Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

T cell activation. II. Activation of human T lymphoma cells by cross-linking of their MHC class I antigens

The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cai2+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Cai2+ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when Cai2+ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CD3-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules.     

MHC class I molecules on CD4 T cells regulate receptor-mediated activation signals

Three T cell populations can be distinguished based on their response to antigen receptor engagement. A sizable fraction dies within hours of TCR ligation, a smaller fraction enters the mitotic cycle, and the remaining T cells merely upregulate the expression of certain cell surface markers. An MHC-I-controlled regulatory mechanism has been identified. MHC I MAbs, or Fab fragments, prevent T cells from mounting a proliferative mitogen response but do not inhibit the mitogen-induced deletion of T cells. IFN-gamma enlarges the fraction of T cells which proliferate in response to mitogen stimulation but, in the presence of MHC I MAb, these cells fail to clonally expand and enter the deletion pathway. Phenotypically, MHC I MAb Fab fragments induce T cells to upregulate the expression of the apoptosis marker CD95, even in the absence of TCR ligand, and prevent the upregulation of costimulatory CD28 molecule expression.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different     

Peptide presentation by MHC class I molecules

The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.     

Negative selection of Tcra-V8+CD8+ T cells by MHC class I molecules

Tcrb-V-specific positive and negative selection of T cells has been well documented. In contrast, nothing is known about Tcra-V-specific selection. Using Tcra-V8-specific KT50 antibody Tcra-V8-specific selection of T cells has been examined. The CD8⁺ T cell subpopulation bearing Tcra-V8 are shown to be negatively selected by major histocompatibility complex (MHC) class I H-2K^d and H-2D^d/L^d molecules. Furthermore, percentages of these T cells are also influenced by Tcra-V haplotypes. Involvement of non-H-2 self (super)antigens in this MHC class I restricted negative selection, however, remains to be determined.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Recognition of class II molecules by human T cells

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.     

Xenogeneic beta 2-microglobulin substitution affects functional binding of MHC class I molecules by CD8+ T cells

NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and beta2-microglobulin (beta2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in beta2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic beta2m to modify the interaction between TCR and MHC-I. Using a human beta2m-transgenic C57BL/6 mouse model, we show that human beta2m supports formation and expression of H-2K(b) and peptide:H-2K(b) complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vbeta repertoire of peripheral CD8+ T cells was skewed in the human beta2m-transgenic mice. Furthermore, the ability of mouse beta2m-restricted CTL to functionally recognize human beta2m+ target cells was diminished compared with their ability to recognize mouse beta2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in beta2m influence the ability of TCR to engage MHC-I during normal T cell physiology.     

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.     

Novel MHC class I-related molecule MR1 affects MHC class I expression in 293T cells

Association with β₂-microglobulin and binding a ligand are necessary conditions for cell surface expression of the antigen presenting molecules. MHC class I-related protein, MR1, is suggested to have an antigen presentation function, nevertheless the physiological ligand(s) is (are) still to be determined. In the present study, by characterising the subcellular deportment of human MR1 transfectants, we have shown its differential mobilisation. Our results demonstrated a preferential association of MR1 with β₂-microglobulin in MHC class I-deficient B cell lines. Furthermore, we have evidenced diminished expression of classical MHC class I molecules in human MR1-transfected 293T cells, showing a possible interaction between MR1 and classical MHC class I molecules.     

OKT4+ cytotoxic T cells can lyse targets via class I molecules and can be blocked by monoclonal antibody against T4 molecules

Human cytotoxic T lymphocytes (CTL) have been shown to recognize either class I or class II major histocompatibility (MHC) products. This recognition has been correlated with the expression of OKT antigens on the surface of the CTL. Thus, OKT4+ CTL have been shown to be reactive with class II products, whereas OKT8+ effectors recognize class I molecules. In this study, responder cells were separated according to their OKT4 or OKT8 cell surface phenotype on a fluorescence-activated cell sorter (FACS). The OKT4+ subsets were stimulated with an LCL mutant that did not express DR and MB/MT but did express SB and class I antigens. After 7 days in culture, the activated subsets were tested on a panel of class I matched or mismatched targets. The cytotoxicity observed could be correlated with the presence of matched class I antigens. In addition, monoclonal antibody (MCA) W6/32, directed at a monomorphic determinant on HLA-A and -B molecules, blocked lysis. Furthermore, six OKT4+ CTL clones were derived from the OKT4+ bulk cultures; three clones were found to be directed at class I molecules whereas the other three recognized class II determinants. The ability of these clones to lyse their relevant targets was blocked by OKT4 MCA, raising questions as to the role of the T4 molecule in antigen class-specific CTL recognition.     

Rethinking peptide supply to MHC class I molecules

The notion that peptides bound to MHC class I molecules are derived mainly from newly synthesized proteins that are defective, and are therefore targeted for immediate degradation, has gained wide acceptance. This model, still entirely hypothetical, has strong intuitive appeal and is consistent with some experimental results, but it is strained by other findings, as well as by established and emerging concepts in protein quality control. While not discounting defectiveness as a driving force for the processing of some proteins, we propose that MHC-class-I-restricted epitopes are derived mainly from nascent proteins that are accessed by the degradation machinery prior to any assessment of fitness, and we outline one way in which this could be accomplished.     

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.