T Cell-dependent Research Articles

Defective B‐cell response to T‐dependent immunization in lupus‐prone mice

Lupus anti-nuclear Ab show the characteristics of Ag-driven T-cell-dependent (TD) humoral responses. If autoAg elicit the same response as exogenous Ag, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone C57BL/6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin (NP-KLH) compared with total Ig, including a smaller percentage of B cells participating in the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells (PC) in the bone marrow. B6.TC PC expressed reduced levels of FcgammaRIIb, which suggests that reduced apoptosis in resident PC prevents the establishment of newly formed NP-specific PC in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous Ag and suggest that low FcgammaRIIb, hypergammaglobulinemia, and high autoAb production would be predictive of a poor response to immunization in lupus patients.

Axon growth and guidance genes identify T‐dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.

PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner

We recently reported that intravenous injections of “empty” PEGylated liposomes without encapsulated or surface coupled proteins elicit a PEG-specific IgM response in rats. In the present study, simultaneous weak anti-PEG IgG and strong anti-PEG IgM responses were detected following intravenous injections of “empty” PEGylated liposomes. The pattern of immune response appears to differ from the classic primary response against T cell-dependent (TD) antigens. The anti-PEG IgM response was detected in T-cell deficient nude BALB/c mice following intravenous injection of “empty” PEGylated liposomes, suggesting that “empty” PEGylated liposomes initiate the immune response against PEG in a T cell-independent manner. In vitro splenic lymphocytes-proliferation assay indicated that TNP-LPS, a typical type 1 T cell-independent (TI) antigen (TI-1 antigen), significantly primed the proliferation, while TNP-Ficoll, a typical type 2 TI antigen (TI-2 antigen), and “empty” PEGylated liposomes did not prime any proliferation under these experimental conditions. In addition, in splenic marginal zone (MZ) B-cell-depleted rats, the anti-PEG IgM response was diminished, while the immune reactions against TNP-BSA (a TD antigen) and TNP-LPS (TI-1 antigen) were not diminished. These results demonstrate that “empty” PEGylated liposomes may promote the immune response against PEG as a result of priming the activation of MZ B cells, as TI-2 antigen promotes a specific IgM response. In conclusion, although the mechanistic details behind the immune reaction against “empty” PEGylated liposomes are not yet clear, the liposomes elicit an anti-PEG IgM response in a T cell-independent manner and appear to be a TI-2 antigen, and splenic MZ B cells may be essential for the immune response against “empty” PEGylated liposomes.

A critical role for DRAK2 in the germinal center reaction and the antibody response (84.1)

Abstract DRAK2 (DAP-related apoptotic kinase-2) protein is a DAP family member. DRAK2 is highly expressed in B and T lymphocytes and it is among the few proteins that are highly enriched in the lymphoid system. To determine whether DRAK2 plays a role in B cell activation and differentiation, we analyzed the germinal center development in the spleen of drak2−/− mice immunized with a T cell-dependent (TD) antigen. Immunofluorescence microscopy and FACS analysis showed the number of germinal centers was three to five-folds less in the drak2−/− mice than in their wild type littermates. Impairment of germinal center development was not due to a deficiency in B cell proliferation, as BrdU uptake in vivo and CFSE staining ex vivo of B cells was comparable in DRAK2-deficient and wild type B cells. 7-AAD and TUNEL staining experiments showed that, the proportion of apoptotic germinal center B and T cells in drak2−/− mice was significantly higher than wild type mice. As a result, the generation of high affinity IgG antibodies was impaired in drak2−/− mice. Specific expression of bcl-xl in T cells of drak2−/− mice rescued germinal center formation. Thus, our findings suggest a novel role of DRAK2 in the germinal center reaction and the maturation of the antibody response. Supported by N.I.H. grants AR 40908, AI 45011 and AI 60573.

