. Finally, in S1PR1-TSS mice with MZ B cells that are unable to traffic antigen to the B cell follicle, anti-FVIII IgM and IgG formation occurs at the same rate as control mice. CONCLUSION: In summary, our findings suggest that MZ B cells are required for anti-FVIII IgM and IgG antibody formation as well as FVIII-specific CD4 T cell proliferation, which requires 3 antigen exposures. The finding that MZ B cells are not required to traffic antigen to the B cell follicle contributes to our hypothesis that MZ B cell production of IgM, with or without direct activation of CD4 T cells contributes to FVIII-specific CD4 T cell activation. These results suggest that targeted approaches designed to prevent anti-FVIII antibody formation may be most effectively achieved by defining key factors that regulate MZ B cell activation following initial FVIII exposure. In doing so, the present findings and future work will likely uncover important pathways to effectively target to reduce, or event prevent anti-FVIII antibody production.

Murine Cytomegalovirus-induced Complement-fixing Antibodies Deposit in Murine Renal Allografts During Acute Rejection.

Human cytomegalovirus (CMV) infection is associated with renal allograft dysfunction and loss, particularly in combination with acute rejection. Emerging literature suggests that non-HLA antibodies may contribute to antibody-mediated rejection, but pathogen-induced antibodies have not been investigated in this context. This study examines the presence of CMV-induced antibodies in murine CMV (MCMV)-infected renal allografts during acute rejection. Intragraft immunoglobulin G (IgG) and complement C3 immunostaining were compared among allogeneic MCMV D-/R-, D+/R-, and D+/R+ renal transplants. Intragraft antibody deposition was examined in B cell-deficient recipients treated with MCMV immune sera. Antibody binding and complement-dependent cytotoxicity (CDC) of D-/R- and D+/R+ sera against infected renal tubular epithelial cells (TECs) were measured in vitro. IgG immunostaining was performed in D+/R+ allografts and native kidneys and in D+/R- allografts treated with ganciclovir to inhibit viral replication. D+/R- and D+/R+ transplants had more abundant IgG and C3 deposition compared with D-/R- recipients. Greater IgG deposition was associated with more severe allograft injury in B cell-deficient recipients treated with MCMV immune sera compared with nonimmune sera. D+/R+ sera induced greater CDC of infected TECs compared with D-/R- sera. Native kidneys had lower IgG deposition compared with allografts, despite similar organ viral loads. Ganciclovir-treated allografts had reduced IgG deposition compared with untreated allografts. In this murine model, complement-fixing antibodies can deposit into MCMV-infected renal allografts, are associated with allograft damage, and can induce CDC of MCMV-infected renal TECs. The allogeneic response and viral replication may also contribute to intragraft antibody deposition.

Marginal Zone B Cells Facilitate RBC Alloimmunization Independent of Direct CD4+ T Cell Activation

