Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire.

The T cell receptor repertoire has a potential for vast diversity. However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection events during development. Such thymic selection affecting TCR V beta gene segment usage has been demonstrated in the mouse. However, similar data has not been forthcoming in man, and little is known about the role of the TCR alpha chain in antigen/major histocompatibility complex (MHC) recognition in any species. Here, we used a monoclonal antibody recognizing the TCR V alpha 12.1 gene product to assess the expression of this gene in the peripheral blood of man. In most individuals tested, the percentage of cells expressing V alpha 12.1 was significantly higher in CD8+ T cells than in CD4+ T cells. That the V alpha gene product itself was responsible for this increased expression in CD8+ T cells was underscored by the lack of substantial skewing of V beta usage in the V alpha 12.1-bearing T cells. Moreover, the skewed expression of V alpha 12.1 was already present at birth, indicating that it was likely to be due to a developmental process rather than the result of exposure to environmental antigens. Based on the established role for CD8 in binding to class I MHC molecules, we suggest that increased expression of V alpha 12.1 on CD8+ T cells points to a role for TCR's using V alpha 12.1 in class I MHC/Ag recognition. These results indicate that V alpha gene usage in the peripheral blood of man is not random, and they support a role for V alpha as a participant in the self-MHC recognition process that shapes the TCR repertoire.

Oligoclonality of human intestinal intraepithelial T cells.

T cells bearing the T cell receptor alpha/beta (TCR-alpha/beta) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithelial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-alpha/beta repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative V beta gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V beta families made up more than 40% of the total V beta transcripts detected in the IEL, whereas there was a more even distribution of V beta gene usage in the paired PBL. The predominant V beta families, especially V beta 1, V beta 2, V beta 3, and V beta 6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V beta 6 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 clones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V beta skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut.

Clonal V alpha 12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Binding of monoclonal anti-native DNA autoantibodies to DNA of varying size and conformation.

A microchemical assay for phosphorus was applied to the measurement of DNA in immune complexes formed with monoclonal or serum anti-DNA autoantibodies and DNA of varying size and conformation. Two monoclonal antibodies were produced by hybridomas derived from spleen cells of autoimmune MRL-lpr/lpr mice and were purified from culture fluid by affinity chromatography on columns of goat anti-mouse Ig-Sepharose. Double-helical DNA fragments were prepared by brief digestion of calf thymus DNA with micrococcal and S1 nucleases and fractionation on Sepharose 4B; their double-stranded structures was confirmed by measurement of thermal denaturation. Immune complexes were formed with monoclonal or serum antibodies and native DNA or DNA fragments or denatured DNA; the complexes were precipitated with goat anti-mouse IgG and washed, and DNA phosphorus content of the precipitates was measured. With one monoclonal autoantibody (H241), there were discontinuous increases in the amount of DNA that could be bound (and decreases in the antigen concn required for half-maximal binding) as the DNA size increased. There were especially marked increases in binding efficiency as fragment size increased from an average of 100 (range 85-105) to an average of 150 (range 105-170) base pairs, and again between 450 (range 360-620) and 600 (range 425-825) base pairs. A second monoclonal antibody (H143) did not show significant variation in binding with DNA fragments larger than 300 base pairs. With smaller fragments, the amount of DNA bound by H143 was reduced, but the DNA concn required for half-maximal binding was not. Affinities of these monoclonal antibodies were within the spectrum of human systemic lupus erythematosus serum IgG anti-DNA autoantibodies. The dependence of binding on mol. wt is important in the evaluation of these monoclonal antibodies as biochemical reagents and as potential participants in formation of immune complexes in vivo.

Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(alpha1,3)Galactose epitope.

BACKGROUND: The cell surface carbohydrate moiety, Gal(alpha1,3)Galactose (alphaGal), has been implicated as the major determinant recognized by more than 80% of human anti-porcine natural antibodies (NAb). An ELISA system was developed for the detection of this subpopulation of porcine cell-reactive NAb using synthetic alphaGal conjugated to bovine serum albumin. METHODS: A screen of 95 human serum samples by this method demonstrated marked variability in the alphaGal reactivity of unrelated donors. The percentage of alphaGal-reactive NAb relative to total immunoglobulin was determined for 10 donors. RESULTS: alphaGal-reactive NAb comprised 1.0-2.4% of total serum IgG, whereas the range was from 3.9% to 8.0% for IgM. CONCLUSIONS: The higher level of alphaGal-reactive IgM suggests that xenoreactive NAbs may be the product of germ-line genes. Two-dimensional gel analysis of affinity-purified alphaGal-reactive NAb from two donors provided evidence suggesting that IgM from this subpopulation of NAb were restricted in protein charge heterogeneity.

Relationship between ABO blood group and levels of Gal alpha,3Galactose-reactive human immunoglobulin G.

BACKGROUND: The terminal Gal alpha1,3Galactose (alphaGal) determinant is present on all porcine glycoproteins and glycolipids, but is not expressed by human cells. Consequently human sera contain anti-alphaGal natural antibodies. The human blood group B antigen [Gal alpha1,3(Fuc1,2)Galactose] is differentiated from the alphaGal epitope by the presence of a fucosyl group. METHODS: To determine whether the expression of the B antigen has any effect on the level of alphaGal-reactive natural antibodies, equal numbers (n=12) of A, B, AB, and O serum samples were evaluated by ELISA and flow cytometry. RESULTS: A significant reduction in IgG alphaGal reactivity was observed with serum samples from B antigen-expressing donors (B, AB) relative to non-B antigen-expressing donors (A, O). CONCLUSIONS: These results are consistent with the possibility that anti-alphaGal antibodies in non-B antigen-expressing individuals include a subset that is reactive with the structurally related B antigen and that this subset is absent in B and AB individuals.

