The double life of a B-1 cell: self-reactivity selects for protective effector functions

During their development, B and T cells with self-reactive antigen receptors are generally deleted from the repertoire to avoid autoimmune diseases. Paradoxically, innate-like B-1 cells in mice are positively selected for self-reactivity and form a pool of long-lived, self-renewing B cells that produce most of the circulating natural IgM antibodies. This Review provides an overview of the developmental processes that shape the B-1 cell pool in mice, outlines the functions of B-1 cells in both the steady state and during host defence, and discusses possible functional B-1 cell homologues that exist in humans.     

B-1 B cell development

CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.     

Cutting edge: TCR revision affects predominantly Foxp3 cells and skews them toward the Th17 lineage

CD4(+) T cells respond to peripheral endogenous superantigen stimulation by undergoing deletion or TCR revision. The latter involves RAG re-expression, TCR gene rearrangement, and expression of a novel TCR. TCR-revised T cells are functional and express a diverse TCR repertoire. Because TCR revision harbors the potential to create self-reactivity, it is important to explore whether T cells known to be self-reactive (regulatory T cells) or those involved in autoimmunity (Th17 cells) arise from TCR revision. Interestingly, we observed that Foxp3(+) cells are excluded from revising their TCR and that only a small fraction of postrevision cells expresses Foxp3. In contrast, Th17 cells are 20 times more frequent among revised than among C57BL/6 CD4(+) T cells, indicating that postrevision cells are biased toward the Th17 lineage. The link between Th17 differentiation and TCR revision might be highly relevant to the role of Th17 cells in promoting autoimmunity.     

Theoretical considerations and probability models for the somatic development of the T-cell repertoire

A theory about the somatic development of the T-cell repertoire is described which explains the high proportion of alloreactive T lymphocytes as a side effect resulting from two biologically important selection processes. It is assumed that the T-cell receptors contain two binding sites X and Y similar to the two V regions of the B-cell receptors and immunoglobulin molecules, but with a different specificity repertoire as a result of the two selection processes. The first selection process is mediated by T-lineage inducer cells, which have already been postulated by Zinkernagel. They are believed to induce only cells with a binding site Y that can bind to major histocompatibility antigens (MHA) on the inducer cells. This selection forces the T-cell system to recognize preferentially cell-bound antigens; and it introduces under certain circumstances a high degree of self-MHA restriction and self-reactivity, and also a low degree of alloreactivity on the basis of cross-reactivity. The subsequent second selection process serves the purpose of self-tolerance induction. This process is expected under certain circumstances to result in a loss self-reactivity combined with a partial loss of self-restriction and selective enrichment of alloreactive T cells. Probability models are presented to illustrate how the two selection processes shape the T-cell repertoire and how the proportion of alloreactive cells is increased under special circumstances. It is possible that some T-cell subclasses are not affected by the second selection and maintain the primary repertoire. The theory is, in contrast to major competing theories, compatible with the assumption that the original V gene repertoires for the T-cell receptors are random.     

Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.     

Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity

Inhibitors of DNA methylation, such as 5-azacytidine, induce gene expression. We have previously reported that cloned T cells treated with 5-azacytidine lose the requirement for Ag and can be activated by autologous HLA-D molecules alone, thus becoming auto-reactive. This phenomenon could potentially mediate an autoimmune disease in vivo. Inasmuch as several drugs are known to cause autoimmune disease, we asked whether they exert the same effects on T cells as 5-azacytidine. We report that hydralazine and procainamide, two drugs associated with a lupus-like autoimmune disease, also inhibit DNA methylation and induce self-reactivity in cloned T cell lines. These results suggest that drug-induced autoimmune disease may be due to activation of as yet unidentified genes through mechanisms involving DNA methylation.     

The structural gene for F liver protein (Flp) maps to chromosome 5 of the mouse

The BXD and AKXL panels of recombinant inbred mouse strains have been typed for the F liver protein alloantigen. The structural gene for F liver protein gene (Flp) is placed on the distal part of chromosome 5, between the known markers Bcd-1 and Gus-s. This excludes the possibility that F liver protein is a major histocompatibility complex molecule, and in turn raises a question about the uniqueness of F and certain other proteins as purgers of self-reactivity among T but not B cells. The typed RI strains have then been used for the immunogenetic studies presented in the succeeding article.     