Background: Blood transfusions decrease complications in patients with bone marrow failure syndromes, hemoglobinopathies, or neoplasia. However, patients who receive this often life-saving treatment can develop alloantibodies against non-ABO red blood cell (RBC) antigens. RBC alloimmunization can have severe clinical consequences, including increased morbidity and mortality secondary to difficulty obtaining compatible RBCs for future transfusion and an increased risk for hemolytic transfusion reactions.Aside from Rh(D) immune globulin, there are no prophylactic treatments available to prevent RBC alloimmunization. Improved understanding of the immune mechanism underlying RBC alloimmunization is central in developing additional prophylactic treatments. Recent studies suggest that marginal zone (MZ) B cells are critical for immune responses to RBC alloantigens. As a result, we sought to determine mechanisms whereby MZ B cells mediate RBC alloimmunization.Methods: RBCs expressing the HOD antigen [hen egg lysozyme, ovalbumin (OVA), and human Duffy] were labeled with DiI and transfused into C57BL/6 recipients, followed by confocal examination for HOD RBC localization by staining sections for CD1d, MadCAM and B220. HOD RBCs were also transfused into C57BL/6 recipients treated with anti-CD4 T cell (clone: GK 1.5), anti-MZ B cell (anti-CD11a and anti-CD49d) or isotype control followed by examination of anti-HOD IgM and IgG antibodies on days 5 and 14 post-transfusion by flow cross-match. To determine the impact of B cells in general and MZ B cells in particular on CD4+ T cell activation following HOD RBC transfusion, CFSE labeled splenocytes from OTII transgenic mice with CD4+ T cells specific for HOD RBCs were adoptively transferred into B-cell deficient μMT mice, MZ B cell depleted C57BL/6 recipients, C57BL/6 controls, and mice genetically deficient in MZ B cells followed by transfusion of HOD RBCs. On days 1 and 3 post-transfusion, OTII proliferation, activation and cytokine profile were assessed using flow cytometric analysis. To examine the role of MZ B cells in antigenic trafficking, C57BL/6 recipients were administered FTY720, which displaces MZ B cells from the marginal sinus, or PBS, followed by HOD RBC transfusion and analysis for anti-HOD antibody formation.Results: HOD RBCs initially localized within the marginal sinus following transfusion, where they displayed significant co-localization with MZ B cells. While depletion of MZ B cells inhibited anti-HOD IgM and IgG antibody formation, CD4+ T cell depletion only prevented anti-HOD IgG antibody development. Despite the requirement of MZ B cells in anti-HOD antibody formation, CFSE-labeled OTII CD4+ T cells showed proliferation as well as evidence of key markers of T cell activation following transfusion of HOD RBCs into MZ B cell depleted, genetically deleted or B cell deficient μMT recipients, strongly suggesting that the requirement of MZ B cells for IgG antibody formation does not reflect MZ B cell-mediated CD4+ T cell activation. Given the localization of HOD RBCs with MZ B cells and the ability of CD4+ T cells to proliferate in the absence of MZ B cells, we next determined whether the requirement of MZ B cells for anti-HOD IgG antibody formation may actually reflect MZ B cell-mediated trafficking of HOD RBCs to the B cell follicle. Consistent with this possibility, MZ B cell displacement from the marginal sinus to the splenic follicle, which prevents MZ B cell-mediated trafficking of antigen, prevented formation of anti-HOD IgM and IgG following HOD RBC transfusion.Conclusion: The ability of CD4+ T cells to undergo proliferation and activation following HOD RBC transfusion in the absence of MZ B cells suggests that the requirement of MZ B cells for antibody formation does not reflect MZ B cell-mediated CD4+ T cell activation. In contrast, the localization of HOD RBCs with MZ B cells, coupled with the ability of MZ B cell displacement to prevent HOD RBC-induced alloimmunization, strongly suggests that MZ B cells regulate RBC alloimmunization by initially trapping and the trafficking RBC alloantigens to the B cell follicle. In doing so, MZ B cells appear to be a key initiator and overall regulator of RBC alloimmunization and therefore a potential target for prophylactic measures designed to inhibit this process. DisclosuresNo relevant conflicts of interest to declare.

B-cell Deficiency Attenuates Transplant Glomerulopathy in a Rat Model of Chronic Active Antibody-mediated Rejection.

Transplant glomerulopathy (TG) is a pathological feature of chronic active antibody-mediated rejection (cAMR) and is associated with renal allograft failure. The specific role of B cells in the pathogenesis of TG is unclear. We used a minor mismatched rat kidney transplant model with B cell-deficient recipients, generated by clustered regularly interspaced short palindromic repeats/Cas9 technology, to investigate the impact of B-cell depletion on the pathogenesis of TG. We hypothesized that B-cell deficiency would prevent TG in the rat kidney transplant model of cAMR. Treatment groups included syngeneic, allogeneic, sensitized allogeneic, and B cell-deficient allogeneic transplant recipients. B cell-deficient recipients demonstrated reduced TG lesions, decreased microvascular inflammation, reduced allograft infiltrating macrophages, and reduced interferon gamma transcripts within the allograft. Allograft transcript levels of interferon gamma, monocyte chemoattractant protein-1, and interleukin-1β correlated with numbers of intragraft macrophages. B cell-deficient recipients lacked circulating donor-specific antibodies and had an increased splenic regulatory T-cell population. In this model of cAMR, B-cell depletion attenuated the development of TG with effects on T cell and innate immunity.

