Common variable immunodeficiency, cross currents, and prevailing winds.

Common variable immunodeficiency (CVID) is a heterogenous disease category created to distinguish late-onset antibody deficiencies from early-onset diseases like agammaglobulinemia or more expansively dysfunctional combined immunodeficiencies. Opinions vary on which affected patients should receive a CVID diagnosis which confuses clinicians and erects reproducibility barriers for researchers. Most experts agree that CVID's most indeliable feature is defective germinal center (GC) production of isotype-switched, affinity-maturated antibodies. Here, we review the biological factors contributing to CVID-associated GC dysfunction including genetic, epigenetic, tolerogenic, microbiome, and regulatory abnormalities. We also discuss the consequences of these biological phenomena to the development of non-infectious disease complications. Finally, we opine on topics and lines of investigation we think hold promise for expanding our mechanistic understanding of this protean condition and for improving the lives of affected patients.

B-cell intrinsic and extrinsic signals that regulate central tolerance of mouse and human B cells.

The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM+ immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here, we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance.

Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.

LAPTM5 mediates immature B cell apoptosis and B cell tolerance by regulating the WWP2-PTEN-AKT pathway.

Elimination of autoreactive developing B cells is an important mechanism to prevent autoantibody production. However, how B cell receptor (BCR) signaling triggers apoptosis of immature B cells remains poorly understood. We show that BCR stimulation up-regulates the expression of the lysosomal-associated transmembrane protein 5 (LAPTM5), which in turn triggers apoptosis of immature B cells through two pathways. LAPTM5 causes BCR internalization, resulting in decreased phosphorylation of SYK and ERK. In addition, LAPTM5 targets the E3 ubiquitin ligase WWP2 for lysosomal degradation, resulting in the accumulation of its substrate PTEN. Elevated PTEN levels suppress AKT phosphorylation, leading to increased FOXO1 expression and up-regulation of the cell cycle inhibitor p27Kip1 and the proapoptotic molecule BIM. In vivo, LAPTM5 is involved in the elimination of autoreactive B cells and its deficiency exacerbates autoantibody production. Our results reveal a previously unidentified mechanism that contributes to immature B cell apoptosis and B cell tolerance.

Developmental changes in the rules for B cell selection.

The autoimmune checkpoint during B cell maturation eliminates self-antigen reactive specificities from the mature B cell repertoire. However, an exception to this rule is illustrated by B-1 cells, an innate-like self-reactive B cell subset that is positively selected into the mature B cell pool in a self-antigen-driven fashion. The mechanisms by which B-1 cells escape central tolerance have puzzled the field for decades. A key clue comes from their restricted developmental window during fetal and neonatal life. Here we use B-1 cells as a prototypic early life derived B cell subset to explore developmental changes in the constraints of B cell selection. We discuss recent advancements in the understanding of the molecular program, centered around the RNA binding protein Lin28b, that licenses self-reactive B-1 cell output during ontogeny. Finally, we speculate on the possible link between the unique rules of early life B cell tolerance and the establishment of B cell - microbial mutualism to propose an integrated model for how developmental and environmental cues come together to create a protective layer of B cell memory involved in neonatal immune imprinting.

Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice.

Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH-DH-JH and Vκ-Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.

MicroRNA control of B cell tolerance, autoimmunity and cancer.

Since the discovery of the first microRNA (miRNA) in 1993, thousands of miRNAs have been identified in humans and mice and many of them have been shown to control a large variety of cellular processes in different cell types including those composing the immune system. MicroRNAs regulate virtually all aspects of immune cell development, differentiation and function. Studies have shown that these molecules are involved in the maintenance of lymphocyte tolerance and, when dysregulated, promote the development of autoimmune diseases. In this review, we focus on the current knowledge about the roles of miRNAs in B cell tolerance and their contribution to autoimmunity, highlighting additional roles for some of these miRNAs in T cell tolerance. Finally, we will comment on miRNAs that promote both autoimmunity and lymphoma.

