Immunological Characterization of the Teleost Adipose Tissue and Its Modulation in Response to Viral Infection and Fat-Content in the Diet

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α⁺ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM⁺ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α⁺ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet.     

Contrasted TCRβ Diversity of CD8+ and CD8? T Cells in Rainbow Trout

Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8⁺ T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8⁺ and CD8⁻ T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8⁺ T cells of naïve fish, it appeared very different in CD8⁻ lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8⁺ T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8⁻ fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8⁺ and CD8⁻ αβ T cells may be subjected to different regulatory patterns in fish and in mammals.     

Recent findings on the structure and function of teleost IgT

As key effector molecules of jawed vertebrate’s adaptive immune system, immunoglobulins are produced by B lymphocytes, either as a secretory form (antibody) or as a membrane form (B cell receptor). Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD. In addition, IgM in these species was thought to be the only immunoglobulin isotype responding to pathogens both in systemic or mucosal compartments. However, the unexpected discovery of IgT, a new teleost immunoglobulin unearthed in 2005, has provided for new opportunities to analyze further roles of teleost immunoglobulins in these two physiologically distinct compartments. The smoke about the potential function of IgT has cleared recently with the finding that this immunoglobulin appears to be specialized in gut mucosal immunity. Significantly, the new capability of measuring not only IgM but also IgT responses will greatly facilitate the evaluation and understanding of fish immune responses as well as the protective effects of fish vaccines. The purpose of this review is to summarize the molecular characterization of new IgT orthologs and subtypes in teleosts, as well as to describe the new findings concerning the protein structure of IgT, the B cells producing it, and its role in mucosal immunity.     

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG⁺ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS⁺ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.     

硬骨鱼新型免疫球蛋白的研究进展

鱼类是最早出现免疫球蛋白的动物,鱼类免疫球蛋白在鱼类的特异性体液免疫应答中发挥重要的作用。一直以来,人们认为在硬骨鱼中仅存在IgM和IgD两种免疫球蛋白,而2005年以来,陆续在斑马鱼、虹鳟鱼及鲤鱼等硬骨鱼中发现了新型免疫球蛋白,分别命名为IgZ、IgT及IgM-IgZ等。这些新型免疫球蛋白不仅在基因结构上很特别,而且呈现出多样性,在不同种的硬骨鱼中的功能也不完全相同,同一种鱼中的IgT也呈现多样性。虽然目前对于鱼类新型免疫球蛋白的研究刚刚起步,对其功能了解较少,但有研究表明IgT在硬骨鱼的粘膜免疫中发挥重要的作用,且认为它与IgA是同源的。该文拟对硬骨鱼中发现的新型免疫球蛋白的结构特点、基因组成和分布模式及功能差异作一简要综述。     

Augmentation of the Antibody Response of Atlantic Salmon by Oral Administration of Alginate-Encapsulated IPNV Antigens

The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1) alginate-encapsulated IPNV antigens (ENCAP); 2) soluble antigens (UNENCAP) or 3) untreated feed (control). This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1^st oral boost and the 2^nd, at 4 weeks after the 2^nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-β and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p<0.04) in serum antibodies following the 1^st boost but not after the 2^nd boost. This coincided with significant up-regulation of CD4 and GATA3 genes. In contrast, serum antibodies in the UNENCAP group decreased both after the 1^st and 2^nd oral boosts. This was associated with significant up-regulation of FOXP3, TGF-β and IL-10 genes. The expression of IgT was not induced in the hindgut after the 1^st oral boost but was significantly up-regulated following the 2^nd one. CD4 and GATA3 mRNA expressions exhibited a similar pattern to IgT in the hindgut. IgM mRNA expression on the other hand was not differentially regulated at any of the times examined. Our findings suggest that 1) Parenteral prime with oil-adjuvanted vaccines followed by oral boost with ENCAP results in augmentation of the systemic immune response; 2) Symmetrical prime and boost (mucosal) with ENCAP results in augmentation of mucosal immune response and 3) Symmetrical priming and boosting (mucosal) with soluble antigens results in the induction of systemic immune tolerance.     

Active idiotypic vaccination in a patient with biclonal follicular lymphoma

Specific immunological responses to the idiotypic epitopes present in the surface immunoglobulin (Ig) of the clonal tumour population can be induced for active immunotherapy in patients with B-cell non-Hodgkin lymphoma (NHL). The clonality of the tumour cells should have important implications for the success of the implemented therapy. Here we report on the case of a patient enrolled in a protocol of active idiotypic immunotherapy in which previous cytofluorometric analysis showed a major IgM⁺, κp⁺ population in the tumoral cell suspensions. However, sequence analysis of both tumour sample and tumour-derived hybrids revealed the presence of two unrelated clones that used different VH and VK gene segments. It was possible to obtain hybridomas secreting these two different IgM, κp idiotypic proteins. The patient was immunised with a mixture of these two idiotypic Igs conjugated to keyhole limpet haemocyanin. Anti-idiotypic antibodies directed against both tumour-associated proteins were detected. This is the first case of anti-idiotypic therapy in a patient with a biclonal NHL. Our work calls attention to the question of clonality in the context of idiotypic vaccination in NHL patients. Received: 18 May 2000 / Accepted: 13 December 2000     

Dietary omega-3 fatty acids enhance the B1 but not the B2 cell immune response in mice with antigen-induced peritonitis