Microbial exposure in early life alters the ontogeny and function of CD8+ T cells

Abstract Evidence has shown that microbial exposure in early life can have long-lasting effects on the health status of an individual. However, the underlying basis of how microbes shape the immune system is not well understood. In this study, we utilized a novel pet shop mouse model and compared the immune development and function of laboratory mice raised in either a clean (SPF) or dirty (pet-shop) environment. When we challenged the two groups with an intracellular pathogen in adulthood, we found that mice raised in the dirty environment mounted a more robust CD8+ T cell response and were highly resistant to infection. Adoptive transfer experiments with CD8+ T cells from clean and dirty mice into T cell deficient recipients indicated that differences in immune susceptibility were linked to changes in the CD8+ T cell compartment. Moreover, the dirty mice had an increased accumulation of antigen-specific CD8+ T cells and produced more effector molecules. To understand how the ontogeny of the CD8+ T cell compartment was altered in the dirty mice, we used our fate-mapping timestamp mice and found a higher proportion of fast-acting fetal-derived CD8+ T cells and fewer slow-acting adult-derived CD8+ T cells present in the dirty mice. We found that immune susceptibility could be normalized between clean and dirty mice by depleting the fetal layer of CD8+ T cells. Collectively, these findings suggest that microbial exposure leads to permanent changes in the immune system by altering the developmental layering of the peripheral T cell compartment. We are currently using a combination of RNA- and ATAC-seq to gain a deeper understanding of how the microbial environment mediates changes to CD8+ T cells at the gene regulatory level.

Pertussis toxin inhibits encephalitogenic T cell infiltration and promotes a B cell driven disease during TH17-EAE

Abstract Pertussis toxin (PTX) is required as a co-adjuvant to induce disease in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the role of PTX during adoptive transfer TH17-EAE and observe alterations in the disease phenotype. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested from MOG35-55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. The recipient mice also received PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX significantly reduced disease severity and delayed onset of paralysis. PTX reduced accumulation of pathogenic T helper cells and myeloid cells in the CNS, which we found to be associated with PTX inhibition of chemokine receptor dependent trafficking. Strikingly, our data shows that PTX promotes the accumulation of B cells in the CNS and elevates the capacity of B cells to present antigen to T cells in the periphery, suggesting an altered disease toward a B cell-dependent pathology. We found that without PTX treatment, the severity of disease was equivalent between WT and μMT mice. In contrast, with PTX treatment, the μMT mice had significantly less disease compared to the WT mice. In conclusion, our data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study the pathologic functions of B cells during multiple sclerosis.

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

B cell-derived IL-1β and IL-6 drive T cell reconstitution following lymphoablation.

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell-derived soluble factors. B cell production of interleukin (IL)-1β and IL-6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL-1β or IL-6 with mAb or the use of recipients lacking mature IL-1β, IL-6, IL-1R, MyD88, or IL-6R impair CD4+ and CD8+ T cell recovery and significantly enhance the graft-prolonging efficacy of lymphoablation. Adoptive co-transfer experiments demonstrate a direct effect of IL-6 but not IL-1β on T lymphocytes. Furthermore, B cells incapable of IL-1β or IL-6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell-derived IL-1β and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.

Pertussis toxin treatment promotes a B cell driven disease during TH17 EAE

Abstract Pertussis toxin (PTX) is an enigmatic co-adjuvant that is required for disease induction in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the functional role of PTX during adoptive transfer TH17-EAE. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested and cultured from MOG35–55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. Recipient mice were treated with PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX delayed disease onset but did not alter disease severity or incidence. We found that PTX reduced trafficking of myeloid cells and inflammatory T helper cells in the CNS. Importantly, PTX treatment increased the number of B cells in the brain with increased antigen presenting function, suggesting an altered disease toward a B cell-dependent pathology. When disease progression was compared between WT and μMT mice treated with PTX or PBS, there was no significant difference in TH17-EAE in the PBS treated groups. However, PTX treatment significantly delayed disease onset, severity and incidence in the μMT mice vs. WT. In conclusion, our current data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype and therefore promotes a B cell driven disease. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study B cell activity during multiple sclerosis.

Induction of germ cell-deficiency in grass puffer by dead end 1 gene knockdown for use as a recipient in surrogate production of tiger puffer

The surrogate broodstock technique produces donor-derived gametes via transplantation of germ cells into surrogate fish. The technique is a promising approach for improving the establishment and management of aquaculture strains with desirable genetic traits. The tiger puffer (Takifugu rubripes) is one of the most popular aquaculture fishes in Japan. However, brood stocks have large body sizes and require a long time to reach sexual maturity. The current study established a sterilization technique using gene knockdown in the grass puffer (T. alboplumbeus), which has a small body size and matures in half the time taken by the tiger puffer, and assessed the possibility of using germ cell-deficient grass puffers as recipients to efficiently produce donor-derived tiger puffer gametes. Dead end 1, which has two transcribed variants, was identified in the grass puffer. Morphants resulting from the microinjection of antisense morpholino oligonucleotides against the transcribed variants, into fertilized eggs, showed germ cell deficiency. Germ cell-deficient morphants exhibited sexual dimorphism with respect to their morphological and gene expression patterns. Testicular germ cells prepared from the testes of tiger puffer were intraperitoneally transplanted into morphant hatchlings. These recipients matured normally and produced functional donor-derived gametes separately from endogenous gametes. Thus, the use of germ cell-deficient recipients that produce only donor-derived gametes improves the efficiency of surrogate production and may accelerate the breeding process in tiger puffer aquaculture.

