The evolution of MHC restrictions in antigen recognition by T cells in a haploidentical bone marrow transplant recipient

We have longitudinally followed the major histocompatibility complex (MHC) restrictions that govern the response of T lymphocytes to specific Ag in a child with severe combined immunodeficiency who was successfully transplanted by using T cell depleted haploidentical maternal bone marrow cells and immunized shortly afterwards with tetanus toxoid (TT) Ag. In the first year post-transplant, monocytes were of both donor and recipient origin whereas T and B cells were of donor origin. Three years after transplant, all monocytes and T and B cells were of donor origin. T lymphocytes taken from the child at that time and depleted in vitro of alloreactivity to paternal Ag proliferated in response to TT presented by maternal as well as paternal monocytes. A TT-specific T cell line established from these cells in the presence of maternal monocytes cooperated with maternal but not with paternal monocytes, whereas a TT-specific T cell line established in the presence of paternal monocytes cooperated with paternal but not with maternal monocytes and with monocytes derived from a paternal uncle who shared the haplotype inherited by the recipient from her father. These results show that long-term memory T cells restricted to recipient MHC Ag not shared with the bone marrow donor continue to circulate long after the disappearance of accessory cells of recipient origin. These T cells could potentially participate in a secondary immune response because they were shown to recognize TT presented by recipient fibroblasts induced to express class II MHC molecules following treatment with IFN-gamma.     

Antigen presentation by EBV-B cells to resting and activated T cells: role of interleukin 1

We have previously demonstrated that Epstein Barr virus-transformed human B lymphocytes (EBV-B cells) present antigen to activated T cells (lines and clones) in a MHC-restricted manner. In the present study, using EBV-nonimmune donors, we demonstrate that EBV-B cells are unable to trigger tetanus toxoid (TT) antigen-specific proliferation in autologous highly purified resting T cells. EBV-B cells from these same individuals were able to present TT to autologous TT-specific activated T cell blasts (Tbl). The inability of EBV-B cells to present TT to resting T cells was not caused by defective antigen processing by EBV-B cells. Thus, paraformaldehyde treatment of antigen-pulsed EBV-B cells did not impair their ability to trigger proliferation of antigen-specific Tbl, and EBV-B cells pulsed with antigen in the presence of autologous TT-specific T cell blasts did not present antigen to resting T cells. Furthermore, antigen-specific proliferation of resting T cells triggered by monocytes was enhanced rather than suppressed by EBV-B cells. The addition of partially purified human IL 1 allowed EBV-B cells to present TT antigen to resting T cells, suggesting that failure to secrete IL 1 contributed to the failure of EBV-B cells to present antigen. IL 1 could not be detected in supernatants of EBV-B cells stimulated with Staphylococcus epidermidis, concanavalin A, and TT antigen in the presence or absence of up to 5% autologous T cells. The differential capacity of EBV-B cells to present antigen to resting T cells vs activated T cells correlated with the T cell requirement for IL 1, because a rabbit antibody to human IL 1 inhibited the monocyte-supported proliferation of resting T cells but not that of activated T cells. These results suggest that the inability of EBV-B cells to present antigen to resting T cells is related to their inability to secrete detectable IL 1.     

Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells

T cell proliferation in response to antigen requires the presence of an antigen-presenting accessory cell. The accessory cell most commonly associated with antigen presentation has been the macrophage (Mo). This study demonstrates that B cells in the form of Epstein Barr virus-transformed B lymphoblasts (EBV-B) are able to present tetanus toxoid (TT) to human T cells and induce proliferation of these cells. T cells freshly isolated from peripheral blood were shown to undergo blast transformation and proliferation in response to TT and irradiated EBV-B cells. Furthermore, TT-reactive T cell blasts were shown to proliferate in the presence of EBV-B cells and TT, but not with other antigens. There was a progressive increase to a plateau in T blast proliferation with increasing numbers of EBV-B cells added to the culture. The concentration of TT required for optimal antigen presentation was similar for EBV-B cells and for Mo. TT-specific cloned T cells also proliferated in response to TT and EBV-B cells and could be continuously grown in culture with TT, interleukin 2-containing supernatant, and EBV-B cells in place of autologous Mo. EBV-B cells pulsed with TT could also act as antigen-presenting cells. The proliferative response of T cell clones to TT in the presence of EBV-B cells was inhibited by antiserum to human p29,34 glycoprotein but not by anti-beta 2-microglobulin. This inhibition was shown to result from interaction with Ia-like determinants on EBV-B cells. These results indicate that B lymphoblastoid cells in man are able, like Mo, to present antigen to T cells in the context of Ia-like determinants.     