Endogenous IL-1R1 Signaling Is Critical for Cognate CD4+ T Cell Help for Induction of In Vivo Type 1 and Type 2 Antipolysaccharide and Antiprotein Ig Isotype Responses to IntactStreptococcus pneumoniae, but Not to a Soluble Pneumococcal Conjugate Vaccine

MyD88(-/-) mice exhibit defective innate, diminished CD4(+) T cell-dependent (TD) type 1, but enhanced type 2, humoral immunity in response to intact Streptococcus pneumoniae (Pn). Because type 1 IL-1R (IL-1R1) signaling is MyD88 dependent, a role for endogenous IL-1 was determined. IL-1R1(-/-), in contrast to MyD88(-/-), mice exhibited relatively intact innate splenic cytokine expression in response to Pn. Nevertheless, IL-1R1(-/-), like MyD88(-/-), mice were more sensitive to killing with live Pn relative to wild-type controls. Although IL-1R1(-/-) mice elicited a normal T cell-independent IgM antipolysaccharide (PS) response to heat-killed Pn, the induction of PS- and protein-specific cognate, but not noncognate, TD type 1 and type 2 IgG isotypes were markedly reduced. Additionally, CD4(+) T cells from Pn-primed IL-1R1(-/-) mice failed to elicit IFN-gamma, IL-5, or IL-13 secretion upon restimulation with Pn in vitro, whereas MyD88(-/-) mice secreted normal levels of IFN-gamma and enhanced levels of IL-5 and IL-13. In contrast, IgG responses to a soluble, pneumococcal protein-PS conjugate, with or without adjuvant, showed little dependence on IL-1R1 and normal CD4(+) T cell priming. These data are the first to demonstrate a nonredundant role for endogenous IL-1 in TD induction of humoral immune responses to an intact pathogen, although not a pathogen-derived soluble conjugate, suggesting that antigenic context is a key determinant for IL-1 dependence. These data further suggest that IL-1 may be critical for preserving CD4(+) Th2 function in the presence, but not absence, of MyD88-dependent signaling via TLRs.

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80–40% in CR2−/− mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2−/− B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

Immunization of Aged Mice with a Pneumococcal Conjugate Vaccine Combined with an Unmethylated CpG-Containing Oligodeoxynucleotide Restores Defective Immunoglobulin G Antipolysaccharide Responses and Specific CD4+-T-Cell Priming to Young Adult Levels

Polysaccharide (PS)-protein conjugate vaccines, in contrast to purified PS vaccines, recruit CD4+-T-cell help and restore defective PS-specific humoral immunity in the immature host. Surprisingly, in the immunocompromised, aged host, anti-PS responses to conjugate vaccines are typically no better than those elicited by purified PS vaccines. Although aging leads to defects in multiple immune cell types, diminished CD4+-T-cell helper function has recently been shown to play a dominant role. We show that in response to immunization with purified pneumococcal capsular PS serotype 14 (PPS14) in saline, the T-cell-independent immunoglobulin G (IgG) anti-PPS14 response in aged mice was comparable to that in young mice. In contrast, the T-cell-dependent IgG anti-PPS14 response to a soluble conjugate of PPS14 and pneumococcal surface protein A (PspA) (PPS14-PspA) in saline was markedly defective. This was associated with defective priming of PspA-specific CD4+ T cells. In contrast, immunization of aged mice with PPS14-PspA combined with an unmethylated CpG-containing oligodeoxynucleotide (CpG-ODN) restored IgG anti-PPS14 responses to young adult levels, which were substantially higher than those observed using purified PPS14. This was associated with enhanced PspA-specific CD4+-T-cell priming. Similarly, intact Streptococcus pneumoniae capsular type 14, which contains Toll-like receptor (TLR) ligands, also induced substantial, though modestly reduced, T-cell-dependent (TD) IgG ant-PPS14 responses in aged mice. Spleen and peritoneal cells from aged and young adult mice made comparable levels of proinflammatory cytokines in response to CpG-ODN, although cells from aged mice secreted higher levels of interleukin-10. Collectively, these data suggest that inclusion of a TLR ligand, as an adjuvant, with a conjugate vaccine can correct defective TD IgG anti-PS responses in elderly patients by augmenting CD4+-T-cell help.

HIV-1 Nef Suppresses T Cell-Dependent Immunoglobulin Class Switching by Inducing Inhibitors of CD40 and IL-4 Receptor Signaling in Bystander B Cells.