P155 THERAPEUTIC EFFECT OF OPS-2071 ON HEALING OF T CELL-MEDIATED COLITIS, A MURINE MODEL OF CROHN’S DISEASE, WITH ANTI-INFLAMMATORY AND ANTIBACTERIAL ACTIVITY

Abstract Background Crohn’s disease (CD) is a form of chronic inflammatory bowel disease (IBD). Although CD is an immune-mediated condition of unknown etiology, many studies suggested that abnormal immune responses against certain intestinal bacteria may trigger development of chronic inflammation. The T cell-mediated colitis model is a well characterized adaptive transfer murine model of chronic small bowel and colonic inflammation that resembles human CD (e.g., diarrhea, a heavily inflamed colon, loss of mucus from goblet cells, Th1/Th17 dominated cytokine profile). OPS-2071, a novel agent synthesized by Otsuka Pharmaceutical Co., Ltd, has broad and strong antibacterial activity with low systemic absorption. In this study, we have evaluated the therapeutic effect of OPS-2071 on murine T cell-mediated colitis and in vitro activity on T cell activation, cytokine production and antibacterial activity. Methods T cell-mediated colitis was induced via naive T cell (CD4+CD62L+CD44-) injection into T and B cell deficient recipient mice. Two weeks following T cell transfer, OPS-2071 or vehicle solution was administered orally for 3 weeks, followed by harvesting of colon tissue and assessment of efficacy evaluation by histological score and inflammatory index (colon weight/ length). In vitro inhibitory action for TNF-α production was assayed by ELISA using LPS-stimulated human peripheral blood cells. The effect on T cell activation and cytokine production (TNF-α, IFN-γ), was examined using human peripheral blood mononuclear cells and, mouse spleen which were stimulated by anti-CD2/CD3/CD28 antibody-loaded beads. In vitro activity against bacteria considered to be the cause of IBD was also tested. Results In the murine T cell transfer model, OPS-2071 significantly reduced both histological score (control: 9.2, OPS: 2.7, p<0.001) and inflammatory index (control: 54.7±2.2 mg/cm, OPS: 27.8±1.7 mg/cm, p<0.001) at a dose of 10 mg/kg. In the in vitro experiments, OPS-2071 suppressed TNF-a production produced by LPS in human whole blood dose-dependently. OPS-2071 also suppressed human and mouse T cell activation and cytokine production and suppressed T cell proliferation. At high OPS-2071 concentrations, these effects were comparable to prednisolone. The Minimum Inhibitory Concentration against IBD related bacteria for OPS-2071, ciprofloxacin, and metronidazole were 0.015 - 0.5, 0.25 - 8, and 0.03 - >128 mg/mL, respectively. Conclusion OPS-2071 demonstrated significant therapeutic effects on colon inflammation in the murine T cell-medicated colitis model, suppressed TNF-a production in vitro and showed in vitro activity against bacteria related to CD. The dual effect of anti-inflammatory effects and antibacterial activity of the novel agent OPS-2071 demonstrated in this study, provide rationale for exploring the impact of this compound on human CD in clinical trials.

CD4+ T lymphocytes produce adiponectin in response to transplants.

Adiponectin is a pleiotropic cytokine with diverse immunomodulatory effects on macrophages and lymphocytes. In the current paradigm, lymphocytes and macrophages respond to adiponectin that is produced by adipocytes and other parenchymal cells. Using a model of chronic arterial inflammation in cardiac transplants, we found that T cells derived from the recipient migrate to the heart and produce adiponectin locally. The evidence that T cells produce significant amounts of adiponectin is based on 3 experimental approaches. First, CD4+ T cells isolated from the blood and spleen after cardiac transplantation express mRNA for adiponectin. Second, reconstitution of T cell-deficient recipients with transgenic CD4+ T cells that express receptors for donor antigens results in arterial infiltrates containing T cells and increased mRNA expression for adiponectin in cardiac transplants. Third, CD4+ T cells isolated from the allograft secrete adiponectin in vitro. Taken together, these data indicate that adiponectin-competent cells originating in the recipient migrate into the transplant. Establishing T cells as a source of adiponectin provides a new dimension, to our knowledge, to the modulatory effects of adiponectin on immune responses.