Galectin-9 bridges human B cells to vascular endothelium while programming regulatory pathways.

Humoral immunity is reliant on efficient recruitment of circulating naïve B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells. Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete. Prior studies on naïve B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology. Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules. Adhesion analyses showed that Gal-9 can bridge human circulating and naïve B cells to vascular endothelial cells (EC), while decelerating transendothelial migration. Moreover, Gal-9 interactions with naïve B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics. Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7's cytosolic adapter EAT-2, which is required for cell activation, was eliminated. Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell - EC interactions while delivering anergic signals to control B cell reactivity.

Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies.

A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies.

B Cell-Mediated Autoimmune Diseases.

Extensive studies have suggested a central role of B cells in the autoimmune pathogenesis, as loss of B cell tolerance results in increased serum levels of autoantibodies, enhanced effector T cell response and tissue damages. Here, we provide an overview of dysregulated B cell responses in the development of autoimmunity. In addition to their presence in the target organs, autoreactive B cells can promote the formation of ectopic lymphoid structures and differentiate into plasma cells that produce large amounts of autoantibodies and cytokines. In animal models that recapitulate the key features of human autoimmune disease, mechanistic studies have indicated two categories of autoantibodies: (1) serological markers for disease diagnosis and prognosis; (2) effector molecules that induce organ hypofunction or damage directly in an epitope-specific manner, or indirectly by activating other immune cell subsets. Moreover, B cell-derived cytokines usually promote the autoreactive T cell response during autoimmune development, but there is compelling evidence that a subpopulation of B cells negatively regulates immune responses, also known as regulatory B cells via secreting anti-inflammatory cytokines (IL-10, IL-35, etc.) or a contact-dependent fashion. Although B cell depletion could eliminate most circulating B cells in the periphery, the clinical outcomes of B cell depletion therapy for autoimmune diseases vary among individuals due to differential activation or survival signals for B cells provided by tissue microenvironment. Thus, therapeutic combinations that target immune checkpoints and B cell activation may represent a promising strategy for the effective treatment of human autoimmune diseases.

Patients with common variable immunodeficiency with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses.

BACKGROUND: The lack of pathogen-protective, isotype-switched antibodies in patients with common variable immunodeficiency (CVID) suggests germinal center (GC) hypoplasia, yet a subset of patients with CVID is paradoxically affected by autoantibody-mediated autoimmune cytopenias (AICs) and lymphadenopathy. OBJECTIVE: We sought to compare the physical characteristics and immunologic output of GC responses in patients with CVID with AIC (CVID+AIC) and without AIC (CVID-AIC). METHODS: We analyzed GC size and shape in excisional lymph node biopsy specimens from 14 patients with CVID+AIC and 4 patients with CVID-AIC. Using paired peripheral blood samples, we determined how AICs specifically affected B-and T-cell compartments and antibody responses in patients with CVID. RESULTS: We found that patients with CVID+AIC displayed irregularly shaped hyperplastic GCs, whereas GCs were scarce and small in patients with CVID-AIC. GC hyperplasia was also evidenced by an increase in numbers of circulating follicular helper T cells, which correlated with decreased regulatory T-cell frequencies and function. In addition, patients with CVID+AIC had serum endotoxemia associated with a dearth of isotype-switched memory B cells that displayed significantly lower somatic hypermutation frequencies than their counterparts with CVID-AIC. Moreover, IgG+ B cells from patients with CVID+AIC expressed VH4-34-encoded antibodies with unmutated Ala-Val-Tyr and Asn-His-Ser motifs, which recognize both erythrocyte I/i self-antigens and commensal bacteria. CONCLUSIONS: Patients with CVID+AIC do not contain mucosal microbiota and exhibit hyperplastic yet inefficient GC responses that favor the production of untolerized IgG+ B-cell clones that recognize both commensal bacteria and hematopoietic I/i self-antigens.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.