Background. Class switch DNA recombination (CSR) from IgM to IgG and IgA is central to immunity against pathogens and requires the activation of B cells by CD4+ T cells through CD40 ligand (CD40L) and cytokines, including IL-4 and IL-10. Viruses evade protective IgG and IgA responses through a number of strategies, including production of soluble proteins that suppress T cell-dependent (TD) class switching in bystander B cells. HIV-1 is thought to impair antigen-specific IgG and IgA production by inducing progressive depletion of CD4+ T cells. Although important, this mechanism is not sufficient to explain the intrinsically poor response of B cells from viremic patients to CD4+ T cell help. This prompted us to explore the B cell-modulating activity of negative factor (Nef), an early HIV-1 protein implicated in immunosuppression. Nef is released in the extracellular environment by infected cells and might perturb the function of bystander B cells by targeting them through its N-terminal myristoylated domain.Methods. Tonsillar tissue sections from healthy donors and lymph nodal tissue sections from 10 HIV-1-infected patients were analyzed for IgD, p24, and Nef expression by immunohistochemistry. Nef expression was also analyzed in IgD+ B cells purified from healthy donors and exposed to a recombinant myristoylated or non-myristoylated Nef protein. These IgD+ B cells as well as an IgD+ B cell line expressing a wild-type Nef transgene or a mutant Nef transgene lacking the myristoylated domain were utilized in CSR assays after further exposure to CD40L, IL-4 and IL-10. CSR and signaling were studied as described1,2,3.Results. We found that HIV-1-infected lymphoid follicles express large amounts of Nef. This viral protein penetrates in bystander B cells both in vivo and in vitro and interferes with the initiation of CSR by CD40L and cytokines without down-regulating CD40 and cytokine receptor expression on B cells. Rather, Nef up-regulates inhibitor of NF-κ B (Iκ B) and suppressor of cytokine signaling (SOCS) proteins, including SOCS1 and SOCS3, in B cells. These powerful inhibitory proteins attenuate the transcriptional activation of germline Ig gene promoters by blocking CD40 and cytokine receptor signaling through NF-κ B and signal transducer and activator of transcription (STAT), respectively. In addition, Nef hampers the up-regulation of key components of the CSR machinery, such as activation-induced cytidine deaminase (AID), and blocks the induction of IgG, IgA and IgE secretion by CD40L and cytokines. Under similar conditions, Nef spares B cell survival, B cell proliferation as well as B cell signaling through mitogen-activated protein kinase p38 and B cell-specific activation protein (BSAP or Pax5).Conclusions. Our findings suggest that HIV-1 inhibits IgG and IgA production not only by impairing CD4+ T cells, but also by turning on powerful CSR inhibitory pathways in bystander B cells through Nef. We propose that Nef-blocking agents might improve protective antibody responses to pathogens and vaccines in HIV-1-infected patients.

Role of Complement Receptor Type 2 and Endogenous Complement in the Humoral Immune Response to Conjugates of Complement C3d and Pneumococcal Serotype 14 Capsular Polysaccharide

Conjugation of the complement fragment C3d to both T-cell-dependent (TD) protein and T-cell-independent type 2 (TI-2) polysaccharide antigens enhances the humoral immune response in mice immunized with either type of antigen. However, the ability of C3d-protein conjugates to enhance the antibody response in mice deficient in complement receptor types 1 and 2 (CR1 and CR2) has raised questions about the role of C3d-CR2 interactions in the adjuvant effect of C3d. In this study, we examined the role of CR2 binding and endogenous complement activation in the antibody response to conjugates of C3d and serotype 14 pneumococcal capsular polysaccharide (PPS14). To block binding of PPS14-C3d conjugates to CR2, mice were immunized with a mixture of vaccine and (CR2)2-immunoglobulin G1 (IgG1). Mice receiving (CR2)2-IgG1 at the time of primary immunization had a marked reduction in the primary anti-PPS14 antibody response but an enhanced secondary anti-PPS14 response, suggesting that C3d-CR2 interactions are required for the primary response but can have negative effects on the memory response. Further, compared with mice receiving PPS14-C3d having a high C3d/PPS14 ratio, mice immunized with PPS14-C3d with low C3d/PPS14 ratios had an enhanced secondary antibody response. Treatment of mice with cobra venom factor to deplete complement had insignificant effects on the antibody response to PPS14-C3d. Experiments with CBA/N xid mice confirmed that PPS14-C3d conjugates retain the characteristics of TI-2 rather than TD antigens. Thus, the adjuvant effect of C3d conjugated to PPS14 requires C3d-CR2 interactions, does not require activation of endogenous complement, and is not mediated by TD carrier effects.

