Early human T cell activation events with engagement of surface MHC class II

Major histocompatibility complex (MHC) class II are expressed on most activated human lymphocytes. They direct antigen presentation events in dendritic cells and B cells (collectively called antigen presenting cells), but the role for MHC class II in human T cells is not well understood. To understand the role of surface MHC class II and to identify the molecules involved in signaling, we have defined the early activation sequence in T cells when MHC class II are engaged by a specific antibody. Specifically, we have characterized the involvement of phosphotyrosine kinases, phospholipase C (PLC), and Ca²⁺ mobilization. With the engagement by either whole anti-class II antibody or its Fab fragments, the enzymatic activity of p56^lck and ZAP-70 increased, but there was no increase in p59^fyn activity. In addition, the intracellular free Ca²⁺ increased, which was due to enhanced influx and not to the mobilization of intracytoplasmic Ca²⁺. These events did not require cross-linking because they were not significantly augmented by the addition of antispecies antibody. The coimmunoprecipitation of tyrosine phosphorylated PLC-γ1 with surface MHC class II suggested that PLC-γ1 could be recruited to MHC class II after engagement. These results show the complexities of the early signals transduced by the engagement of surface MHC class II on T cells. J. Cell. Biochem. 70:346–353, 1998. © 1998 Wiley-Liss, Inc.     

A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.     

Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages

Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4(+) T cells and MHC class II(+) APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4(+) dendritic cells (DC) and macrophages (Mphi) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and Mphi. DC efficiently presented partially degraded, but not native CII alpha-chains, while Mphi presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and Mphi presented soluble HCgp39, and activated Mphi from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and Mphi to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.     

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.     

Cognate B cell signaling via MHC class II: differential regulation of B cell antigen receptor and MHC class II/Ig-alpha beta signaling by CD22

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-alphabeta dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-alphabeta signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo

Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.     

Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes

We describe the direct detection of radiolabeled antigen fragments bound to class II MHC molecules following immunoglobulin-mediated endocytosis and processing of native antigen in B lymphoblastoid cells. Tris-Tricine SDS gels revealed six distinct iodinated processing products that could be detected on class II MHC 1 hr after antigen endocytosis and persisted for at least 20 hr. These physiological processed antigen-class II complexes were remarkably stable, as judged by the fact that class II alpha beta dimers, which remain associated in SDS, became labeled with the same set of processed peptides. Using a lectin-binding assay, we show that these physiological processing products bind to the newly maturing population of MHC molecules rather than binding to the preexisting cell surface population; in contrast, an exogenous peptide binds predominantly to the latter population. A direct T cell-independent assay for processed peptide-MHC complex formation should facilitate additional studies on the exogenous antigen processing pathway.     

A biochemical characterization of feline MHC products: Unusually high expression of class 11 antigens on peripheral blood lymphocytes

The polymorphism of feline MHC antigens was examined using biochemical methods. The following observations were made: (1) feline class I and II antigens are polymorphic. Their biochemical features were established using rabbit and mouse reagents directed against human MHC products; they resemble those observed for other mammalian species; (2) the expression of class II antigens in unstimulated cat peripheral blood lymphocytes (PBLs) appears to be unusually high. Cat PBLs express far more class II than class I antigens, whereas in human Epstein-Barr virus-transformed lines, which are known to express relatively large amounts of class II antigens, the situation is reversed.Abbreviations used in this paper EBV Epstein-Barr virus - FLA feline lymphocyte antigen - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction - PBL peripheral blood lymphocyte - RT room temperature - TX-114 Triton X-114 - 1-D IEF one-dimensional isoclectric focusing - 2-D SDS-PAGE twodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Characteristic biochemical differences in human T and B lymphocytes separated on nylon wool

T and B lymphocytes from human tonsils were separated on a nylon wool column. T. cells are enriched in the nonadherent, B cells in the adherent fraction. Several enzymes and other markers were tested in separated and non separated lymphocyte populations. Certain enzymes and other properties can be used as T or B lymphocyte markers because of their preferential occurrence and because of the advantages of their estimation (simple, quick methods, objective evaluation). The following characteristics were considered as markers on the basis of our results: (I) acid phosphatase, Na+-K+-activated ATPase, BAEE-peptidase and chromium labeling in T lymphocytes; (II) 5'-nucleotidase, FITC-IgG binding, N-acetyl-D-glucosaminidase, thymidine and valine incorporation in B lymphocytes.     

Mycobacterium tuberculosis heat shock fusion protein enhances class I MHC cross-processing and -presentation by B lymphocytes

Exogenous heat shock protein (HSP):peptide complexes are processed for cross-presentation of HSP-chaperoned peptides on class I MHC (MHC-I) molecules. Fusion proteins containing HSP and Ag sequences facilitate MHC-I cross-presentation of linked antigenic epitopes. Processing of HSP-associated Ag has been attributed to dendritic cells and macrophages. We now provide the first evidence to show processing of HSP-associated Ag for MHC-I cross-presentation by B lymphocytes. Fusion of OVA sequence (rOVA, containing OVA(230-359) sequence) to Mycobacterium tuberculosis HSP70 greatly enhanced rOVA processing and MHC-I cross-presentation of OVA(257-264):K(b) complexes by B cells. Enhanced processing was dependent on linkage of rOVA sequence to HSP70. M. tuberculosis HSP70-OVA fusion protein enhanced cross-processing by a CD91-dependent process that was independent of TLR4 and MyD88. The enhancement occurred through a post-Golgi, proteasome-independent mechanism. These results indicate that HSPs enhance delivery and cross-processing of HSP-linked Ag by B cells, which could provide a novel contribution to the generation of CD8(+) T cell responses. HSP fusion proteins have potential advantages for use in vaccines to enhance priming of CD8(+) T cell responses.     

Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP.

Cells of the mouse B cell clone, CH12.LX, receive Ag-dependent differentiative signals through their surface membrane class II molecules. The present study was performed to determine the role of class II cross-linking and cAMP in the successful delivery of these signals. Delivery of differentiative signals by anti-Ek mAb was increased by further cross-linking with a secondary anti-isotype antibody. Intact or (Fab')2, but not Fab forms of anti-Ek successfully delivered the Ag-dependent differentiative signal. Inability of monovalent Fab fragments to deliver the signal could not be attributed to an inability to adequately bind Ek molecules. The requirement for cAMP for class II-mediated signaling was also examined, because previous studies have implicated elevated cAMP levels as necessary for class II signaling. Both Ag-dependent, Ek-mediated differentiation and the Ek-mediated inhibition of Ag-independent LPS-induced differentiation were inhibited by the adenyl cyclase inhibitor 2'5'ddA, although elevation of cAMP was not in itself sufficient to deliver the differentiative signal. Inhibition of LPS-induced differentiation could be mediated by mAb binding to either Ek, Abk, or Abb on CH12.LX or an Ab-bearing transfectant, CH12.ABB1. This inhibition was abrogated by 2'5'ddA in the case of Ek or Abb, both of which deliver Ag-dependent differentiative signals to CH12.LX cells. In the case of Abk, which does not deliver such signals to CH12.LX, 2'5'ddA did not abrogate anti-Abk-mediated inhibition of the LPS response. The effects of 2'5'ddA were reversed by the cAMP analog, dibutyryl cAMP, and Ag-dependent-induced differentiation of CH12.LX or CH12.ABB1 was accompanied by an increase in cAMP levels.     

Involvement of ERK, p38 MAP kinase, and PKC in MHC class II-mediated signal transduction in a resting B cell line

Substantial evidence suggests that MHC class II molecules play a critical role in transducing signals during B cell activation and differentiation. In addition, we previously found that cross-linking of MHC class II molecules using anti-MHC class II antibodies inhibited NF-kappaB activation in resting B cells isolated from mouse spleen. In this study, we investigated the mechanism of anti-MHC class II antibody-mediated inhibition of LPS-induced NF-kappaB activation using a resting B cell line, 38B9. We found that treatment with a corresponding anti-MHC class II antibody reduced the activation of NF-kappaB in LPS-stimulated 38B9 cells, treatment of the antibody mediated down-regulation of PKC and ERK/p38 MAP kinase pathways, and treatment with PKC inhibitors caused down-regulation of ERK and p38 MAP kinase activities in LPS-stimulated 38B9 cells. Our results suggest that the PKC and ERK/p38 MAP kinase pathways are regulated by anti-MHC class II antibodies, and that MHC class II molecules are actively involved in the signal transduction pathway in the resting B cell line, 38B9. Consequently, disruption of these pathways might contribute to the inhibition of LPS-induced NF-kappaB activation in 38B9 cells.     

Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II- invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.     

Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes.

⁴⁵Ca²⁺ uptake was detected within minutes following addition of T- and B-cell² mitogens to mouse lymphocytes. The T-cell mitogens (Con A and PHA) gave an ~twofold increase in ⁴⁵Ca²⁺ uptake (representing an influx of ~ 130 amol per lymphocyte, corresponding to an increase in average cellular Ca²⁺ of ~0.95 mM). B-cell mitogens which gave the largest ⁴⁵Ca²⁺ uptake (~twofold) were purified LPS preparations from Salmonella minnesota R595 and Escherichia coli 0111:2125. The ⁴⁵Ca²⁺ uptake by rabbit splenocytes using specific anti-b4 allotype antiserum was comparable to that obtained with the two purified LPS preparations. A23187, in low nontoxic doses, gave an ~sixfold increase in ⁴⁵Ca²⁺ uptake with mouse T cells. The ⁴⁵Ca²⁺ uptake was modulated by cyclic nucleotides showing a “yin-yang” effect. The results suggest a possible entry of ⁴⁵Ca²⁺ from the extracellular medium through “gated Ca²⁺ channels” in the plasma membrane into the cytosol by passive diffusion. The Ca²⁺ may be sequestered in the mitochondria, and the excess Ca²⁺ is later effluxed into the extracellular medium. The fact that ⁴⁵Ca²⁺ uptake appears to be one of the earliest events occurring after ligand binding to the cell, together with the demonstration of a Ca²⁺-dependent glucose uptake and a requirement for extracellular Ca²⁺ for DNA synthesis, suggest that, as it is now known to function in many other cellular responses, Ca²⁺ may operate as a second messenger for lymphocyte activation.