A truncated T cell receptor repertoire reveals underlying immunogenicity of an antigenic determinant.

Induction of T cell responses to an antigenic peptide that is known to bind a major histocompatibility complex molecule is a function of either the T cell receptor (TCR) repertoire or regulatory influences by CD8 or CD4 regulatory T cells. We have tested the hypothesis that a lack of 10 TCR V beta gene segments in V beta a mice may result in an incomplete repertoire of regulatory T cells involved in maintaining peripheral tolerance. Such a hole in the repertoire of regulatory cells could result in expression of T cell responses to antigenic determinants that normally remain undetected in mice with a wild-type repertoire of TCR V beta gene segments. We show here that H-2d mice respond to the peptide 74-96 of hen egg-white lysozyme (HEL) when they are of V beta a haplotype at their TCR locus. The wild-type (V beta b) H-2d mice with their complete set of 20 TCR V beta gene segments fail to respond to HEL 74-96. The 74-96-specific T cell responsiveness was revealed in the wild-type (V beta b) mice when they were treated in vivo with anti-CD8 antibody, implicating the existence of regulatory cells that prevent expression of T cell responses specific for peptide 74-96. This is a demonstration that holes in the regulatory T cell repertoire can, in certain circumstances, become beneficial to the host, for example, in susceptibility against pathogens.

DM determines the cryptic and immunodominant fate of T cell epitopes.

The ability of the immune system to focus T cell responses against a select number of potential epitopes of a complex antigen is termed immunodominance. Epitopes that trigger potent T cell activation, after in vivo priming, are classified as immunodominant. By contrast, determinants that fail to elicit any response are called cryptic. DM, a major histocompatibility complex (MHC) heterodimer, plays a pivotal role in the presentation of MHC class II-restricted epitopes by catalyzing the exchange of class II-associated invariant chain peptide with the antigen-derived peptides within the MHC class II binding groove. Using L cells transfected with genes for MHC class II, invariant chain, and DM, we have studied the contribution of DM in the presentation of two cryptic (peptide 11-25 and peptide 20-35) and one dominant (peptide 106-116) epitope of hen egg white lysozyme (HEL). Cells lacking DM heterodimers efficiently display the determinants HEL 11-25 and HEL 20-35 to T cells. Strikingly, however, cells expressing DM are severely compromised in their ability to present the cryptic HEL 11-25/A(d) and 20-35/A(d) epitopes. DM-mediated antagonism of HEL 11-25/A(d) and 20-35/A(d) presentation could thus be central to 11-25/A(d) and 20-35/A(d) being cryptic epitopes in the HEL system. Interestingly, the display of the immunodominant epitope of HEL, 106-116/E(d), and of a dominant epitope of sperm whale myoglobin (SWM), 102-118/A(d), is entirely dependent on the expression of DM. Thus, cells lacking DM molecules are unable to efficiently express HEL 106-116/E(d) and SWM 102-118/A(d) determinants. We conclude that the DM heterodimers direct the immunodominant and cryptic fate of antigenic epitopes in vivo.

In a small multideterminant peptide, each determinant is recognized by a different V beta gene segment.

Given the vast potential for diversification of the T cell receptor (TCR) repertoire and the fact that V(a) beta mice exist in the wild, it would have been predicted that in spite of the absence of 10 TCR V beta gene segments, V(a) beta mice would still have been able to produce an antigen-specific T cell response to all determinants. We have recently shown that Vb beta mice, with a wild-type TCR V beta repertoire, respond to peptide 110-121 of sperm whale myoglobin, with a majority of T cells expressing TCR V beta 8.2 and restricted to a hybrid I-A(d)/I-E(d) major histocompatibility complex molecule, and a smaller number of T cells expressing TCR V beta 8.1 and restricted to the I-A(d) molecule. However, V(a) beta mice, lacking members of the TCR V beta 8 gene family, responded only with I-A(d)-restricted T cells. Thus, it appeared that the I-A(d)-restricted response was less constrained, or more plastic. We now show that the two separate panels of I-A(d)-restricted T cell hybrids derived from V(a) beta or Vb beta mice in fact recognize distinct determinants within the same peptide 110-121. The determinant recognized by V(a) beta T cells is NH2 terminal (core: 110-118) with an absolute requirement for the residue Ala-110 for a successful interaction with TCRs. On the other hand, Vb beta T cells recognize the COOH-terminal region (core: 112-118) on the same peptide with an absolute requirement for COOH-terminal residue 118. In the dominance hierarchy displayed by the three distinct determinants of peptide 110-121, V(a) beta mice cannot recognize the two most dominant: the hybrid I-A(d)/I-E(d)-restricted determinant and the COOH-terminal, I-A(d)-restricted determinant. They instead respond with T cells specific for a third, distinctly NH2-terminal determinant. Our results show a strict association between recognition of a particular specificity and TCR V beta usage. This evidence suggests that even when a small peptide induces a heterogenous group of TCR V beta S, this need not be considered evidence for plasticity. Rather, at the level of individual determinants within the peptide, the results can point in the opposite direction, towards serious constraints in recognition at the level of V beta expression.