Roles of CD4+T-Cell-Independent and -Dependent Antibody Responses in the Control of Influenza Virus Infection: Evidence for Noncognate CD4+T-Cell Activities That Enhance the Therapeutic Activity of Antiviral Antibodies

Previous studies have indicated that B cells make a significant contribution to the resolution of influenza virus infection. To determine how B cells participate in the control of the infection, we transferred intact, major histocompatibility complex class II (MHC-II)-negative or B-cell receptor (BCR)-transgenic spleen cells into B-cell-deficient and CD8(+) T-cell-depleted muMT mice, termed muMT(-8), and tested them for ability to recover from infection. muMT(-8) mice that received no spleen cells invariably succumbed to the infection within 20 days, indicating that CD4(+) T-cell activities had no significant therapeutic activity on their own; in fact, they were harmful and decreased survival time. Interestingly, however, they became beneficial in the presence of antiviral antibody (Ab). Injection of MHC-II((-/-)) spleen cells, which can provide CD4(+) T-cell-independent (TI) but not T-cell-dependent (TD) activities, delayed mortality but only rarely resulted in clearance of the infection. By contrast, 80% of muMT(-8) mice injected with normal spleen cells survived and resolved the infection. Transfer of BCR-transgenic spleen cells, which contained approximately 10 times fewer virus-specific precursor B cells than normal spleen cells, had no significant impact on the course of the infection. Taken together, the results suggest that B cells contribute to the control of the infection mainly through production of virus-specific Abs and that the TD Ab response is therapeutically more effective than the TI response. In addition, CD4(+) T cells appear to contribute, apart from promoting the TD Ab response, by improving the therapeutic activity of Ab-mediated effector mechanisms.

IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis.

During atherogenesis, LDL is oxidized, generating various oxidation-specific neoepitopes, such as malondialdehyde-modified (MDA-modified) LDL (MDA-LDL) or the phosphorylcholine (PC) headgroup of oxidized phospholipids (OxPLs). These epitopes are recognized by both adaptive T cell-dependent (TD) and innate T cell-independent type 2 (TI-2) immune responses. We previously showed that immunization of mice with MDA-LDL induces a TD response and atheroprotection. In addition, a PC-based immunization strategy that leads to a TI-2 expansion of innate B-1 cells and secretion of T15/EO6 clonotype natural IgM antibodies, which bind the PC of OxPLs within oxidized LDL (OxLDL), also reduces atherogenesis. T15/EO6 antibodies inhibit OxLDL uptake by macrophages. We now report that immunization with MDA-LDL, which does not contain OxPL, unexpectedly led to the expansion of T15/EO6 antibodies. MDA-LDL immunization caused a preferential expansion of MDA-LDL-specific Th2 cells that prominently secreted IL-5. In turn, IL-5 provided noncognate stimulation to innate B-1 cells, leading to increased secretion of T15/EO6 IgM. Using a bone marrow transplant model, we also demonstrated that IL-5 deficiency led to decreased titers of T15/EO6 and accelerated atherosclerosis. Thus, IL-5 links adaptive and natural immunity specific to epitopes of OxLDL and protects from atherosclerosis, in part by stimulating the expansion of atheroprotective natural IgM specific for OxLDL.

Abnormal T cell–dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero

In allogeneic hematopoietic stem cell transplant recipients, restoration of humoral immunity is delayed and can remain impaired for years. In many severe combined immune deficiency (SCID) patients given haploidentical bone marrow (BM), lesions in humoral immunity are exacerbated by poor engraftment of donor B cells. The nature of these defects is important to understand as they render patients susceptible to infection. Previous work in mice suggested that in utero transplantation (IUT) of allogeneic BM might offer several advantages for the correction of primary immune deficiencies. In SCID mice given fully allogeneic BM in utero, the lymphoid compartment was restored with minimal evidence of graft-versus-host disease (GVHD). The present report examines B-cell reconstitution and function in mice that have received allogeneic IUT. Results are compared with those of adult mice given total body irradiation (TBI) followed by transplantation with allogeneic BM. In addition to enumerating the various B-cell subsets present in BM, spleen, and peritoneal cavity (PC), B-cell competence was assessed by challenging mice with T cell-independent (TI) and T cell-dependent (TD) antigens. The results demonstrated that all B-cell subsets in the BM and periphery were restored in allogeneic IUT and TBI mice, as were antibody responses after TI challenge. Upon immunization with TD antigens, however, IUT and TBI mice exhibited suboptimal responses as measured by the capacity to isotype switch and generate germinal center (GC) B cells. Thus, although allogeneic BM transplantation results in complete recovery of the B-cell compartment, certain elements of the humoral response remain defective.

Reconstitution of functional human B lymphocytes in NOD/SCID mice engrafted with ex vivo expanded CD34(+) cord blood cells.

