Oligoclonal expansions of mucosal T cells in Crohn's disease predominate in NKG2D-expressing CD4 T cells.

Crohn's disease (CD) is an inflammatory pathology of the mucosal intestine that results from uncontrolled immune response towards commensal microbes. Clonal expansions of T cells have been found in patients with CD suggesting an antigen-specific stimulation of pathogenic T cells. Here we show, using T-cell receptor repertoire analysis by real-time PCR, that oligoclonal expansions are found in both CD8+ and CD4+ T cells in the blood and intestinal mucosa of CD patients. The majority of CD4+ T-cell-expanded clones are CD4+NKG2D+ T cells. These clonal expansions were found in both inflamed and neighboring healthy tissue and were persisting during the course of the disease. The presence of these CD4+NKG2D+ T-cell clones at the macroscopically normal edge of the surgical resection might be predictive of inflammation relapse post surgery.

The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families.

To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/alpha-galactosylceramide, which specifically identify the previously described CD1d autoreactive Valpha14-Jalpha18/Vbeta8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Valpha14 T cells, we found that, like Valpha14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide-specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, gammadelta T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non-cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.

CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain.

Endosomal trafficking is an essential component of the CD1 pathway of lipid antigen presentation to T cells. We demonstrate that CD1d access to endosomal compartments is under dual regulation by an intrinsic tyrosine-based motif, which governs intense recycling between the plasma membrane and the endosome, and by the invariant chain, with which CD1d associates in the endoplasmic reticulum. Both pathways independently enhance antigen presentation to V(alpha)14(+) NKT cells, the main subset of CD1d-restricted T cells. These results reveal the complexity of CD1d trafficking and suggest that the invariant chain was a component of ancestral antigen presentation pathways prior to the evolution of MHC and CD1.

In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers.

The CD1 family of major histocompatibility complex (MHC)-like molecules specializes in presenting lipid and glycolipid antigens to alpha/beta T lymphocytes, but little is known about the size of the CD1-restricted T cell population or the frequency of T lymphocytes specific for a given glycolipid antigen. Here, we report the generation and use of mouse CD1d1-glycolipid tetramers to visualize CD1d-restricted T cells. In contrast with previous BIAcore-based estimates of very short half-lives for CD1d-glycolipid complexes, we found that the dissociation rate of several different CD1d-glycolipid complexes was very slow. Fluorescent tetramers of mouse CD1d1 complexed with alpha-galactosylceramide (alphaGalCer), the antigen recognized by mouse Valpha14-Jalpha281/Vbeta8 and human Valpha24-JalphaQ/Vbeta11 natural killer T (NKT) cell T cell receptors (TCRs), allowed us for the first time to accurately describe, based on TCR specificity, the entire population of NKT cells in vivo and to identify a previously unrecognized population of NK1.1-negative "NKT" cells, which expressed a different pattern of integrins. In contrast, natural killer (NK) cells failed to bind the tetramers either empty or loaded with alphaGalCer, suggesting the absence of a CD1d-specific, antigen-nonspecific NK receptor. Mouse CD1d1-alphaGalCer tetramers also stained human NKT cells, indicating that they will be useful for probing a range of mouse and human conditions such as insulin-dependent diabetes mellitus, tumor rejection, and infectious diseases where NKT cells play an important role.

Unaltered phenotype, tissue distribution and function of Valpha14(+) NKT cells in germ-free mice.

The expression pattern of mouse CD1d and the tissue distribution of CD1d-restricted Valpha14-Jalpha281 NKT cells suggest that the liver and the marginal zone of the spleen might be preferred sites of activation of this potent innate pathway of early cytokine secretion. Because these tissues are particularly involved with the filtration of blood-borne pathogens, and because NKT cells with an activated / memory phenotype accumulate over the first weeks of life and their CD1 ligands bind microbial glycolipids, it has been hypothesized that expansion of the NKT cell subset may be driven by exposure to the microbial environment. To test this hypothesis, we analyzed the frequency, surface phenotype and functional properties of NKT cells in normal and in germ-free C57BL / 6 mice. Surprisingly, we found that the NKT cell subset develops in the presence or absence of a microbial environment. Although these results do not rule out the possibility that NKT cells exert a protective function against some microbial agents, they demonstrate that non microbial ligands, possibly self-antigens are sufficient for the generation, maturation and peripheral accumulation of NKT cells.