Recognition of multiple peptide cores by a single T cell receptor.

We present evidence that a single T cell clone can recognize at least five different overlapping peptides, each with its distinct core structure, in the context of the same major histocompatibility complex (MHC) molecule. Distinct core residues are crucial for triggering the T cell receptor (TCR) in each case. These results suggest that the TCR (a) has multiple sets of contact residues for alternative peptide-MHC ligands, the binding to any one of which can trigger the cell; and/or (b) is able to attach to the peptide-MHC complex in more than one orientation. In this sense, the TCR is a multisubsite structure capable of being stimulated by a variety of peptide ligands associated with the same MHC molecules.

Childhood leprosy: profiles from a leprosy referral hospital in West Bengal, India.

Monitoring childhood leprosy in terms of incidence and occurrence of deformities are crucial for better control and understanding the transmission of the disease. In this paper, a profile of all new untreated leprosy patients below 15 years of age who reported at a Leprosy Referral Centre in West Bengal during 2004-2006 are described. Of 151 children studied, 84 (55.6%) were males, 33% were multibacillary and of them, 30% were smear positive. 16% had already developed grade 2 disability (WHO). Multiple nerve involvement was seen in a quarter of children. These findings highlight the seriousness of leprosy among children and the great need to address these issues urgently. Awareness, active case detection especially among contacts and motivation are the essential needs of the hour to prevent tragedy of deformed children due to a totally manageable disease.

Genotypic analysis of Mycobacterium leprae strains from different regions of India on the basis of rpoT.

Mycobacterium leprae strains from Indian leprosy patients were analyzed using the six base tandem repeat, GACATC, in rpoT gene as genetic marker. DNA was extracted from slit-skin smears and nasal swabs of new untreated as well as treated leprosy patients living in different regions of India. PCR amplification of rpoT gene and sequencing of amplicons showed the presence of two genotype of M. leprae in this study, 73.4% having three copies (ancient Indian type) and 26.6% contain 4 copies (considered to be Japanese and Korean). These genotypes along with other short tandem repeats may help in studying the historical spread of disease and the strains of M. leprae disseminated by various human races that migrated to India from other places of Asia and European countries during our history.

Extent and correlates of leprosy stigma in rural India.

Representative random samples of leprosy patients (599) and community members (2399) from rural areas of Uttar Pradesh, West Bengal and Chhattisgarh states of India were interviewed by trained field investigators during 2006, using two separate 5-point scales to assess the extent and correlates of leprosy stigma. Varying degrees of stigma were faced by the affected persons within the family and outside in all the States, restricting their social participation and sharing of common facilities. The community members also confirmed the existence of a high level of stigma. Low educational and economic status, older age-groups, and presence of deformities enhance both perceived and enacted stigma.

Presence of pro-oxidants in plasma of patients suffering from falciparum malaria.

Haemolysis is the major cause of anaemia in acute Plasmodium falciparum malaria, destroying both parasitized and non-parasitized erythrocytes. Oxidative stress on erythrocytes is considered an important mechanism of haemolysis. Since non-parasitized erythrocytes are also destroyed, the extracellular environment of the erythrocyte may be a contributor to the oxidative stress. To examine the influence of extracellular factors on oxidative stress and haemolysis, baseline values of erythrocyte thio-barbituric acid-reactive substance (ETBAR) and haemolytic indices such as plasma haemoglobin and lactate dehydrogenase (LDH) were estimated in 19 children in Orissa (India) with acute P. falciparum malaria (haemoglobin level < or = 70 g/L). The indices were measured after incubating cross-matched isogroup adult control erythrocytes with patient's plasma, and patient's erythrocytes with adult control plasma both in presence of and in absence of t-butyl hydroperoxide (t-BHP). The procedure was repeated in the blood of 19 age- and sex-matched non-malarial children. Baseline plasma LDH, haemoglobin and ETBAR concentrations were significantly greater in malaria patients than non-malarial children (P < 0.001 for all). Post-incubation values of ETBAR and plasma haemoglobin were significantly higher (P < 0.05) when adult control erythrocytes were incubated with patient plasma, and plasma haemoglobin was significantly higher (P < 0.05) in incubates of patient erythrocytes with adult control plasma, than their respective pre-incubation values when incubated in absence of t-BHP. These differences were not noticed in the incubates of non-malarial children with healthy adult control samples. When incubated in presence of t-BHP all the post-incubation values in the patients were significantly higher than their respective pre-incubation values and post-incubation values without t-BHP (P < 0.001). In non-malarial control samples, only ETBAR concentration was higher than their respective pre-incubation and post-incubation values without t-BHP (P < 0.01). All the values for post-incubation samples with t-BHP were significantly higher in patients than controls (P < 0.001). In post-incubation samples of control erythrocytes and patient plasma in presence of t-BHP, ETBAR correlated inversely with pre-incubation haptoglobin values (P < 0.001). Thus, plasma of acute malaria patients appears to contain pro-oxidants, which may contribute to extracellular oxidative stress on both parasitized and non-parasitized erythrocytes.

