Mechanisms of induction of primary virus-specific cytotoxic T lymphocyte responses.

We have investigated the ability of various antigen-presenting cell (APC) types to induce primary anti-viral cytotoxic T lymphocyte (CTL) responses by single in vitro stimulation. Of these APC types, only dendritic cells (DC) and RMA-S lymphoma cells could induce primary CTL responses, but by divergent mechanisms. DC were capable of generating primary virus-specific CTL, either by presenting viral peptide or processed infectious virus. In contrast, RMA-S cells could not present endogenous antigen, e.g. after virus infection, but this cell line very efficiently presented exogenous viral peptides to induce primary virus-specific CTL in vitro. Spleen cells, lipopolysaccharide-induced B cell blasts or the non-mutated RMA cells did not have the ability to trigger unprimed T cells by single in vitro stimulation. We have investigated several characteristics important for primary CTL response induction by DC and RMA-S cells (summarized in Fig. 6). Primary CTL response induction by DC or RMA-S cells was blocked by anti-LFA-1 or anti-CD8 monoclonal antibodies (mAb). DC rapidly aggregated with unprimed T cells, which was independent of LFA-1 and CD8 molecules. RMA-S cells did not form conjugates with unprimed T cells. Despite their abundant major histocompatibility complex (MHC) class I cell-surface expression, DC did not bind much exogenously added viral peptide. In contrast, the MHC class I molecules on RMA-S cells bound a large quantity of exogenously administered peptide. Powerful adhesion by DC and high expression of relevant MHC/peptide complexes on RMA-S cells are important features in the initial contact with unprimed T lymphocytes. In a later stage of contact, both DC and RMA-S cells activate LFA-1 (and CD8) molecules at the T cell surface to strengthen and maintain the contact between T cell and APC.     

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2D^b using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind D^b. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to D^b. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the D^b pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited D^b-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all D^b-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, D^b-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to D^b, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.     

In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.     

The alpha 3 domain of major histocompatibility complex class I molecules plays a critical role in cytotoxic T lymphocyte stimulation

Allogeneic major histocompatibility complex class I molecules induce strong cytotoxic T lymphocyte (CTL) responses whereas xenogeneic molecules do not. We have tested a series of mouse/human hybrid molecules for their ability to stimulate mouse CTL. The molecules with murine alpha 3 domains consistently stimulated stronger CTL responses than those with human alpha 3 domains, independent of the species origin of the N-terminal alpha 1 or alpha 2 domains. We have found that the ability of class I molecules to induce strong cytotoxic responses correlates positively with their ability to stimulate expansion of the CD8+CD4-T cell subset. The results indicate that mouse T cells can recognize class I molecules with human alpha 1 and/or alpha 2 domains, but for efficient stimulation of these T cells it is important that the immunizing molecule contains a murine alpha 3 domain. We suggest that T cell priming requires an efficient interaction of CD8 with the class I alpha 3 domain, and this shows some species restriction.     

CD8 T cells from major histocompatibility complex class II-deficient mice respond vigorously to class II molecules in a primary mixed lymphocyte reaction

Mature CD4⁺ and CD8⁺ T cells are restricted by major histocompatibility complex (MHC) class II and class I molecules, respectively. In a primary mixed lymphocyte reaction (MLR), CD8⁺ T cells from C57BL/6 (B6) mice can respond to allo-class I molecules, but not allo-class II molecules. However, a significant fraction of CD8⁺ T cells from C57BL/6 class II-deficient (B6Aα^?) mice violate this rule by responding vigorously in a MLR to class II molecules. The frequency of responding cells is ～ 50 % of that of B6 CD8⁺ T cells responding to B6bm1 allo-class I molecules. This response requires neither appropriate co-receptor, i.e. CD4, nor exogenous lymphokines, indicating that interactions between the T cell receptors (TCR) and class II molecules are remarkably efficient. Since these CD8⁺ T cells are positively selected by class I molecules in the thymus of class II-deficient mice, these CD8⁺ T cells should interact with both classes of MHC molecules. The absence of thymic negative selection by class II molecules may result in the production of these CD8⁺ T cells. The data imply that a substantial fraction of CD4⁺CD8⁺ double-positive thymocytes in wild-type mice interacts with both classes of MHC molecules prior to thymic selection.     