Breach of autoreactive B cell tolerance by post-translationally modified proteins.

OBJECTIVES: Over 50% of patients with rheumatoid arthritis (RA) harbour a variety of anti-modified protein antibodies (AMPA) against different post-translationally modified (PTM) proteins, including anti-carbamylated protein (anti-CarP) antibodies. At present, it is unknown how AMPA are generated and how autoreactive B cell responses against PTM proteins are induced. Here we studied whether PTM foreign antigens can breach B cell tolerance towards PTM self-proteins. METHODS: Serum reactivity towards five carbamylated proteins was determined for 160 patients with RA and 40 healthy individuals. Antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic joint tissue. Mice were immunised with carbamylated or non-modified (auto)antigens and analysed for autoantibody responses. RESULTS: We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self-proteins and modified non-self-proteins. Studies in mice show that anti-CarP antibody responses recognising carbamylated self-proteins are induced by immunisation with carbamylated self-proteins and by immunisation with carbamylated proteins of non-self-origin. Similar to the data observed with sera from patients with RA, the murine anti-CarP antibody response was, both at the monoclonal level and the polyclonal level, highly cross-reactive towards multiple carbamylated proteins, including carbamylated self-proteins. CONCLUSIONS: Self-reactive AMPA responses can be induced by exposure to foreign proteins containing PTM. These data show how autoreactive B cell responses against PTM self-proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B cell tolerance.

Rehabilitation or the death penalty: autoimmune B cells in the dock.

CD20-based monoclonal antibodies have become established as treatments for lymphoma, rheumatoid arthritis, systemic lupus erythematosus, vasculitis and dermatomyositis, with the principle therapeutic mechanism relating to B-cell depletion through effector cell engagement. An article by Brühl et al. in this issue of the European Journal of Immunology [Eur. J. Immunol. 2015. 45: 705-715] reveals a fundamentally distinct mechanism of silencing autoimmune B-cell responses. Rather than B-cell depletion, the authors use anti-CD79b antibodies to induce B-cell tolerance and suppress humoral immune responses against collagen to prevent the development of arthritis in mice. Here we highlight the differences in the mechanisms used by anti-CD20 and anti-CD79b Ab therapy and discuss why depletion of B cells may not be required to treat autoimmune arthritis and other B-cell-associated pathologies.

IL-17-producing CD4(+) T cells contribute to the loss of B-cell tolerance in experimental autoimmune myasthenia gravis.

The role of Th17 cells in the pathogenesis of autoantibody-mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR-specific CD4(+) T cells is producing IL-17. IL-17(ko) mice developed fewer or no EAMG symptoms, although the frequencies of tAChR-specific CD4(+) T cells secreting IL-2, IFN-γ, or IL-21, and the percentage of FoxP3(+) Treg cells were similar to WT mice. Even though the total anti-tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL-17(ko) as compared to WT mice. Most importantly, pathogenic anti-murine AChR antibodies were significantly lower in IL-17(ko) mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR ß/δ(ko) mice with WT or IL-17(ko) CD4(+) T cells. In conclusion, we show that the level of IgG2b and the loss of B-cell tolerance, which results in pathogenic anti-murine AChR-specific antibodies, are dependent on IL-17 production by CD4(+) T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody-mediated autoimmunity.

Glycolipid antigens and autoantibodies in autoimmune neuropathies.

Autoantibodies to glycans present on glycolipids mediate the postinfectious paralytic disease, Guillain-Barré syndrome (GBS). These glycans are also found on lipo-oligosaccharides (LOSs) of GBS-inducing microbes, suggesting molecular mimicry as a mechanism for disease induction. How B lymphocyte tolerance to self-glycans is regulated during the initiation phase of the disease is currently under investigation. The discovery of antiglycolipid antibodies that bind to heteromeric glycolipid complexes has generated new insights in this field. Heteromeric complexes are structurally distinct glycolipids that interact to form new molecular shapes capable of either enhancing or attenuating recognition by autoantibodies. Although the principles emerging from this phenomenon have a substantial impact on diagnostics methods, they also raise intriguing questions about the diversity of innate antibody repertoires, mechanisms of tolerance, and autoantibody targeting of neural membranes.