Functional capacity of B cells developed from ex vivo expanded hematopoietic stem cells has not been fully evaluated. Therefore, we investigated the antigen-specific antibody production in human B cells maturated from ex vivo expanded cord blood (CB) CD34(+) cells in NOD/Shi-scid (NOD/SCID) mice. CB CD34(+) cells were cultured for 5 days in the presence of human cytokines and the murine stromal cell line HESS-5, and transplanted into irradiated NOD/SCID mice. These mice, reconstituted with human hematopoietic cells, were challenged with T-cell-independent (TI) or T-cell-dependent (TD) antigens after CD19(+) cells appeared at 6 weeks. Three months later, anti-dinitrophenol (DNP)-specific antibody was detected in both mice immunized with DNP-Ficoll (TI) and those immunized with DNP-keyhole limpet hemocyanin or DNP-ovalbumin (TD). The anti-DNP antibody was mainly immunoglobulin M, but a small amount of immunoglobulin G also was detected. In the spleen, the majority of CD19(+) cells expressed mature B-cell markers such as CD40, immunoglobulin M, immunoglobulin D, cytoplasmic Cmu, and light chains kappa, and lambda. These results indicate that human B cells develop from CD34(+) cells in NOD/SCID mice to produce antigen-specific antibody with in vivo primary stimulation. This system provides a powerful and versatile tool for studying the entire process of human B-lymphocyte development and producing specific human monoclonal antibodies.

BCR signal through alpha 4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation.

alpha 4 potentially mediates BCR signals through a rapamycin-sensitive TOR pathway. To investigate a potential role for alpha 4 in B cell activation, the alpha 4 gene was disrupted conditionally in B cells by mating male CD19-Cre mice with female alpha 4-floxed mice. CD19-Cre+/alpha 4flox mice showed loss of alpha 4 protein in B lineage cells and a decreased number of phenotypically normal mature B cells. Compared to normal B cells, alpha 4(-) B cells showed a decreased proliferation in response to the B cell stimulants (anti-IgM antibody plus IL-4, anti-CD40 mAb and lipopolysaccharide), and a reduced S6 kinase activation and rapamycin sensitivity. While CD19-Cre+/alpha 4flox mice showed impaired antibody responses to both T cell-independent and T cell-dependent (TD) antigens, the TD antigen response was markedly impaired as demonstrated by reduced isotype switching, reduced germinal center formation and reduced V region somatic hypermutation. These results show that alpha 4 plays a pivotal role in antigen-specific signal transduction during B cell activation and differentiation in vivo.

Antibody repertoires in infants and adults: effects of T-independent and T-dependent immunizations.

Polysaccharide(PS)-encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis are among the most prevalent bacterial pathogens of humans. Infections caused by these organisms are both common (otitis media, sinusitis) and severe (meningitis, bacteremia). Antibodies directed against the capsular PS of encapsulated bacteria prevent infection by promoting opsonophagocytic killing. Most bacterial PS, however, are type II T-cell-independent (TI-2) antigens that are poorly immunogenic in young children at highest risk of developing disease. Conjugation of bacterial PS to a protein carrier converts the immune response to a T-cell-dependent (TD) form and significantly improves the immunogenicity of PS, especially in infants. H. influenzae type b (Hib) is a major cause of invasive infection in non-immune children. The medical importance of this pathogen and the availability of both TI-2 and TD Hib PS vaccine formulations have made the human anti-Hib-PS immune response an excellent model for the study of the biology of these B cell responses.

B cell immunopoiesis: visualizing the impact of CD40 engagement on the course of T cell-independent immune responses in an Ig transgenic system.

This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.

CD5-positive and CD5-negative human B cells converge to an indistinguishable population on signalling through B-cell receptors and CD40.