The role of the human major histocompatibility complex in cytotoxic T-cell responses to virus-infected cells

The human major histocompatibility complex (HLA) has been demonstrated to play two roles in the generation and expression of cytotoxic T-lymphocyte responses to virusinfected cells: (1) cytotoxic T cells can only recognize viral antigens in conjunction with antigens encoded by HLA-A and -B genes; and (2) HLA-linked genes may control the capacity to generate T-cell responses to a given virus or to virus in conjunction with particular self HLA-A and -B antigens. Analysis of T-cell responses generatedin vivo to Epstein-Barr virus suggests that human T cells may recognize virus in conjunction with antigens other than the class I HLA polymorphic specificities.     

Graft rejection by T cells not restricted by conventional major histocompatibility complex molecules

The appropriate crosses of mice lacking conventional major histocompatibility complex (MHC) class I or class II molecules generate single- and double-deficient offspring. These were used as donors for skin grafts across major plus minor, or just minor, histocompatibility differences. Surprisingly, in the two circumstances, there was a rapid rejection of grafts lacking both MHC class I and class II molecules. Rejection was mediated by thymically derived CD4⁺ T cells of the host. We provide evidence that these T cells recognize an unconventional ligand, capable of activating a pre-formed T cell compartment but incapable of positively selecting it. The existence of this unexpected rejection phenomenon should serve to caution those aiming to engineer “universal donor” cells by simply abrogating expression of MHC class I and class II molecules.     

Transection of major histocompatibility complex class I-induced neurites by cytotoxic T lymphocytes.

Damage to neurites with transection of axons and spheroid formation is commonly noted in the central nervous system during viral and autoimmune diseases such as multiple sclerosis, but it remains open whether such changes are caused primarily by immune mechanisms or whether they are secondary to inflammation. The present experiments explored whether neurites can be directly attacked by cytotoxic T lymphocytes (CTLs). Cultured murine neurons induced by interferon-gamma and tetrodotoxin to express major histocompatibility complex class I were pulsed with a dominant peptide of the lymphochoriomeningitis virus envelope glycoprotein (GP33) and then confronted with GP33-specific CD8(+) CTLs. Within 3 hours the neurites developed cytoskeleton breaks with adjacent solitary neuritic spheroids, as documented by confocal examination of the cytoskeletal marker beta-tubulin III. At the same time cytoskeleton staining of the neuronal somata showed no damage. The CTLs selectively attacked neurites and induced segmental membrane disruption 5 to 30 minutes after the establishment of peptide-specific CTL-neurite contact, as directly visualized by live confocal imaging. Thus, major histocompatibility complex class I/peptide-restricted CD8(+) T lymphocytes can induce lesions to neurites, which might be responsible for axonal damage during neuroinflammatory diseases.     

Analysis of immunological tolerance to major histocompatibility complex antigens. I. High frequencies of tolerogen-specific cytotoxic T lymphocyte precursors in mice neonatally tolerized to class I major histocompatibility complex antigens

Injection of (CBA X A)F1 cells into neonatal CBA mice rendered them tolerant to skin grafts of (CBA X A)F1 origin. Limiting dilution analysis revealed a very low frequency of tolerogen-inducible cytotoxic T lymphocyte precursors (CTL-P) in spleens of tolerant mice. Two in vitro procedures allowed, however, the induction of tolerogen-specific CTL-P of high frequencies in tolerant mice: (a) the "by-pass" activation of spleen cells from tolerant mice by concanavalin A under short-term bulk culture conditions followed by culture of limiting numbers of activated responder cells, and (b) absorption of spleen cells from tolerant mice on monolayers of tolerogen-activated T cells from normal syngeneic mice. Furthermore, spleen cells from tolerant mice, recently challenged with a tolerogen-bearing skin graft, specifically suppressed the activation of tolerogen-reactive splenic CTL-P from normal CBA mice under limiting dilution conditions. These data confirm the presence of tolerogen-specific CTL-P of high frequency in tolerant mice and suggest their functional inactivation through a suppressive mechanism.     