Neonatal CD8+ T cells provide innate immune protection against unrelated pathogens

Abstract We previously found that neonatal and adult CD8+ T cells adopt different fates after infection with specific pathogens because they are derived from a distinct progenitor cell that is distinguished by expression of Lin28b. In this report, we investigated if CD8+ T cells respond differently to unrelated pathogens in early life. To start, we compared gene expression profiles of neonatal and adult CD8+ T cells after stimulation with innate cytokines (IL-12 + IL-18). Interestingly, we found that neonatal CD8+ T cells are more responsive and express a distinct cytokine profile compared to their adult counterparts. To examine the biological significance of these age-related differences, we next adoptively transferred monoclonal populations of neonatal or adult CD8+ T cells into T cell-deficient recipients and compared their ability to mediate immune protection against an irrelevant strain of Listeria monocytogenes. Surprisingly, we found that neonatal donor cell displayed increased proliferation and effector functions, and the pathogen burden was significantly reduced when compared to recipients containing adult donor cells. We also found that Lin28b may be responsible for the enhanced responsiveness to innate cytokines in early life, as ectopic expression of Lin28b in adult cells enabled them to initiate a program of bystander activation that was analogous to neonates. Collectively, these findings indicate that neonatal CD8+ T cells possess innate-like functions during infections at the cost of generating memory, and these unique behaviors may be driven by high levels of Lin28b. We currently are employing ATAC-seq to determine whether Lin28b changes the program of bystander activation by altering the chromatin landscape.

Marginal Zone B Cells Regulate RBC Alloimmunization Toward Distinct RBC Alloantigens

Background: While red blood cell (RBC) transfusion can be beneficial, exposure to allogeneic RBCs can result in the development of RBC alloantibodies that can make it difficult to obtain compatible RBCs for future transfusions. Aside from phenotype matching protocols, no strategy currently exists that is capable of preventing RBC alloimmunization following therapeutic transfusion. As RBC alloantigens represent diverse determinants capable of driving distinct immune pathways, common immunological nodes must be identified in order to successfully prevent RBC alloimmunization against a variety of different alloantigens. Recent results demonstrate that marginal zone (MZ) B cells mediate anti-KEL antibody formation in the complete absence of CD4 T cells. However, whether MZ B cells similarly regulate RBC alloantibody formation against other RBC alloantigens remains unknown. As a result, we examined the role of MZ B cells and CD4 T cells in the development of RBC alloantibodies following exposure to the HOD (hen egg lysozyme, ovalbumin and duffy) antigen.Methods: Each recipient was transfused with HOD or KEL RBCs following either MZ B cell or CD4 T cell depletion using a cocktail of MZ B cell (anti-CD11a and anti-CD49d) or anti-CD4 depleting antibody, 4 and 2 days prior to transfusion. Control groups received isotype control injections in parallel. MZ B cell deficient (CD19cre/+ X Notch2flx/flx) and CD4 T cell deficient (MHC class II knockout) recipients were also used to examine the role of MZ B cells and CD4 T cells, respectively. Serum collected on days 5 and 14 post-transfusion was evaluated for anti-HOD or anti-KEL antibodies by incubating HOD or KEL RBCs with serum, followed by detection of bound antibodies using anti-IgM and anti-IgG and subsequent flow cytometric analysis. Evaluation of antibody engagement and overall survival of HOD or KEL RBCs was accomplished by labeling RBCs with the lipophilic dye, DiI, prior to transfusion, followed by examination for bound antibody and RBC clearance on days 5 and 14 post-transfusion by flow cytometry.Results: Similar to the ability of MZ B cell depletion to reduce anti-KEL antibody formation following KEL RBC exposure, depletion of MZ B cells significantly reduced anti-HOD IgM and IgG antibodies following HOD RBC transfusion. In contrast, injection of recipients with isotype control antibodies in parallel failed to prevent alloantibody formation following HOD or KEL RBC transfusion. Similar results were obtained following HOD or KEL RBC transfusion into recipients genetically deficient in MZ B cells. In contrast, although MZ B cells were required for HOD and KEL RBC-alloantibody formation, manipulation of CD4 T cells differentially impacted the ability of each antigen to induce alloantibodies. While transfusion of HOD or KEL RBCs resulted in robust IgM alloantibodies in the absence of CD4 T cells, depletion or genetic elimination of CD4 T cells significantly inhibited anti-HOD IgG antibody formation, while failing to impact IgG anti-KEL antibody formation. Consistent with this, while manipulation of CD4 T cells protected HOD RBCs from antibody deposition and subsequent RBC clearance, this same approach failed to similarly protect KEL RBCs following transfusion. In contrast, depletion of MZ B cells not only prevented detectable alloantibody production, but also completely protected HOD or KEL RBCs from antibody deposition and subsequent RBC clearance.Conclusion: These results suggest that while MZ B cells mediate a robust IgM antibody response following either KEL or HOD antigen exposure, MZ B cells appear to possess the capacity to orchestrate unique downstream IgG responses through CD4 T cell dependent and independent pathways contingent on target alloantigen. As a result, while manipulation of CD4 T cells may prevent alloantibody formation against some antigens, targeting this immune population inadequately prevents RBC alloantibody formation against all RBC antigens. As chronic transfusion therapy exposes recipients to a wide variety of alloantigens, these results suggest that MZ B cells may represent a central initiating node that governs RBC alloimmunization against a variety of RBC alloantigens, and may therefore serve as a useful target in preventing alloantibody formation in chronically transfused individuals. DisclosuresNo relevant conflicts of interest to declare.