Paternal antigen-bearing cells transferred during insemination do not stimulate anti-paternal CD8+ T cells: role of estradiol in locally inhibiting CD8+ T cell responses

Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T cells are suppressed during pregnancy in some but not all mouse models. Since semen has been shown to mediate immune modulation, we tested whether exposure to paternal Ag during insemination activated or tolerized anti-paternal CD8+ T cells. The uterine lumen of mated female mice contained male MHC I+ cells that stimulated effector, but not naive, CD8+ T cells ex vivo. Maternal MHC class I+ myeloid cells fluxed into the uterine lumen in response to mating and cross-presented male H-Y Ag to effector, but not naive, CD8+ T cells ex vivo. However, neither unprimed nor previously primed TCR-transgenic CD8+ T cells specific for either paternal MHC I or H-Y Ag proliferated in vivo after mating. These T cells subsequently responded normally to i.p. challenge, implicating ignorance rather than anergy as the main reason for the lack of response. CD8+ T cells responded to either peptide Ag or male cells delivered intravaginally in ovariectomized mice, but this response was inhibited by systemic estradiol (inducing an estrus-like state). Subcutaneous Ag induced responses in both cases. Allogeneic dendritic cells did not induce responses intravaginally even in ovariectomized mice in the absence of estradiol. These results suggest that inhibition of antiallogeneic responses is restricted both locally to the reproductive tract and temporally to the estrous phase of the menstrual cycle, potentially decreasing the risk of maternal immunization against paternal Ags during insemination.     

Natural killer cell clones can efficiently process and present protein antigens.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.     

Induction of IgG and IgE synthesis in normal B cells by autoreactive T cell clones

During the course of generating tetanus toxoid (TT)-specific T cell clones frm an HLA-DR2,7 donor, four clones were obtained which proliferated in the presence of autologous monocytes alone without the addition of TT antigen. This proliferation was specifically inhibited by anti-HLA-DR framework mouse monoclonal antibody, and appeared to be HLA-DR-restricted. Two of the clones proliferated in response to HLA-DR2-bearing monocytes, and the other two clones proliferated in response to HLA-DR7-bearing monocytes. The capacity of these four autoreactive human T cell helper clones to induce IgE synthesis in B cells was studied. All four clones stimulated autologous peripheral blood B cells to synthesize IgE and IgG antibody. Induction of IgE synthesis in B cells by the autoreactive T cell clones followed the same pattern of HLA-DR restriction which governed the proliferative response of these clones. These results suggest that the interaction of autoreactive helper T cells with B cell HLA-DR antigens may be important in the activation of IgE immune responses in humans.     

Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells

The T cell response to microbial T cell mitogens (MTM) such as enterotoxins from Staphylococcus aureus (SE) and the soluble mitogen from Mycoplasma arthritidis, resemble the minor lymphocyte stimulatory locus (Mls) response in several aspects. An important feature of the Mls response is it restriction to CD4+ cells. This study demonstrates that in contrast to Mls, the MTM response includes both CD4+ and CD8+ subsets. Both CD4+ and CD8+ cells expanded in IL-2 after stimulation with SEB showed preferential expression of T cell receptors bearing V beta 8 domains. Mouse and human target cells could be lysed in the presence of MTM both by MTM-stimulated CD8+ lymphocytes and by MHC class I-restricted CTL clones of defined Ag specificity. MTM-induced lysis required the expression of MHC class II, but not class I Ag, on the target cells. Inhibition studies of SEB and Ag-dependent cytolysis by CTL clones underlined the crucial role of CD3 and LFA-1 in both instances, but showed CD8 dependence only for AG-dependent cytolysis. Together these findings suggest important differences between the putative MTM-mediated interaction of TCR with MHC molecules and the classical TCR/MHC interaction involved in MHC-restricted Ag recognition.     

A SCID patient reconstituted with HLA-incompatible fetal stem cells as a model for studying transplantation tolerance.