TNF receptor superfamily member 13b (TNFRSF13B) hemizygosity reveals transmembrane activator and CAML interactor haploinsufficiency at later stages of B-cell development.

BACKGROUND: Heterozygous C104R or A181E TNF receptor superfamily member 13b (TNFRSF13B) mutations impair removal of autoreactive B cells, weaken B-cell activation, and convey to patients with common variable immune deficiency (CVID) an increased risk for autoimmunity. How mutant transmembrane activator and CAML interactor (TACI) influences wild-type TACI function is unclear; different models suggest either a dominant negative effect or haploinsufficiency. OBJECTIVE: We investigated potential TACI haploinsufficiency by analyzing patients with antibody-deficient Smith-Magenis syndrome (SMS) who possess only 1 TNFRSF13B allele and antibody-deficient patients carrying one c.204insA TNFRSF13B null mutation. METHODS: We tested the reactivity of antibodies isolated from single B cells from patients with SMS and patients with a c.204insA TNFRSF13B mutation and compared them with counterparts from patients with CVID with heterozygous C104R or A181E TNFRSF13B missense mutations. We also assessed whether loss of a TNFRSF13B allele induced haploinsufficiency in naive and memory B cells and recapitulated abnormal immunologic features typical of patients with CVID with heterozygous TNFRSF13B missense mutations. RESULTS: We found that loss of a TNFRSF13B allele does not affect TACI expression, activation responses, or establishment of central B-cell tolerance in naive B cells. Additionally, patients with SMS and those with a c.204insA TNFRSF13B mutation display normal regulatory T-cell function and peripheral B-cell tolerance. The lack of a TNFRSF13B allele did result in decreased TACI expression on memory B cells, resulting in impaired activation and antibody secretion. CONCLUSION: TNFRSF13B hemizygosity does not recapitulate autoimmune features of CVID-associated C104R and A181E TNFRSF13B mutations, which likely encode dominant negative products, but instead reveals selective TACI haploinsufficiency at later stages of B-cell development.

Postnatal xenogeneic B-cell tolerance in swine following in utero intraportal antigen exposure.

BACKGROUND: The objective of this study was to investigate the humoral immune response to xenogeneic antigens administered during the fetal state utilizing a baboon-to-pig model. METHODS: Nine fetuses from an alpha-1,3-galactosyltransferase gene knockout (GalT-KO) MGH-miniature swine sow underwent transuterine ultrasound-guided intraportal injection of T-cell depleted baboon bone marrow (B-BM) at mid-gestation. Two juvenile GalT-KO swine undergoing direct B-BM intraportal injection were used as controls. RESULTS: Postnatal humoral tolerance was induced in the long-term surviving piglets as demonstrated by the absence of any antibody response to baboon donor cells. In addition, a second intraportal B-BM administration at 2.5 months post-birth led to no antibody formation despite re-exposure to xenogeneic antigens. This B-cell unresponsiveness was abrogated only when the animal was exposed subcutaneously to third-party xenogeneic and allogeneic antigens, suggesting that the previously achieved humoral non-responsiveness was donor specific. In comparison, the two juvenile GalT-KO control swine demonstrated increasing anti-baboon IgM and IgG levels following intraportal injection. CONCLUSIONS: In summary, xenogeneic B-cell tolerance was induced through in utero intraportal exposure to donor cells and this tolerance persisted following postnatal rechallenge with donor B-BM, but was lost on exposure to third-party antigen, possibly as a result of cross-reactive antibody formation.