Anti-idiotypic T cells suppress rejection of renal allografts in rats

Kidney allografts between inbred rats differing at the major histocompatibility complex (MHC) are normally rejected, usually within 10 to 12 days. In many strain combinations, however, permanent graft acceptance can be induced by either immunological enhancement or a short course of immunosuppressive chemotherapy. In both cases, prolonged graft survival is accompanied by the appearance in the spleen of a population of suppressor cells. When transferred to a syngeneic host, these cells abrogate or strikingly diminish the rejection response elicited by a renal allograft of the same genotype as the original kidney donor. We have now examined the properties of these suppressor cells and have detected a subpopulation that proliferates in vitro when stimulated by irradiated syngeneic T blasts reactive to MHC alloantigens of the kidney donor strain. Comparable proliferation, however, is not induced either by syngeneic blasts reactive to a third strain or by polyclonal syngeneic blasts. These results support the hypothesis that this subpopulation is anti-idiotypic, with specificity for the idiotypes carried by syngeneic T cells stimulated by the kidney allograft. Such anti-idiotypic cells could function as suppressors.     

Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.     

Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope

In autoimmune type 1 diabetes, pathogenic T lymphocytes are associated with the specific destruction of insulin-producing beta-islet cells. Identification of the autoantigens involved in triggering this process is a central question. Here we examined T cells from pancreatic draining lymph nodes, the site of islet-cell-specific self-antigen presentation. We cloned single T cells in a non-biased manner from pancreatic draining lymph nodes of subjects with type 1 diabetes and from non-diabetic controls. A high degree of T-cell clonal expansion was observed in pancreatic lymph nodes from long-term diabetic patients but not from control subjects. The oligoclonally expanded T cells from diabetic subjects with DR4, a susceptibility allele for type 1 diabetes, recognized the insulin A 1-15 epitope restricted by DR4. These results identify insulin-reactive, clonally expanded T cells from the site of autoinflammatory drainage in long-term type 1 diabetics, indicating that insulin may indeed be the target antigen causing autoimmune diabetes.     

Minor but not major histocompatibility antigens of thymus epithelium tolerize precursors of cytolytic T cells

Treatment of fetal thymuses with 2-deoxyguanosine depletes these organs of many haematopoietic cells, and if such thymuses are transplanted into allogeneic athymic nude mice, intrathymic development of cytolytic T-lymphocyte precursors (CTL-P) occurs, including those which are specific for class I major histocompatibility complex (MHC) antigens expressed by the thymus epithelium. Thus, T cells from BALB/c (H-2d) nude mice transplanted with allogeneic C57BL/6 (H-2b) thymic epithelium can be stimulated in vitro to produce CTL specific for H-2b class I MHC antigens. We report here that thymocytes and lymph node T cells from such mice are responsive in mixed leukocyte reaction in the absence of exogenous growth factors, indicating that lack of tolerance is manifest at the level of CTL-P and proliferating T cells. We also show that T cells from such mice are tolerant to minor histocompatibility antigens of the thymus donor in the context of MHC antigens of the recipient. The results indicate that haematopoietic rather than epithelial cells tolerize CTL-P and that donor-type minor but not major histocompatability antigens can be presented in tolerogenic form by haematopoietic cells expressing recipient-type MHC antigens.     

Responses to the H-2Kba mutant involve recognition of syngeneic Ia molecules

Conventional antigens appear to be recognized by T lymphocytes only when associated with major histocompatibility complex (MHC) antigens. Using antigen-specific proliferation as a model for helper T lymphocytes, it has been demonstrated that Ly1+T cells recognize antigen presented in association with syngeneic Ia molecules. In contrast to responses to conventional antigens, however, a large number of studies have suggested that the stimulation of alloreactive Ly1+T cells, and helper T cells specific for allogeneic cytotoxic T lymphocyte (CTL) responses, involve the direct recognition of Ia alloantigens. For the generation of optimal allogeneic CTL activity it has been proposed that Ly1+T cells recognize allo-Ia antigens directly and provide help to pre-CTLs that respond to allo-H-2K and/or D determinants. Thus, the B6.C.H-2bm1 mutant (bm1, formerly referred to as Hz1), which is believed to consist of a substitution of two amino acids in the H-2Kb antigen, has presented a paradox, for it can stimulate strong mixed lymphocyte culture (MLC), graft versus host and CTL responses by T cells of H-2b haplotype mice in the apparent absence of any alloantigenic differences in the I region. We now present evidence that the stimulation of proliferative and helper T cells by the mutant B6.C.H-2bm1 results from the H-2Kba antigen being recognized in the context of syngeneic Ia determinants. Thus responses to both conventional antigens and allogeneic MHC gene products may proceed via the recognition of antigen in the context of self Ia molecules.     