On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens

Although there are numerous minor histocompatibility antigens (MiHA), T cell responses leading to graft-versus-host (GVH) and graft-versus-tumor effects involve only a small number of immunodominant MiHA. The goal of the present study was to analyze at the cellular and molecular levels the mechanisms responsible for MiHA immunodominance. Cytotoxic T lymphocytes (CTL) generated in eight combinations of H2^b strains of mice were tested against syngeneic targets sensitized with HPLC-fractionated peptides eluted from immunizing cells. The number of dominant MiHA was found to range from as little as two up to ten depending on the strain combination used. The nature of dominant MiHA was influenced by both the antigen profile of the antigen-presenting cells (APC) and the repertoire of responding CTL. When C57BL/6 dominant MiHA (B6^dom) and H-Y were presented on separate APC, they showed similar immunogenicity. In contrast, when they were presented on the same APC, B6^dom MiHA totally dominated H-Y. B6^dom MiHA did not suppress anti-H-Y responses by acting as T cell receptor antagonists for anti-H-Y CTL, nor were anti-B6^dom CTL precursors more abundant than anti-H-Y CTL precursors. Dominance resulted from competition for the APC surface between anti-B6^dom and anti-H-Y CTL; the crucial difference between the dominant and the dominated MiHA appears to depend on the differential avidity of their respective CTL for APC. The only B6^dom epitope thus far identified is the nonapeptide AAPDNRETF presented by H2-D^b. We found that compared with other known D^b-binding peptides, AAPDNRETF is expressed at very high levels on the cell surface, binds to the D^b molecule with very high affinity, and dissociates very slowly from its presenting class I molecule. These data indicate that one cannot predict which MiHA will be dominant or dominated based simply on their respective immunogenicity when presented on separate APC. Indeed, the avidity of T cell/APC interactions appears to determine which antigen(s) will trigger T cell responses when numerous epitopes are presented by the same APC.     

Co-receptor-independent signal transduction in a mismatched CD8+ major histocompatibility complex class II-specific allogeneic cytotoxic T lymphocyte

The contribution of co-receptors in signal transduction upon T cell receptor (TCR)-mediated recognition of major histocompatibility complex (MHC) class II antigen by mature T lymphocytes expressing TCR derived from the apparently co-receptor-independent, I-A^k-specific allogeneic CD8⁺ CTL clone QM11 has been examined. Mature double-negative, CD8⁺ and CD4⁺ bulk T cell lines and clones expressing TCRQM11 were developed from TCRQM11 transgenic mice. All these T cells, irrespective of co-receptor expression, showed specific lytic activity on cells expressing I-A^k. Furthermore, co-receptorless mutants were obtained from a CD4⁺ and CD8⁺ clone. The responses of these co-receptorless mutants upon specific recognition of the alloantigen, as judged by cytolytic activity, granule exocytosis, lymphokine production, proliferation, and tyrosine phos-phorylation of the ξ chain, were comparable to those of the original clones. Thus, the results proved the co-receptor independence of the recognition of I-A^k by TCRQM11 and further indicated there is no indispensable unique signal transduced by co-receptors. However, when the amount of the available antigen was limited by anti-I-A^k antibody, the CD4⁺ T cell clone showed a remarkable resistance to the inhibition whereas the mismatched CD8⁺ clone was readily inhibitable. The anti-I-A^k-resistant component of the CD4⁺ clone showed dependency on the CD4 molecule. Taken collectively, the results indicate that the role played by a co-receptor molecule in mature T cells is purely quantitative amplification of the signal through the formation of a TCR/MHC/co-receptor ternary complex, and also indicate that the role of co-receptor molecules as TCR-independent adhesion molecules is at best minimal.     