Whether CD5 on B cells marks a subset functionally distinct from the conventional CD5 negative (CD5neg) adult population or is more an indicator of activation, remains contentious. Here we have investigated whether CD5 positive (CD5pos) and CD5neg B cells can be distinguished in terms of their response to surrogate signals aimed to model, in vitro, T-cell dependent (TD) and T-independent (TI) encounters with antigen in vivo: the predominantly CD5pos B-cell population found in cord blood, CD5 B cells positively selected from tonsils and their CD5neg counterparts, were compared. Neonatal B cells displayed a near-identical phenotype to that of adult CD5pos B cells, being characterized by uniform immunoglobulin M (IgM), immunoglobulin D (IgD), CD23 and CD44 coexpression. When cultured with anti-IgM maintained at high density on CD32-tranfected mouse L cells to model TI responses or on CD40 ligand (CD40L)-bearing L cells (with or without captured anti-IgM) to model TD encounters, DNA synthesis was stimulated to a similar extent in all three populations. Focusing on CD5 and CD23, we found that - although the signals delivered promoted distinct profiles of expression - under each condition of activation, the phenotypes that emerged for adult CD5pos and CD5neg B cells were remarkably similar. Neonatal B cells displayed a greater diminution in CD5 expression than adult CD5pos B cells following CD40 signals but otherwise the two populations again behaved similarly. The inclusion of interleukin-4 (IL-4) to cultures where cells were costimulated via surface (s)IgM and CD40 resulted in a complete loss of CD5 expression and a corresponding hyperexpression of CD23, irrespective of the population studied. The near-identical response of CD5pos and CD5neg B cells to surrogate TD or TI signals in vitro and their convergence to indistinguishable phenotypes is wholly supportive of CD5 being a fluctuating marker of activation rather than it delineating functionally distinct subsets.

Anti-galactose-α(1,3) galactose antibody production in α1,3-galactosyltransferase gene knockout mice after xeno and allo transplantation

Antibodies (Abs) that mediate the hyperacute rejection and acute vascular rejection/delayed xenograft rejection of pig organs in humans and Old World primates are predominantly directed at a single carbohydrate epitope, galactose-α1,3-galactose (α1,3Gal). The T-cell dependence of elicited anti-α1,3Gal Ab responses in humans and Old World primates is controversial. In this study we have characterized anti-α1,3Gal Ab production in mice with disrupted α1,3-galactosyltransferase genes (GT-Ko mice) and determined the T-cell dependence of anti-α1,3Gal Ab responses, following xenograft and allograft transplantation. GT-Ko mice produce natural anti-α1,3Gal IgM and IgG in an age-dependent manner, however, these Abs could not elicit hyperacute rejection nor affect the rate of cardiac xenograft (3–5 days) or allograft rejection (7–9 days). Transplantation of xenogeneic Lewis rats hearts elicited modest anti-α1,3Gal Ab, but vigorous xenoAb responses. The anti-α1,3Gal Ab response was restricted to the IgM and IgG3 subclass while the xenoAb response comprised IgM and all four IgG subclasses. Transplantation of allogeneic C3H hearts elicited weak anti-α1,3Gal Ab responses that were primarily IgM, but vigorous alloAb responses. Despite the restriction of elicited anti-α1,3Gal Ab responses to the IgM and IgG3 isotypes, these responses are T-cell dependent. The ability of allografts to elicit weak anti-α1,3Gal but strong allo-Ab responses, can be explained by the dependence of α1,3Gal-specific B cells on cognate help from T cells.

Route of administration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors.

Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.

Differential expression of the B cell-restricted molecule CD22 on neonatal B lymphocytes depending upon antigen stimulation.

Newborns respond poorly to certain antigens and produce mainly IgM antibodies. By flow cytometry we analyzed on neonatal and adult B cells the expression of CD22, a B cell receptor (BCR)-associated membrane molecule, known as negative modulator of BCR signaling. After T cell-independent (TI-)stimulation with anti-mu F(ab')(2) fragments we found a dramatic decrease in the percentage of neonatal CD22(+) B cells and CD22 mean fluorescence intensity (MFI) shift, whereas adult B cells remained unaffected. Survival and proliferation rates of neonatal B cells were higher compared to adult B cells whereas the degrees of apoptosis and necrosis were comparable. Surprisingly, after stimulation with lower doses of anti-mu apoptosis as well as proliferation increased significantly in contrast to adult B cells. T cell-dependent (TD)-stimulation with anti-CD40 monoclonal antibody and IL-4 resulted in a dramatic increase in the percentage of CD22(+) neonatal B cells in contrast to unaffected adult B cells. CD22 MFI shifts showed no significant changes, respectively. The survival rate was higher for adult B cells, whereas apoptosis and cell death were comparable. These results suggest that TI antigens lower the neonatal BCR signaling threshold via down-regulation of CD22, resulting in hyperresponsive B cells apt to premature apoptosis. On the other hand, up-regulation of CD22 after TD stimulation may allow increased inhibiting influence of CD22 on neonatal BCR signaling, impairing B cell activation and differentiation.