We studied a severe combined immunodeficiency (SCID) patient who received transplantations with completely HLA-mismatched fetal liver and thymus from two different donors. The patient is now 14 years old, healthy and shows normal immunoresponses to recall antigens. His T cells are of donor origin, whereas the monocytes, B cells, and natural killer (NK) cells are of the recipient. The successful immunological reconstitution raised questions as to how T and B cells could collaborate across an HLA barrier and how tolerance was achieved. We have shown that tetanus toxin-specific T cell clones isolated from this patient recognized this antigen in the context of host and not of donor HLA-DR, indicating that those cells were educated in the host environment, presumably the thymus. Despite this, an unexpectedly high frequency of host-reactive clones was found that could recognize MHC antigens of the host. It was particularly striking that CD8+ CTL clones were obtained that recognized class I MHC antigens on the host cells. Nevertheless, the patient did not show any sign of acute or chronic graft-versus-host disease (GVHD). These data indicated that no or only incomplete clonal deletion had taken place in this patient and suggest the presence of a peripheral suppressor mechanism. Thus far, we have no indication for the existence of suppressor T cells. Inasmuch as it was found that host-reactive T cells fail to produce IL-4, which is exceptional for CD4+ T cells, we are exploring the possibility that abnormal cytokine production patterns of host-reactive T cells are associated with suppression of these cells in vivo.     

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.     

Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines

Murine L cells expressing the products of transfected HLA-DR1 genes functioned as APC for two influenza-specific, human Th cell clones with comparable efficiency to a DR1-expressing human lymphoblastoid cell line. In order to investigate the restriction specificity of the two Th clones, a transfectant expressing the species-mismatched MHC class II dimer DR1:I-E was tested as an APC. Both T cells showed no loss of Ag sensitivity due to substitution of the murine chain. One of the Th clones, TLC 72, showed even greater degeneracy by responding to Ag in the context of I-Ek. Taking into account the lower level of MHC class II expression on the I-Ek transfectant, there is remarkably little loss of efficiency of Ag-induced T cell activation due to the substitution of I-E for DR as restriction element. The Ag-specific responses of both clones were inhibited by anti-CD4 antibody when DR-transfected L cells or human lymphoblastoid cells were used as APC. This inhibition was also seen when Ag was presented to TLC72 by the I-Ek-expressing transfectant. Whether this inhibition is the result of negative signaling or of blocking an interaction between human CD4 and I-Ek is discussed. Similarly the inhibitory effects of mAb against the T cell accessory molecule LFA/1 were the same for both clones when either the transfectants or the lymphoblastoid cell line were used as APC, suggesting that L cells may express a molecule that is capable of acting as a ligand for human LFA/1. The results presented here further illustrate the value of transfectants in analyzing T cell recognition and accessory cell requirements. The patterns of degeneracy of MHC restriction exhibited by these clones provides a platform for a more detailed analysis of key residues involved in MHC class II-restricted T cell Ag recognition.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.     

A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4+ T cells

The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.     

Antigen presentation by human dermal fibroblasts: activation of resting T lymphocytes

We have shown that human dermal fibroblasts, exposed to interferon-gamma (IFN-gamma) to induce surface class II major histocompatibility complex (MHC) antigens, were capable of presenting tetanus toxoid (TT) antigen to human TT-specific T cell clones. Antigen presentation by fibroblasts was antigen dependent, required HLA-DR expression by fibroblasts, and was MHC restricted. In contrast, we now report that IFN-gamma-treated fibroblasts are unable to present TT antigen to purified resting T cells obtained from the peripheral blood of TT-immune donors. In addition, although IFN-gamma-treated fibroblasts were able to stimulate alloreactive T cell clones, they were unable by themselves to stimulate primary allogeneic responses in resting T cells. The failure of fibroblasts to stimulate resting T cells was not due to suppressor effects by fibroblasts, because induction of TT and alloantigen responses in resting T cells by monocytes was not inhibited by the presence of fibroblasts. On the contrary, IFN-treated fibroblasts were synergistic with small numbers of monocytes in activating resting T cells. In addition, the failure of antigen presentation by fibroblasts to resting T cells was reversed by the addition of recombinant human interleukin 2 (rIL 2) to cultures, but not of purified human interleukin 1 (IL 1). These results emphasize that the requirements for activation of resting T cells differ from those of T cell clones. Although fibroblasts can efficiently present antigen to T cell clones, antigen presentation by fibroblasts to resting T cells requires the addition of exogenous IL 2. It is postulated that fibroblasts differ from classical antigen-presenting cells in that fibroblasts are incapable of stimulating the production of IL 2 in resting T cells.     