Existence of T cells manifesting self-reactivity indistinguishable from alloreactivity

The studies reported here were designed to analyze the phenotypic characteristics of self-reactive T lymphocytes induced in culture by allogeneic effect factor (AEF), as well as the control of their functional activities by the major histocompatibility complex (MHC). Unprimed T cells cultured with AEF in the absence of exogenous stimulating target cells become activated against self-antigens, as evidenced by their ability to manifest two distinct activities. First, such cells could lyse syngeneic target cells. This cytolytic activity was directed against H-2K antigens and was mediated by Lyt-2+ T cells. Second, the AEF-activated T cells could be stimulated in a secondary culture to high levels of proliferative activity by irradiated syngeneic spleen cells. The stimulator cells in this syngeneic mixed lymphocyte reaction (MLR) were found to be Thy-1-negative, Ia-positive splenic adherent cells. Stimulation in the secondary syngeneic MLR was provided by I-region specificities, and the majority of the proliferating cells were Lyt-1+ cells. Finally, AEF-induced T cells were effective in serving as effectors of graft-vs-host reactions in vivo in syngeneic recipients. These results prove that, under appropriate conditions, murine T lymphocytes can display aggressive patterns of self-reactivity that are similar in both quantity and quality to the classical patterns of alloreactivity and may have great significance for our understanding of MHC recognition processes.     

In vivo assessment of the relative contributions of deletion, anergy, and editing to B cell self-tolerance

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.     

Interferon-gamma receptor polymorphisms determine strain differences in accessibility of activated lymphocyte NK-triggering antigens to recognition by self-reactive NK cells

The "NK-triggering-antigen regulator" (Nktar) gene is a locus identified in the C57BL/6 genome which regulates the ability of unlabeled activated Con A blasts to compete for recognition of labeled syngeneic Con A blasts by BALB/c NK cells. Linkage analysis on Con A blasts from (BALB/c x CByB6F1) N2 backcross progeny for (1) relative level of competitive inhibition of BALB/c NK lysis of syngeneic Con A blasts and (2) genotypes at polymorphic microsatellite markers distributed throughout the mouse genome mapped the Nktar gene locus to a 5-cM region of chromosome 10 containing the interferon-gamma receptor (Ifngr) gene locus. N2 Con A blasts exhibited an inverse relationship between (a) their cell surface density of IFN-gammaR molecules detected by FACS with monoclonal anti-CD119 and (b) their cold target inhibition of BALB/c NK self-reactivity. Con A blasts from Ifngr(-/-) knockout mice showed a relatively high level of inhibition of BALB/c NK self-lysis and a relatively low level of class I MHC, which were both reversed by transient transfection with the Ifngr gene. Sequencing studies showed that Balb/c Ifngr encodes a Gly(69) whereas C57BL/6 Ifngr encodes Glu(69) due to a difference at nucleotide 284. Sequencing studies of N2 progeny demonstrated 100% concordance between their Nktar inhibitory phenotype and their Ifngr genotype. These findings demonstrate that the Nktar and Ifngr loci are identical. They further indicate that polymorphisms related to the Ifngr locus and affecting expression of cell surface IFN-gammaR may underlie genetic differences in the availability of NK-triggering antigens (NKTAgs) to recognition by self-reactive BALB/c NK cells by differentially affecting the ability of IFN-gammaR molecules to mediate up-regulation of NKTAg-masking class I molecules.     

Self-reactive, T cell receptor-gamma delta+, lymphocytes from the intestinal epithelium of weanling mice.

Intraepithelial T lymphocytes (IEL) are dispersed throughout the intestinal epithelial lining but their role in cellular immune defense is unknown. Their location suggests that their highly activated state may be due to constant exposure to bacterial Ag. To study IEL specificity and function we have prepared a panel of IEL-T cell hybridomas from both adult and weanling C57B1/6 mice. Many of these expressed TCR-gamma delta, a cell type rare in peripheral lymph nodes and spleen but predominant at epithelial surfaces. We have identified a subset of gamma delta T cells from weanling mice which is self reactive, i.e., these hybrids secrete IL-2 spontaneously, without antigenic stimulation or a requirement for APC. Self-reactive TCR-gamma delta+ hybrids and lines, all of which bear a particular TCR (V gamma 1.1C gamma 4V delta 6), have previously been derived from neonatal thymus and the skin. Northern blot and immunoprecipitation analyses suggest that the self-reactive IEL hybrids also bear a C gamma 4/V delta 6 TCR. Antibody inhibition experiments showed that the self-reactivity of the IEL hybrids is TCR mediated. Spontaneous IL-2 production was blocked by soluble anti-CD3 and anti-TCR-gamma delta antibodies but not by antibodies to the TCR-alpha beta. The self-reactive IEL hybrids lack class II MHC and the class I-like proteins CD1 and TLA but express class I MHC. IEL hybrids may also require the vitronectin receptor as an accessory molecule for their activation because spontaneous IL-2 production is blocked by antibody to the vitronectin receptor as well as by the extracellular matrix protein active site peptide RGDS, but not the control peptide RGES. V gamma 1.1C gamma 4V delta 6 T cells in the thymus, skin, and intestine may represent a small and unique subpopulation of lymphocytes with a potential for autoimmune reactivity at peripheral sites.     