CD1d-restricted mouse V alpha 14 and human V alpha 24 T cells: lymphocytes of innate immunity.

Mouse V alpha 14 T cells and their human homologs, V alpha 24 T cells, are prominent subsets of CD1d-restricted T cells. Here we discuss their striking similarities to B-1 B cells and gammadelta T cells and propose that these immune cells mediate various innate strategies in response to endogenous or exogenous danger signals.

Modifications of Igalpha and Igbeta expression as a function of B lineage differentiation.

Transcription of the mb1 and B29 genes is initiated when lymphoid progenitors enter the B cell differentiation pathway, and their transmembrane Igalpha and Igbeta products constitute essential signaling components of pre-B and B cell antigen receptors. We analyzed Igalpha/Igbeta biosynthesis, heterogeneity, and molecular interactions as a function of human B lineage differentiation in cell lines representative of the pro-B, pre-B, and B cell stages. All B lineage representatives produced a 36-kDa Igbeta form and three principal Igalpha forms, transient 33/40-kDa species and a mature 44-kDa glycoprotein. Deglycosylation revealed a major Igalpha core protein of 25 kDa and a minor 21-kDa Igalpha protein, apparently the product of an alternatively spliced mRNA. In pro-B cells, the Igalpha and Igbeta molecules existed primarily in separate unassembled pools, exhibited an immature glycosylation pattern, did not associate with surrogate light chain proteins, and were retained intracellularly. Their unanticipated association with the Lyn protein-tyrosine kinase nevertheless suggests functional potential for the Igalpha/Igbeta molecules in pro-B cells. Greater heterogeneity of the Igalpha and Igbeta molecules in pre-B and B cell lines was attributable to increased glycosylation complexity. Finally, the Igalpha/Igbeta heterodimers associated with fully assembled IgM molecules as a terminal event in B cell receptor assembly.

IL-7 sensitizes human pre-B cells but not pro-B cells to Fas/APO-1 (CD95)-mediated apoptosis.

Homeostasis of human B cell development is maintained by a complex network of cytoplasmic and surface expressed molecules. Abnormalities in this process may result in the expansion of malignant B cell precursors in B lineage acute lymphoblastic leukaemia (ALL). ALL cells share surface antigens with normal early precursor B cells. We have studied here the role of Fas/APO-1 (CD95) antigen on leukaemic precursor B cell line growth and survival, and the modulation of its effects by signals involved in normal early B cell development. Four ALL cell lines representative of the early steps of B cell differentiation are shown to express surface Fas/APO-1 (CD95) antigen and to undergo apoptosis in the presence of anti-Fas cross-linking antibodies. This effect is strongly enhanced when pre-B, but not pro-B cells, are pretreated with IL-7 but not with IL-2, IL-3, IL-4 or IL-10. Furthermore, pre-B cell death induced by anti-Fas antibodies in combination with IL-7 is increased upon pre-B receptor but not CD19 cross-linking. Bcl-2 and Bax protein expression is not influenced by IL-7 or pre-BR stimulation in either pro-B or pre-B cell lines. These results indicate that signals involved in normal early B cell development can modulate the Fas (CD95)-mediated apoptosis of leukaemic precursor B cells.

Structural analysis of human antibodies to proteinase 3 from patients with Wegener granulomatosis.

We determined the structure of five IgM autoAbs to proteinase-3 (PR3). These Abs are highly specific for Wegener's granulomatosis (WG) and may be involved in the pathogenesis of vasculitis in WG. Five clonal lymphoblastoid cell lines secreting Abs to PR3 were derived from four patients' B cells. From 3 to 5% of supernatants from wells contained detectable anti-PR3 Abs, indicating that anti-neutrophil cytoplasmic Ab specificity represents a sizable part of the IgM B cell repertoire in patients with WG. Mu heavy chains of WG1, WG4-1, and WG4-2 clones belonged to the VH3 subgroup. WG4-1 and WG4-2 heavy chains were identical, indicating an oligoclonal expansion of autoreactive B cells in this patient. WG4-1 (and WG4-2) used the VH3-23 V(H) gene, the product of which was shown to directly bind PR3. Heavy chains of WG2 and WG3 derived from VH4-59 and VH1-2 genes, respectively. Comparison with germline sequences showed that three of the five V(H) genes from clonal lines were somatically mutated with a R:S ratio in complementarity-determining regions of 3:0, 5:1, and 5:1, respectively. Three kappa light chains derived from the Vkappa4 gene, and one derived from a Vkappa1 gene. In these four Vkappa genes, there were overall R:S ratios of mutation of 8:1 and 0:7 in complementarity-determining regions and framework regions, respectively. These data suggest that the production of these autoantibodies, which are increasingly important in the diagnosis and management of WG, are influenced by an Ag-driven process.