A minor transplantation antigen detected by MHC-restricted cytotoxic T lymphocytes during graft-versus-host disease

Transplantation of bone marrow can give rise to graft-versus-host disease when donor T lymphocytes, mismatched with the host for major histocompatability (MHC) antigens, become sensitized and attack host tissues. However, graft-versus-host disease can also arise between donor and host with compatible MHC antigens but mismatched for a minor histocompatability antigen. We report here on the occurrence of severe acute graft-versus-host disease in a male patient with acute myeloid leukaemia who had received bone marrow matched for MHC (HLA) antigens from his sister. Strong cytotoxicity of the posttransplantation (that is, donor) lymphocytes against the patient's pretransplantation lymphocytes was found. Thus, the transplanted lymphocytes differed in a non-HLA antigen from the patient. The possible role of this strong cytotoxic minor histocompatability antigen in the development of graft-versus-host disease in man is being evaluated. Furthermore, with the use of cytotoxic T-cell lines, derived from the patient's 6 day effector cells, we are now able to type for it before grafting.     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.     

T cells become licensed in the lung to enter the central nervous system

The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.     

T-cell recognition of a chimaeric class II/class I MHC molecule and the role of L3T4

In addition to expressing clonally distributed antigen-specific and major histocompatibility complex (MHC)-restricted receptors, T cells also express non-clonally distributed surface molecules that are involved in T-cell function. Among the most intriguing of the latter are L3T4 and Lyt 2, which are expressed on individual T lymphocytes in striking, though not absolute, concordance with their restriction by either class II or class I MHC determinants, and which are thought to contribute to the overall avidity of T-cell interactions by binding to monomorphic determinants on class II and class I MHC molecules, respectively. To examine the ability of T cells to recognize a single class II domain in the absence of the remainder of the Ia molecule, as well as to evaluate the structural basis for the putative interaction of L3T4 with Ia, a recombinant class II/class I murine MHC gene was constructed and introduced into mouse L cells. Here we demonstrate that a subset of class II allospecific cytotoxic T lymphocytes (CTL) can specifically recognize and lyse L-cell transfectants expressing an isolated polymorphic A beta 1 domain, and that anti-L3T4 antibody can block such killing, a result inconsistent with the highly conserved membrane-proximal domains of Ia acting as unique target sites for L3T4 binding.     

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.     

Developmental regulation of T-cell receptor gene expression

In contrast to B cells or their antibody products, T lymphocytes have a dual specificity, for both the eliciting foreign antigen and for polymorphic determinants on cell surface glycoproteins encoded in the major histocompatibility complex (MHC restriction). The recent identification of T-cell receptor glycoproteins as well as the genes encoding T-cell receptor subunits will help to elucidate whether MHC proteins and foreign antigens are recognized by two T-cell receptors or by a single receptor. An important feature of MHC restriction is that it appears to be largely acquired by a differentiating T-cell population under the influence of MHC antigens expressed in the thymus, suggesting that precursor T cells are selected on the basis of their reactivity with MHC determinants expressed in the host thymus. To understand this process of 'thymus education', knowledge of the developmental regulation of T-cell receptor gene expression is necessary. Here we report that whereas messenger RNAs encoding the beta-and gamma-subunits are relatively abundant in immature thymocytes, alpha mRNA levels are very low. Interestingly, whereas alpha mRNA levels increase during further development and beta mRNA levels stay roughly constant, gamma mRNA falls to very low levels in mature T cells, suggesting a role for the gamma gene in T-cell differentiation.     

Predictable acquisition of a new MHC recognition specificity following expression of a transfected T-cell receptor beta-chain gene

Activation of mature T lymphocytes requires specific corecognition of antigen together with membrane-associated glycoprotein products of the major histocompatibility complex (MHC). This dual specificity is determined by a single receptor structure consisting of a clone-specific alpha beta heterodimer. Because both the alpha and beta subunits possess unique combining-site-containing V regions, it remains an open issue as to what contribution each of the two chains of the receptor makes to the antigen versus MHC recognition specificities of the complete dimer present on any given T cell or in the T-cell pool as a whole. In the present work, we have used DNA-mediated gene transfer to express a new alpha or beta chain in a recipient murine T-cell hybridoma possessing a related antigen but distinct MHC specificity compared to the receptor-gene donor. Our results demonstrate that a beta-gene transfected hybridoma expresses new receptors with a predictable hybrid specificity, establishing that the beta chain has the predominant role in MHC molecule recognition in this model.     

ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum

The ability of killer T cells carrying the CD8 antigen to detect tumours or intracellular pathogens requires an extensive display of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of potential target cells. These peptides are derived from almost all intracellular proteins and reveal the presence of foreign pathogens and mutations. How cells produce thousands of distinct peptides cleaved to the precise lengths required for binding different MHC class I molecules remains unknown. The peptides are cleaved from endogenously synthesized proteins by the proteasome in the cytoplasm and then trimmed by an unknown aminopeptidase in the endoplasmic reticulum (ER). Here we identify ERAAP, the aminopeptidase associated with antigen processing in the ER. ERAAP has a broad substrate specificity, and its expression is strongly upregulated by interferon-gamma. Reducing the expression of ERAAP through RNA interference prevents the trimming of peptides for MHC class I molecules in the ER and greatly reduces the expression of MHC class I molecules on the cell surface. Thus, ERAAP is the missing link between the products of cytosolic processing and the final peptides presented by MHC class I molecules on the cell surface.     

T-cell specificity for H-2 and Ir gene phenotype correlates with the phenotype of thymic antigen-presenting cells

Experiments with chimaeric animals have demonstrated that the H-2 restriction specificity and immune response (Ir) gene phenotype of the T cell is acquired during development in the thymus. The mechanism by which this process occurs is unclear. One level of obligate expression of H-2 and Ir gene products is on the surface of antigen-presenting cells (APCs) which come from bone marrow precursors. We have now examined the turnover of APCs in the thymuses of F1 leads to parent (P) radiation-induced bone marrow chimaeras and found that APCs of donor phenotype appear at about 2 months after reconstitution. If the peripheral T-cell population is depleted after this time, new T cells emerging from the parental thymus (containing F1 APCs) behaving like F1 T cells, suggesting that cells from the bone marrow can influence thymic-directed T-cell differentiation. The thymic APC is an attractive condidate to play such a part in the development of the T-cell repertoire.     

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.     

Interaction of peptide antigens and class II major histocompatibility complex antigens

T lymphocytes require a foreign antigen to be presented on a cell surface in association with a self-transplantation antigen before they can recognize it effectively. This phenomenon is known as major histocompatibility complex (MHC) restriction. It is not clear how an incalculably large number of foreign proteins form unique complexes with a very limited number of MHC molecules. We studied the recognition properties of T cells specific for a peptide derived from bacteriophage lambda cI protein. Analogues of this peptide, as well as peptides derived from other unrelated antigens which can be presented in the context of the same MHC molecule, can competitively inhibit activation of these T cells by the cI peptide. Furthermore, these unrelated antigens can stimulate cI-specific T cells if certain specific amino-acid residues are replaced. Here we suggest a model in which all antigens give rise to peptides that can bind to the same site on the MHC molecule. T-cell recognition of this site (which is presumed to be polymorphic) with or without antigen bound can explain self-selection in the thymus and MHC restriction.     

Self-tolerance alters T-cell receptor expression in an antigen-specific MHC restricted immune response

The influence of major histocompatibility complex (MHC) gene products on the T-lymphocyte alpha beta receptor (TCR) repertoire is well documented, but how specificity is also generated for a diverse array of foreign peptide antigens is unknown. One proposed mechanism is that the TCR repertoire is selected by the recognition of processed self-antigens bound to MHC molecules. Here, we examine the influence of non-MHC-encoded self-antigens on the TCR repertoire expressed in an antigen-specific immune response. Most pigeon cytochrome c-specific, Ek alpha Ek beta (Ek) Ia-restricted T cells from B10.A mice express a product of the V alpha 11 gene family in association with a V beta 3 gene-encoded protein. We therefore examined V alpha 11 and V beta 3 gene expression in cytochrome c-specific T-cell lines derived from various mouse strains with different non-MHC genetic backgrounds. T cells from several strains failed to express any V beta 3 due to tolerance induced by Mlsc-encoded self-antigens. Variable levels of V alpha 11 messenger RNA (mRNA) were expressed by antigen-specific T cells from all the strains. In one strain V beta 3 was expressed in the relative absence of V alpha 11. These results directly demonstrate that self-tolerance alters TCR gene usage in the immune response to a foreign antigen, and indicate that TCR V alpha and V beta proteins may, in part, be independently selected.