The effects of omega-3 fatty acids on the adaptive immune response have mainly been analysed in vitro with varying results. How omega-3 fatty acids affect the adaptive immune response in vivo is largely unknown. This study examined the effects of dietary fish oil on the adaptive immune response in antigen-induced inflammation in mice, focusing on its effects on B cells and B cell subsets. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and peritonitis induced by intraperitoneal injection of mBSA. Serum, spleen and peritoneal exudate were collected prior to and at different time points after induction of peritonitis. Serum levels of mBSA-specific antibodies were determined by ELISA and the number of peritoneal and splenic lymphocytes by flow cytometry. The levels of germinal center B cells and IgM⁺, IgG⁺ and CD138⁺ cells in spleen were evaluated by immunoenzyme staining. Mice fed the fish oil diet had more peritoneal B1 cells, more IgM⁺ cells in spleen and higher levels of serum mBSA-specific IgM antibodies compared with that in mice fed the control diet. However, dietary fish oil did not affect the number of peritoneal B2 cells, splenic IgG⁺ or CD138⁺ cells or serum levels of mBSA-specific IgG antibodies in mice with mBSA-induced peritonitis. These results indicate that dietary fish oil can enhance the adaptive immune response, specifically the B1 cell response, which may lead to better protection against secondary infection as well as improvement in reaching homeostasis following antigenic challenge.     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Antigen-specific CD8⁺ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8⁺ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8⁺ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8⁺ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8⁺ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.     

A TCRβ Repertoire Signature Can Predict Experimental Cerebral Malaria

Cerebral Malaria (CM) is associated with a pathogenic T cell response. Mice infected by P. berghei ANKA clone 1.49 (PbA) developing CM (CM⁺) present an altered PBL TCR repertoire, partly due to recurrently expanded T cell clones, as compared to non-infected and CM^- infected mice. To analyse the relationship between repertoire alteration and CM, we performed a kinetic analysis of the TRBV repertoire during the course of the infection until CM-related death in PbA-infected mice. The repertoires of PBL, splenocytes and brain lymphocytes were compared between infected and non-infected mice using a high-throughput CDR3 spectratyping method. We observed a modification of the whole TCR repertoire in the spleen and blood of infected mice, from the fifth and the sixth day post-infection, respectively, while only three TRBV were significantly perturbed in the brain of infected mice. Using multivariate analysis and statistical modelling, we identified a unique TCRβ signature discriminating CM⁺ from CTR mice, enriched during the course of the infection in the spleen and the blood and predicting CM onset. These results highlight a dynamic modification and compartmentalization of the TCR diversity during the course of PbA infection, and provide a novel method to identify disease-associated TCRβ signature as diagnostic and prognostic biomarkers.     

T-cell receptor Vβ repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8⁺ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8⁺ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8⁺ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8⁺T-lymphocyte subsets showed high frequencies of LDE CD8⁺T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8⁺ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8⁺ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8⁺ T-cell clones that are selected during CL immune responses, suggesting that CD8⁺ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8⁺T-cells are associated with larger lesions.     

Impairment of Pneumococcal Antigen Specific Isotype-Switched Igg Memory B-Cell Immunity in HIV Infected Malawian Adults

Pneumococcal disease is associated with a particularly high morbidity and mortality amongst adults in HIV endemic countries. Our previous findings implicating a B-cell defect in HIV-infected children from the same population led us to comprehensively characterize B-cell subsets in minimally symptomatic HIV-infected Malawian adults and investigate the isotype-switched IgG memory B-cell immune response to the pneumococcus. We show that similar to vertically acquired HIV-infected Malawian children, horizontally acquired HIV infection in these adults is associated with IgM memory B-cell (CD19⁺ CD27⁺ IgM⁺ IgD⁺) depletion, B-cell activation and impairment of specific IgG B-cell memory to a range of pneumococcal proteins. Our data suggest that HIV infection affects both T-cell independent and T-cell dependent B-cell maturation, potentially leading to impairment of humoral responses to extracellular pathogens such as the pneumococcus, and thus leaving this population susceptible to invasive disease.     

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBL^hi) or without (MBL^lo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.

Methodology/Principal Findings

For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBL^lo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBL^hi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)⁺ clonal B-cells.

Conclusions/Significance

These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBL^lo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.     

Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.     

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection

Advancements in high-throughput sequencing (HTS) of antibody repertoires (Ig-Seq) have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale. However, currently, only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions, possibly limited by inadequate sequencing depth and throughput. To better understand how HIV-1 infection would impact humoral immune system, in this study, we systematically analyzed the differences between the IgM (HIV-IgM) and IgG (HIV-IgG) heavy chain repertoires of HIV-1 infected patients, as well as between antibody repertoires of HIV-1 patients and healthy donors (HH). Notably, the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries, and the diversity of unique clones in HIV-IgG remarkably reduced. In aspect of somatic mutation rates of CDR1 and CDR2, the HIV-IgG repertoire was higher than HIV-IgM. Besides, the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire, presumably caused by the great number of novel VDJ rearrangement patterns, especially a massive use of IGHJ6. Moreover, some of the B cell clonotypes had numerous clones, and somatic variants were detected within the clonotype lineage in HIV-IgG, indicating HIV-1 neutralizing activities. The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.     

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene

The catfish IGH locus is large (∼1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3′ end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3′ C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.     

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4⁺ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.