Down-regulation of class II major histocompatibility complex molecules on antigen presenting cells after interaction with helper T cells

The recognition of antigenic peptides by CD4⁺ helper T cells is demonstrated here to result in a dramatic (up to 90%) decrease in expression of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APC). The reduction is selective to the class II isotype presenting the antigen, but if affects both allelic forms of the same isotype in heterozygous APC. The observed MHC down-regulation requires a specific T cell receptor-peptide-class II interaction, a direct contact between T cell and APC, and the involvement of CD2 molecules. These findings have important implications for the regulation of immune response, self tolerance, and autoimmunity.     

Antigen-specific suppression of human antibody responses by allogeneic T cells. III. Role of the major histocompatibility complex

Specific antibody responses obtained in vitro from human blood mononuclear cells (PBM) were profoundly suppressed by allogeneic T cells. Experiments carried out with combinations of cells from HLA identical siblings, and HLA identical but unrelated donors, showed that suppression depended upon HLA incompatibility between responding PBM and allogeneic Ts. In order to map the specific HLA loci concerned, a series of experiments were undertaken using combinations of cells from a large number of HLA typed donors. Significant suppression was found to occur in every combination of HLA incompatible cells tested, including those with nonidentity at HLA-A, B, DR, A and DR, or B and DR, suggesting that suppression can be generated by nonidentity at class I or class II loci. With some HLA-A homozygous donors, however, a dominant role for class I (HLA-A) antigens was indicated by the finding of one-directional suppression in combinations where the HLA-A locus was seen as foreign by one partner only (A3,----A2,3; and A2----A2,26). Similar one-directional suppression was also seen with cells from a pair of siblings who were HLA identical except for a single A locus antigen arising from an HLA-A/B recombination (A3,----A3,1). These results indicate an important, but not exclusive role for class I MHC antigens in the activation of allogeneic Ts. The way in which this occurs is unknown, but one possibility is that it results from the activation of normal antigen-specific Ts by the interaction of their receptors for self-MHC with cross-reacting alloantigens.     

Leishmania major infection in mice primes for specific major histocompatibility complex class I-restricted CD8+ cytotoxic T cell responses

This report shows that lymphoid tissues of mice which have resolved a primary infection with Leishmania major contain parasite-specific major histocompatibility complex (MHC) class I-restricted cytolytic CD8⁺ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen-presenting cells pulsed with a cyanogen bromide digest of L. major. In H-2^b mice, two distinct populations of CD8⁺ T cells were identified which both lysed target cells pulsed with L. major-derived peptides but were restricted by a different H-2^b class I gene product. Interestingly, these two populations appear to recognize different parasite-derived peptides. It is noteworthy that one K°-restricted CD8⁺ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major-derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite-specific MHC class I restricted cytolytic CD8⁺ T cells for the elimination of L. major by the infected host remains to be determined.     

Identification of tumor antigen-specific cytotoxic T lymphocytes cross-recognizing allogeneic major histocompatibility class I molecules

Adoptive immunotherapy of cancer utilizes tumor antigen-specific cytotoxic T lymphocytes (CTL) as mediators of a targeted anti-tumor effect. In this study, we show that such CTL can be able to cross-recognize allogeneic major histocompatibility complex (MHC) molecules in a phenomenon of molecular mimicry. A self histo-leukocyte antigen (HLA) A*0201-restricted CTL specific for peptide MT27-35 from the human differentiation antigen Melan-A/MART-1 was shown to cross-recognize allogeneic A*0220 molecules which differ from syngeneic A*0201 for a single amino acid substitution at position 66 of the antigen-binding groove. A*0220 molecules were recognized on a variety of human cells of different histological origin but not on COS-7 cells. A second self-A*0201-restricted CTL, specific for peptide D10/6-271 encoded by the tumor-specific DAM-gene family, was shown to cross-recognize allogeneic B*3701 molecules which differ from syngeneic A*0201 by 32 amino acids in the peptide antigen-binding cleft. B*3701 molecules were recognized on a variety of cell types including COS-7 cells. These data raise new safety issues for clinical trials of cancer immunotherapy using adoptive transfer of in vitro generated, allogeneic CTL with specific anti-tumor activity.     

Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex

We aimed to study the effects of mesenchymal stem cells (MSCs) on alloreactivity and effects of T-cell activation on human peripheral blood lymphocytes (PBLs) in vitro. MSCs were expanded from the bone marrow of healthy subjects. MSCs isolated from second to third passage were positive for CD166, CD105, CD44, CD29, SH-3 and SH-4, but negative for CD34 and CD45. MSCs cultured in osteogenic, adipogenic or chondrogenic media differentiated, respectively, into osteocytes, adipocytes or chondrocytes. MSC added to PBL cultures had various effects, ranging from slight inhibition to stimulation of DNA synthesis. The stimulation index (SI = (PBL + MSC)/PBL) varied between 0.2 and 7.3. The SI was not affected by the MSC dose or by the addition of allogeneic or autologous MSCs to the lymphocytes. Suppression of proliferative activity was observed in all experiments after the addition of 10,000-40,000 MSCs to mixed lymphocyte cultures (MLCs). Lymphocyte proliferation was 10-90%, compared with a control MLC run in parallel without MSCs. In contrast, the addition of fewer MSCs (10-1000 cells) led to a less consistent suppression or a marked lymphocyte proliferation in several experiments, ranging from 40 to 190% of the maximal lymphocyte proliferation in control MLCs. The ability to inhibit or stimulate T-cell alloresponses appeared to be independent of the major histocompatibility complex, as results were similar using 'third party' MSCs or MSCs that were autologous to the responder or stimulating PBLs. The strongest inhibitory effect was seen if MSCs were added at the beginning of the 6 day culture, and the effect declined if MSCs were added on day 3 or 5. Marked inhibitory effects of allogeneic and autologous MSCs (15,000) were also noted after mitogenic lymphocyte stimulation by phytohaemagglutinin (median lymphocyte proliferation of 30% of controls), Concanavalin A (56%) and protein A (65%). Little, if any, inhibition occurred after stimulation with pokeweed mitogen. Low numbers of MSCs (150 cells) were unable to inhibit mitogen-induced T-cell responses. MSCs have significant immune modulatory effects on MLCs and after mitogenic stimulation of PBL. High numbers of MSCs suppress alloreactive T cells, whereas very low numbers clearly stimulated lymphocyte proliferation in some experiments. The effect of a larger number of MSCs on MLCs seems more dependent on cell dose than histocompatibility and could result from an 'overload' of a stimulatory mechanism.     

In vitro primary induction of cytotoxic T cells against virus-infected syngeneic cells.

An in vitro method is described for primary induction of murine cytotoxic T cells against syngeneic cells infected with ectromelia or lymphocytic choriomeningitis (LCM) virus. Cytotoxicity was assayed by 51Cr release from macrophage or L929 target cells. Cytotoxic activity was sensitive to anti-theta and complement and was expressed only against target cells infected with the same virus and sharing H-2K or H-2D genes with the infected stimulator cells. The crucial factors in generating responses were mouse strain, responder: stimulator ratio, nature of infected stimulator cells, and presence of sufficient macrophages. C57BL/6 cells were less demanding than CBA/H and BALB/c cells. Under optimal conditions defined here, the in vitro response had similar kinetics and potency to the primary response in the spleen in vivo.     

T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding

T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-31g). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4⁺ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4⁺ T cell response.