A natural serum autoantibody specific for the Thy-1 glycoprotein (anti-Thy-1 autoantibody [ATA]) is produced by B-1 cells that are positively selected by self-antigen. Here, using ATA micro kappa transgenic mice we show that cells with this B cell receptor are negatively selected during bone marrow (BM) development. In a Thy-1 null environment, BM ATA B cells progress to a normal follicular stage in spleen. However, in a self-antigen-positive environment, development is arrested at an immature stage in the spleen, concomitant with induction of CD5. Such cells are tolerant and short-lived, different from B-1. Nonetheless, ATA-positive selection was evident by self-antigen-dependent high serum ATA production, comprising approximately 90% of serum immunoglobulin M in ATA micro kappa mice. Splenectomy did not eliminate ATA production and transfer of tolerant splenic B cells did not induce it. These findings demonstrate that B-1 positive selection, resulting in the production of natural serum ATA, arises independently from the major pathway of BM B cell development and selection.

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Valpha14Jalpha281(+) NKT cells without affecting recognition by Valpha14(-) NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Valpha14(+) (but not Valpha14(-)) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Abstract CD1d-dependent accumulation of αβ T cells bearing a canonical Vα14Jα281 α-chain (Vα14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Vα14+ T cells do not exhibit significant autoreactivity despite high reactivity to α-galactosylceramide presented by CD1d (α-GalCer/CD1d). To assess the role of TCRβ chain in determining the α-GalCer/CD1d vs autoreactive specificity of Vα14+ T cells, we conducted TCRα or TCRβ chain transduction experiments. In this study we demonstrate, by combining different TCRβ chains with the Vα14 α-chain in retrovirally transduced T cell lines, that the Vα14 α-chain plays a primary role, necessary but not sufficient for imparting α-GalCer/CD1d recognition. β-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Vα14+ T cells, since transduction of TCRαβ chains from a high CD1d autoreactive Vα14+ T cell line conferred the α-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Vα14+ T cell reactivity is due to both β-chain diversity and control mechanism(s) beyond primary TCR structure.

CD1d-dependent accumulation of alphabeta T cells bearing a canonical Valpha14Jalpha281 alpha-chain (Valpha14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Valpha14+ T cells do not exhibit significant autoreactivity despite high reactivity to alpha-galactosylceramide presented by CD1d (alpha-GalCer/CD1d). To assess the role of TCRbeta chain in determining the alpha-GalCer/CD1d vs autoreactive specificity of Valpha14+ T cells, we conducted TCRalpha or TCRbeta chain transduction experiments. In this study we demonstrate, by combining different TCRbeta chains with the Valpha14 alpha-chain in retrovirally transduced T cell lines, that the Valpha14 alpha-chain plays a primary role, necessary but not sufficient for imparting alpha-GalCer/CD1d recognition. beta-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Valpha14+ T cells, since transduction of TCRalphabeta chains from a high CD1d autoreactive Valpha14+ T cell line conferred the alpha-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Valpha14+ T cell reactivity is due to both beta-chain diversity and control mechanism(s) beyond primary TCR structure.

Summary: Here we review three areas in B‐cell development in the mouse, with a focus on relevance to B‐1/CD5+ B cells. Multiparameter flow cytometry has allowed the dissection of intermediate stages of developing B cells, both in fetal liver and bone marrow. In the first area, we present recent work that has delineated a fraction of pre‐pro‐B cells, committed to the B lineage, but lacking any immunoglobulin rearrangements. Next, the role of the pre‐B‐cell receptor in B‐cell repertoire selection has become clear in the past few years, but we present work suggesting that the action of this process during fetal life is different, resulting in selection of a very distinct repertoire compared with adult. Finally, we describe a new VH3609 antithymocyte Ig transgenic mouse model system that has provided the first definitive evidence for the role of self‐antigen in development and maintenance of natural autoreactive B cells.

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid alphagalactosylceramide (alpha-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the alpha helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and alpha-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that alpha-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) alpha chains and different beta chains respond differently to alpha-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR beta in defining natural killer T cell specificity, despite the more restricted diversity of the alpha chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR alpha and beta chains survey a surface composed of both mCD1.1 and the carbohydrate portion of alpha-GalCer.

cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1 ؉ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4 ؉ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10 ؊ naive cells from adult mice respond poorly compared with 6C10 ؉ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4 ؉ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope. T he development and differentiation of T cells is accompanied by alteration of surface Ag expression, generating significant heterogeneity in the periphery (1–7). Although most commonly recognized phenotypic heterogeneity among CD4 ϩ or CD8 ϩ T cells is a result of antigenic activation (5), there are several surface proteins expressed differentially in the pool of cells considered to be mature Ag nonexposed T cells (" naive " T cells). In mice, these surface proteins include several GPI-linked molecules, such as Qa-2 (8), Ly-6C (6, 9), Ly-6A/E (6, 10), and the Thy-1/CD90-dependent 6C10 (3, 11–13). GPI-linked proteins are clustered in specialized glycolipid-enriched membrane mi-crodomains called caveolae (14 –18). Because several GPI-linked proteins associate with src-family tyrosine kinases, p56 lck and p59 fyn , and cross-linking of these proteins leads to tyrosine phos-phorylation of intracellular substrates (19 –21), resulting in activation (22–25), they could function as signaling molecules on T cells or could modulate TCR-mediated cell activation (22, 26 –30). Thus, there is increasing interest in the possibility that such phe-notypic heterogeneity reflects functional diversification of peripheral T cells, predetermining cell fate following activation. 6C10 is of particular interest due to its association with CD4 ϩ T cell function and with the GPI-linked protein Thy-1. The 6C10 determinant is recognized by the mouse IgM monoclonal anti-thymocyte/T cell autoantibody SM6C10 as a natural autoantibody (11). 6C10 expression is dependent on surface Thy-1/CD90 gly-coprotein expression, and it is found at high levels on immature thymocytes of all inbred mouse strains, similar to Thy-1 (11). However, although Thy-1 is expressed by nearly all T cells in the spleen or lymph nodes, 6C10 is expressed only by a fraction of peripheral T cells, mostly by CD4 ϩ T …

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40-80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1- variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10- naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.

Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene–encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.