[Analysis of expression of the molecules Igalpha and Igbeta in human pro-B leukemic lines]. / Analyse de l'expression des molécules Ig alpha et Ig beta dans les lignées leucémiques pro-B humaines.

Ig alpha and Ig beta are two glycosylated transmembrane proteins of the Ig superfamily that are encoded by the B cell-specific genes mb-1 and B29, respectively. Ig alpha/Ig beta heterodimers may associate with the mu/surrogate light chains (psi LC) complex and with membrane Immunoglobulins on the surface of pre-B and B cells, respectively. They play a crucial role in the signal transduction that follows pre-B and B cell receptor cross-linking. Previous works have shown that mb-1 and B29 transcripts are expressed in normal mouse and human pro-B cells. However, little is known about the expression of Ig alpha and Ig beta molecules in pro-B cells. To address this issue we first analysed the expression of the Ig alpha and Ig beta molecules in the RS4; 11 and Nalm16 human pro-B cell lines using specific monoclonal antibodies. We found that both cell lines expressed Ig alpha and Ig beta but this expression was limited to the cytoplasm compartment. Three forms (44, 40 and 36 kDa) of the Ig alpha molecule and a single form (36 kDa) of the Ig beta molecule were detected in these lines. The heterogeneity of the Ig alpha molecule was partly related to the presence of a truncated Ig alpha protein which is likely the product of a short mb-1 transcript expressed in these cell lines. This short transcript is generated by alternative splicing of the mb1 mRNA with loss of exon 2. Ig alpha heterogeneity was also related to the expression of different glycosylated forms of the Ig alpha molecule. Only a minor fraction of the Ig alpha and Ig beta molecules associate with each other to form Ig alpha/Ig beta heterodimers and Ig beta homodimers. In these pro-B cell lines Ig alpha and Ig beta were found to associate with the lyn tyrosine kinase, suggesting that they may play some functional role at this B cell differentiation stage. Transfection of muHC gene in the Nalm16 cells results in the assembly of the pre-B receptor and in its expression on the cell surface. The level of surface expression of the pre-B receptor was found to correlate with the level of muHC and psi LC synthesis and with the level of association of the different components of the pre-B receptor with each other. Analysis of the 697 and Nalm6 pre-B cells and of the Ramos B cells indicated that heterogeneity of Ig alpha and Ig beta increases as a function of B cell differentiation.

Fate of surrogate light chains in B lineage cells.

Biosynthesis of the immunoglobulin (Ig) receptor components and their assembly were examined in cell lines representative of early stages in human B lineage development. In pro-B cells, the nascent surrogate light chain proteins form a complex that transiently associates in the endoplasmic reticulum with a spectrum of unidentified proteins (40, 60, and 98 kD) and Bip, a heat shock protein family member. Lacking companion heavy chains, the surrogate light chains in pro-B cells do not associate with either the Ig(alpha) or Ig(beta) signal transduction units, undergo rapid degradation, and fail to reach the pro-B cell surface. In pre-B cells, by contrast, a significant portion of the surrogate light chain proteins associate with mu heavy chains, Ig(alpha), and Ig(beta) to form a stable receptor complex with a relatively long half-life. Early in this assembly process, Bip/GRP78, calnexin, GRP94, and a protein of approximately 17 kD differentially bind to the nascent mu heavy chains. The 17-kD intermediate is gradually replaced by the surrogate light chain protein complex, and the Ig(alpha) and Ig(beta) chains bind progressively to the mu heavy chains during the complex and relatively inefficient process of pre-B receptor assembly. The results suggest that, in humans, heavy chain association is essential for surrogate light chain survival and transport to the cell surface as an integral receptor component.