Production of germ cell‐deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor-derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell-deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell-deficient rainbow trout did not show a male-biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell-deficient and control recipients. The donor-derived germ cells showed significantly higher proliferation in the gonads of germ cell-deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell-deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell-deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end-knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+ CD45RB high T Cells into Immunodeficient Mice.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4(+)CD45RB(high) T cells into T and B cell deficient recipient mice. The CD4(+)CD45RB(high) T cell population that largely consists of naïve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Itk signals promote neuroinflammation by regulating CD4+ T-cell activation and trafficking.

Here we demonstrate that interleukin-2-inducible T-cell kinase (Itk) signaling in cluster of differentiation 4-positive (CD4(+)) T cells promotes experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). We show that Itk(-/-) mice exhibit reduced disease severity, and transfer of Itk(-/-) CD4(+) T cells into T cell-deficient recipients results in lower disease severity. We observed a significant reduction of CD4(+) T cells in the CNS of Itk(-/-) mice or recipients of Itk(-/-) CD4(+) T cells during EAE, which is consistent with attenuated disease. Itk(-/-) CD4(+) T cells exhibit defective response to myelin antigen stimulation attributable to displacement of filamentous actin from the CD4(+) coreceptor. This results in inadequate transmigration of Itk(-/-) CD4(+) T cells into the CNS and across brain endothelial barriers in vitro. Finally, Itk(-/-) CD4(+) T cells show significant reduction in production of T-helper 1 (Th1) and Th17 cytokines and exhibit skewed T effector/T regulatory cell ratios. These results indicate that signaling by Itk promotes autoimmunity and CNS inflammation, suggesting that it may be a viable target for treatment of MS.

CD8+ T Cells Mediate Antibody-Independent Platelet Clearance Following Transfusion