Human anti-porcine xenogeneic T cell response. Evidence for allelic specificity of mixed leukocyte reaction and for both direct and indirect pathways of recognition.

Partially inbred, MHC homozygous miniature swine have been used to study the nature of the human xenogeneic cellular immune response to swine Ags in vitro. Human T cells responded to xeno-MHC Ags in MLR at least as well as they did to allo-MHC Ags and appeared to share similar requirements for APC of either stimulator (direct pathway) or responder (indirect pathway) derivation. In addition, mAb-blocking experiments indicated that the majority of the primary human anti-pig xeno-response was directed toward porcine MHC class II Ags and involved interaction with the human CD4 accessory molecule. Finally, the availability of intra-MHC recombinant haplotypes in our herds has made it possible to map genetically the antigenic specificities recognized in human anti-swine cellular responses. For this purpose, T cell clones were generated from human anti-swine MLR cultures and were tested for reactivity to stimulator cells from MHC homozygous and recombinant haplotypes. Clear evidence for antixenogeneic MHC Ags was observed. In all cases in which allelic differences between haplotypes were detected (the majority of clones), the reactivity could be mapped to the class II region of stimulator haplotypes. In addition, cross-reactivity between haplotypes was consistent with known sequence similarities between DR beta-chains. Our results indicate, therefore, that the human anti-porcine T cell response is similar in strength and specificity to an allogeneic response, and that the TCR repertoire, accessory molecule interactions, and cytokine production required for both direct and indirect pathways of recognition in the human anti-porcine MHC class II responses are functionally intact.

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies.

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.

The recurrent expression of variable region segments in human IgM anti-DNA autoantibodies.

RNA sequences for the V regions of human hybridoma-produced autoantibodies were determined by primer extension with reverse transcriptase. The sequencing of IgM autoantibodies from a leprosy patient revealed examples of recurrent use of V region gene segments in different autoantibodies from this patient and a previously studied patient with SLE. Moreover, several gene segments used in these autoantibodies show little alteration from germ-line sequences. mAb TH3, from a patient with leprosy, binds denatured DNA and poly(dT). The center of its H chain CDR35 has a sequence identical to that found previously in two anti-DNA antibodies from a lupus patient; these identities and their overlapping with two other published sequences define a human D-gene segment of approximately 25 nucleotides. Autoantibody TH9, from a leprosy patient, does not bind DNA. Its VH sequence has 87% identity with a VHI anti-DNA antibody, but differs from it markedly in the CDR1 region. TH9 also has a different H chain CDR3. The closely related JH4 or JH5 gene segments are expressed in five lupus or leprosy autoantibodies. In four of the antibodies, examples of V kappa 1, V kappa 3, or V kappa 4 and J kappa 2, or J kappa 5 segments were found. Two distinct leprosy-derived anti-DNA antibodies, 8E10 and TH3, share a completely identical V kappa sequence. This sequence differs in only two positions from that of a germ-line RF L chain gene. Several gene segments that are close to the germ line in sequence encode Ig V regions with autoantibody reactivity. These results provide a base line for determining whether these genes are precursors of more highly diversified antibodies that may be pathogenic in patients with SLE.

Abnormal T-cell expansion and V-gene usage in myasthenia gravis patients.

To determine the extent of V-gene heterogeneity of blood T lymphocytes in patients suffering from Myasthenia Gravis (MG), we used eight recently available monoclonal antibodies (MoAb), directed against different V alpha and V beta gene products of the variable part of the T-cell receptor (TCR), covering approximately 25% of the alpha/beta T cells in normal peripheral blood (PBL) of healthy individuals. Using a two-colour immunofluorescence method, we could calculate the expression of alpha/beta V segments within the two major T-cell subsets, CD4-/CD8+ and CD4+/CD8- lymphocytes. Twenty-seven per cent (4/15) of the MG patients had T cells showing signs of abnormal expansion. Furthermore, among these expanded T cells, a restricted V beta 12 gene expansion could be seen, in three out of four patients. No correlation between TCR V-gene usage and HLA haplotypes (HLA-A, -B, -DR and -DQ) could be seen. Our data suggest that the majority of MG patients have abnormally expanded T-cell clones. The relevance of these findings is discussed.

A persistent T cell expansion in the peripheral blood of a normal adult male: a new clinical entity?

A dramatic and persistent T cell expansion in a healthy adult male was initially identified, using anti-T cell receptor for antigen (TCR)-specific MoAbs. The expanded T cells were found to be expressing TCR containing V alpha 12.1 and V beta 5.2, and they composed approximately one third of all the CD8+ T cells. The cells were shown to be not only non-activated (HLA-DR-, IL-2R-) but also of 'virgin' cell type (CD45RA+/CD45RO-) and they persisted over the observation period of more than one and a half years. Various T and B cell markers, and all other laboratory and physical parameters analysed, were normal. The expanded CD8+ T cells were further characterized by polymerase chain reaction (PCR) amplification, using V beta- and C beta-specific primers, followed by hybridization with J beta-specific probes. Close to 90% of the V alpha 12.1+ V beta 5.2+ T cells were found to utilize the J beta 2.5 gene segment, thus strongly suggesting the expanded T cells to be monoclonal. The condition may constitute a T cell counterpart to 'monoclonal gammopathy of undetermined significance' (MGUS), and by analogy we suggest it should be designated 'monoclonal T cell expansion of undetermined significance' (MTUS).

HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.