Indirect recognition of porcine swine leukocyte Ag class I molecules expressed on islets by human CD4+ T lymphocytes

Xenotransplantation of porcine islets is considered a viable alternative treatment for type 1 diabetes mellitus. Therefore, we characterized human PBL responding to porcine islets both in vitro by coculture and in vivo using SCID mice reconstituted with human PBLs (HuPBL-SCID) and transplanted with porcine islets. T cell lines generated in vitro and graft-infiltrating T cells obtained from HuPBL-SCID mice were CD4+-proliferated specifically to porcine islets cultured with autologous APC. This proliferation was abrogated by an anti-human class II Ab. These T cell lines also proliferated to purified swine leukocyte Ag (SLA) class I molecules in the presence of self-APC, indicating that the primary xenoantigens recognized are peptides derived from SLA. This CD4+ T cell line lysed porcine islets but not splenocytes. CD4+ T cell clones with Th0, Th1, and Th2 cytokine profiles were isolated. The Th0 and Th1 clones lysed porcine islets, whereas the Th2 clone that secreted a large amount of IL-4 was not lytic. These results demonstrate that human T cells responding to porcine islets are primarily CD4+ and recognize porcine xenoantigens by the indirect Ag pathway presentation. These activated T cells produce cytokines that lyse islets. Furthermore, we demonstrate that the major porcine xenoantigens recognized are SLA class I molecules.     

Defining the requirements for peptide recognition in gene therapy-induced T cell tolerance

Expression of a retrovirally transduced MHC class I Ag, H-2K(b) (K(b)), in bone marrow-derived cells leads to specific prolongation of K(b) disparate skin grafts. To examine the extent to which peptides derived from K(b) contribute to the induction of tolerance, retroviruses carrying mutant K(b) genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of K(b) showed prolongation of K(b) disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant K(b) molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from K(b) through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

Cutting edge: B cells promote CD8+ T cell activation in MRL-Fas(lpr) mice independently of MHC class I antigen presentation

Spontaneous CD8+ T cell activation in MRL-Faslpr mice is B cell dependent. It is unclear whether this B-dependent activation is mediated by direct Ag presentation via MHC class I proteins (i.e., cross-presentation) or whether activation occurs by an indirect mechanism, e.g., via effects on CD4+ cells. To determine how CD8+ T cell activation is promoted by B cells, we created mixed bone marrow chimeras where direct MHC class I Ag presentation by B cells was abrogated while other leukocyte compartments could express MHC class I. Surprisingly, despite the absence of B cell class I-restricted Ag presentation, CD8+ T cell activation was intact in the chimeric mice. Therefore, the spontaneous B cell-dependent CD8+ T cell activation that occurs in systemic autoimmunity is not due to direct presentation by B cells to CD8+ T cells.     

Kinetics of MHC-antigen complex formation on antigen-presenting cells

With the use of flow cytometry, we recorded changes in intracellular ionized calcium [Ca2+]i of Indo-1 loaded T cells that were triggered by contact with APC. This rapid readout of TCR perturbation enabled us to monitor the formation of stimulatory Ag-MHC complexes on EBV-transformed B cells that were either pulsed with native tetanus toxoid (TT) or with a 12-amino-acid fragment of this protein. Neither unpulsed APC nor Ag-specific APC that were pulsed with native Ag and kept at +4 degrees C were able to induce changes in basal T cell [Ca2+]i in TT-specific T cell clones. After 1 h at 37 degrees C, however, the Ag-pulsed APC were able to induce a three-to-fourfold increase in [Ca2+]i. This length of time appeared to be almost independent of the concentration of Ag with which the APC were pulsed, suggesting that the lag time was due more to intracellular transit than to association of the processed Ag with the MHC molecule. Furthermore, the same lag time and independence of Ag concentration were found when the EBV-transformed B cells were pulsed with a mouse-anti-transferrin receptor mAb and tested for their capacity to trigger a T cell clone specific for processed mouse Ig. This indicates that, in addition to surface Ig, other receptors that are internalized can function in the same fashion in the uptake and processing of a soluble Ag. In contrast to what was found with intact native Ag, no lag time was observed when the APC were pulsed with high concentrations of a 12-amino-acid peptide, containing the amino acid sequence recognized by a TT-specific T cell clone, suggesting that the formation of MHC-peptide complexes occurs instantly. Pulsing with a lower peptide concentration, however, caused the appearance of a time-dependent increase in efficacy of Ag presentation, suggesting a slow accumulation of MHC-peptide complexes on the B cell membrane.