Background: Alloimmunization to major histocompatibility complex (MHC) antigens is the leading immunological barrier to transplantation and platelet transfusion therapy. In the context of platelet transfusion medicine, alloimmunization to MHC antigens can substantially diminish the therapeutic efficacy of subsequent blood transfusions. Presently, MHC alloantibodies are recognized as the primary mediators of immune-mediated platelet clearance. However, individuals previously exposed to MHC alloantigens can display significant platelet clearance in the absence of detectable anti-platelet antibodies. Given the ability of cellular immunity to mediate rejection of solid organ allografts across MHC differences, we hypothesized that cellular immunity likewise facilitates the clearance of platelets in MHC alloimmunized recipients.Methods: FVB (H-2Kq) MHC-immunized or non-immunized C57BL/6 (H-2Kb) recipients were transfused with filter leukoreduced platelet rich plasma (PRP) isolated from C57BL/6 donors expressing green fluorescent protein (GFP) under a H-2Kb promoter (B6 GFP) or PRP obtained from a FVB X B6 GFP F1 cross, which generates GFP+ H-2Kq+ platelets. Following transfusion, mice were bled at 10 minutes, 1 hour, 2 hours or 24 hours to calculate the percentage of GFP+ transfused platelets remaining using flow cytometric analysis. To test the hypothesis that platelet clearance can occur in an antibody-independent process, FVB H-2Kq MHC-immunized or non-immunized μMT recipients were similarly transfused with B6 GFP or FVB X B6 GFP PRP. CD8+ T cells in immunized μMT recipients were depleted by i.p. injection of anti-CD8 (clone 2.43) antibody for 3 consecutive days prior to transfusion. NK cells were similarly depleted by injection of anti-NK1.1 (clone PK-136) antibody, 1 day prior to transfusion. CD4, CD8 T cell and NK cell depletion was evaluated in the peripheral blood prior to platelet transfusion, and compared to non-treated and isotype control treated recipients.Results: While GFP+ platelets were readily detected following transfusion into non-immunized or MHC matched recipients, transfusion of FVB-B6 platelets into H-2Kq MHC-immunized C57BL/6 recipients resulted in rapid clearance within the first hour following transfusion. In contrast, though little platelet clearance was detected in H-2Kq MHC-immunized μMT recipients within an hour following transfusion, very few transfused platelets were observed after 24 hours. Moreover, despite platelet clearance, anti-MHC antibodies were not detectable in μMT recipients. To determine the cellular component responsible for clearance in alloimmunized μMT recipients, CD8+ T cells or NK cells were depleted prior to transfusion. NK cell depletion failed to impact platelet clearance in alloimmunized μMT recipients; however, depletion of CD8+ T cells prior to platelet transfusion abrogated platelet clearance in immunized μMT recipients.Conclusion: Immunized recipients rapidly clear platelets in an alloantigen specific fashion following platelet transfusion in a murine model. Though platelet clearance occurred at a faster rate in intact immunized C57BL/6 recipients, B cell deficient MHC-immunized μMT recipients possessed the capacity to induce significant clearance at later time points. Evaluation of the cellular populations responsible for antibody-independent clearance in μMT recipients strongly suggest that CD8+ T cells play a key role in antibody-independent immune-mediated platelet clearance. These results suggest that, in addition to antibodies, CD8+ T cells can play a role in the development of platelet refractoriness and may contribute to platelet non-responsiveness in patients with little to no detectable anti-platelet alloantibodies. DisclosuresNo relevant conflicts of interest to declare.

TIM-4 expression by B cells identifies an inflammatory B effector 1 subset that promotes allograft rejection and inhibits tumor metastases (IRC3P.471)

Abstract B cells, like T cells, give rise to polarized subsets, termed Be1, Be2 and Breg that produce IFNγ, IL-4 and IL-10, respectively. We previously reported that TIM-1 is an inclusive marker for Be2 cells and Breg that can transfer allograft tolerance. We now show that TIM-4, a TIM-1 ligand, is expressed by a distinct subpopulation (~6-10%) of B cells. TIM-4 B cells are enriched for IFNγ (2.5%) and express relatively less IL-4 (1%) and IL-10 (4%) while TIM-1 B cells are enriched for IL-4 (6.5%) and IL-10 (14%) and express less IFNγ (1%). Accordingly, transfer of TIM-4 B cells to B cell deficient (JhD) islet allograft recipients accelerated rejection (MST 11d) vs. controls (no B cells; MST 15d) or TIM-1 B cell transfer (MST 70d), p<0.05. TIM-4 B cell-mediated rejection required IFNγ as transfer of IFNγ-/- TIM-4 B cells modestly prolonged MST (15d to 25d, p<0.05). In this model, transfer of TIM-4 B cells enhanced IFN-γ+ T cells (1.5X) while decreasing IL-4+, IL-10+ and Foxp3+ T cells (1.2-1.8X) whereas TIM-1 B cells increased IL-4+, IL-10+ and Foxp3+ T cells (1.2-1.7X) while decreasing IFNγ+ T cells (1.4X) vs. no B cell controls (p<0.05 for each). Moreover, transfer of TIM-4 B cells into μMT mice decreased B16 melanoma lung metastases ~ 2-fold, while TIM-1 B cells increased lung metastases ~2-fold vs. controls (p < 0.0001). Thus, we provide strong evidence that TIM-4 expression by B cells identifies a distinct Be1 inflammatory subset that can directly impact T cells responses.