IL-2 Mediates CD4+ T Cell Help in the Breakdown of Memory-Like CD8+ T Cell Tolerance under Lymphopenic Conditions

Background

Lymphopenia results in the proliferation and differentiation of naïve T cells into memory-like cells in the apparent absence of antigenic stimulation. This response, at least in part due to a greater availability of cytokines, is thought to promote anti-self responses. Although potentially autoreactive memory-like CD8⁺ T cells generated in a lymphopenic environment are subject to the mechanisms of peripheral tolerance, they can induce autoimmunity in the presence of antigen-specific memory-like CD4⁺ T helper cells.

Methodology/Principal Findings

Here, we studied the mechanisms underlying CD4 help under lymphopenic conditions in transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that the self-reactivity mediated by the cooperation of memory-like CD8⁺ and CD4⁺ T cells was not abrogated by CD40L blockade. In contrast, treatment with anti-IL-2 antibodies inhibited the onset of autoimmunity. IL-2 neutralization prevented the CD4-mediated differentiation of memory-like CD8⁺ T cells into pathogenic effectors in response to self-antigen cross-presentation. Furthermore, in the absence of helper cells, induction of IL-2 signaling by an IL-2 immune complex was sufficient to promote memory-like CD8⁺ T cell self-reactivity.

Conclusions/Significance

IL-2 mediates the cooperation of memory-like CD4⁺ and CD8⁺ T cells in the breakdown of cross-tolerance, resulting in effector cytotoxic T lymphocyte differentiation and the induction of autoimmune disease.     

The immunoglobulin heavy chain constant region affects kinetic and thermodynamic parameters of antibody variable region interactions with antigen

A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG(1), IgG(2a), and IgG(2b) switch variants of murine monoclonal antibody (mAb) 3E5 IgG(3) with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.     

Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors

The importance of IL-2Rbeta function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rbeta-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rbeta function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R beta-chain in IL-2Rbeta-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Ralpha expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rbeta provides an essential signal during thymic development to regulate self-reactivity.     

B cells expressing a natural polyreactive autoantibody have a distinct phenotype and are overrepresented in immunoglobulin heavy chain transgenic mice

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the lambda1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/lambda1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward lambda1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/lambda1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/lambda1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.     

Dual T cell receptor expressing CD8+ T cells with tumor- and self-specificity can inhibit tumor growth without causing severe autoimmunity

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp(33)), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.     

Discrepancy between in vitro measurable and in vivo virus neutralizing cytotoxic T cell reactivities. Low T cell receptor specificity and avidity sufficient for in vitro proliferation or cytotoxicity to peptide-coated target cells but not for in vivo protection.

The TCR-alpha beta of CTL recognize peptide Ag in association with MHC class I molecules. TCR binding should be highly specific to guarantee pathogen specificity and to avoid self-reactivity. Therefore, the in vivo relevance of T cells exhibiting cross-reactivities in vitro and the respective role of the TCR affinities involved are not clear. To analyze high and low avidity T cell activities both in vitro and in vivo, we investigated primary and clonal CTL responses specific for the lymphocytic choriomeningitis virus nucleoprotein 118-126 epitope in association with the two closely related H-2Ld or H-2Lq molecules. As expected, we found highly specific class I-allele-restricted CTL responses when antiviral protection or immunopathology in vivo and lysis of virus infected target cells in vitro were analyzed. In contrast, the CTL were MHC crossreactive and thus considerably less discriminatory against targets expressing high MHC-peptide densities and in proliferation assays. The data show that relatively high TCR avidities are required for virus neutralization in vivo, in contrast to in vitro analyses of peptide-coated target cells or proliferative T cell responses that may engage TCR of low avidity and broad specificity and therefore may not reflect biologically relevant TCR avidities.     

How pregnancy can affect autoimmune diseases progression?

Autoimmune disorders are characterized by tissue damage, caused by self-reactivity of different effectors mechanisms of the immune system, namely antibodies and T cells. Their occurrence may be associated with genetic and/or environmental predisposition and to some extent, have implications for fertility and obstetrics. The relationship between autoimmunity and reproduction is bidirectional. This review only addresses the impact of pregnancy on autoimmune diseases and not the influence of autoimmunity on pregnancy development. Th17/Th1-type cells are aggressive and pathogenic in many autoimmune disorders and inflammatory diseases. The immunology of pregnancy underlies the role of Th2-type cytokines to maintain the tolerance of the mother towards the fetal semi-allograft. Non-specific factors, including hormonal changes, favor a switch to Th2-type cytokine profile. In pregnancy Th2, Th17/Th2 and Treg cells accumulate in the decidua but may also be present in the mother’s circulation and can regulate autoimmune responses influencing the progression of autoimmune diseases.