Evidence for erythrocyte lipid peroxidation in acute falciparum malaria.

To assess the extent of oxidative stress in erythrocytes of patients with acute Plasmodium falciparum malaria, erythrocyte thiobarbituric acid-reactive substance (ETBAR), and intracellular, membrane and extracellular antioxidants were estimated in 102 cases of P. falciparum malaria and 50 control subjects. The mean concentration of ETBAR was significantly higher (P < 0.001) and many of the antioxidants were significantly lower in patients than controls. Among the erythrocyte antioxidants, catalase, reduced glutathione (GSH) and tocopherol were significantly lower in the patients (P < 0.05, 0.001, 0.001, respectively). Erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were not reduced to a statistically significant level. Similarly, the plasma antioxidants ascorbate and albumin were significantly lower (P < 0.001) but not urate. ETBAR correlated inversely with erythrocyte GSH and tocopherol (P < 0.001), and plasma ascorbate and albumin (P < 0.001) but not with the erythrocyte enzymic antioxidants. However, on multiple regression analysis only tocopherol correlated strongly with ETBAR, followed by GSH and plasma ascorbate. ETBAR also correlated well with haemolytic indices such as haemoglobin, plasma unconjugated bilirubin and haptoglobin concentrations (P < 0.001, for all). On follow-up after 2 weeks, ETBAR and different antioxidants reached near control levels. These observations indicate an enhanced oxidative stress on erythrocytes in acute falciparum malaria that may contribute substantially to haemolysis and anaemia.

Preferential restriction of minor alloantigen-specific suppressor T cells to I-E rather than I-A molecules.

Suppressor T (Ts) cells specific for minor alloantigens can be generated by intravenous administration of high doses (10(8) cells) of alloantigenic spleen cells. Such Ts cells have previously been shown to inhibit the in vivo induction of CTL responses and are now shown to suppress in vitro proliferative responses to specific minor antigens. This report demonstrates that the activity of such antigen-specific Ts cells in four different strain combinations involving two haplotypes is blocked by anti-I-E antibody. The proliferative helper T cells (Th) in the same strain combinations are restricted to I-A molecules. Whether our results reflect a more general bias of suppressor T cells to be restricted to I-E molecules is discussed.

Characteristics of histamine receptors present on suppressor T cells in "healthy individuals".

Twelve to 30% histamine receptor bearing cells were detectable in the peripheral blood mononuclear cells of healthy tuberculin sensitive individuals. The number of binding sites per cell ranged from 2.1 X 10(4) to 5.08 X 10(4) (mean 2.5 X 10(4)) with an affinity ranging from 2.5 X 10(-6) M to 10.9 X 10(-6) M (mean 3.6 X 10(-6) M). The histamine receptors on these cells were found to be of H2 type as indicated by the abrogation of binding of 3H-histamine by cimetidine. It was further confirmed that histamine receptor bearing cells in the peripheral blood belonged to a T cell subset which formed rosettes with AET treated sheep erythrocytes and had receptors for Fc portion of IgG and phenotype markers of T3 and T8. Deletion of such cells by means of affinity chromatography on histamine bound Sepharose columns, led to enhanced antigen induced lymphoproliferation indicating the suppressor nature of these T cells.

The positively selected T cell repertoire: is it exclusively restricted to the selecting MHC?

Transgenic lines of mice showing compartmentalized expression of MHC I-E in different thymic microenvironments and nearly normal expression in the periphery were used to ask whether the mature T cell repertoire is restricted exclusively to the selecting MHC molecules expressed on thymic cortical epithelial cells. We show that upon in vivo priming, mice with a T cell repertoire selected in the thymic cortex on MHC I-A, in the absence of MHC I-E, display a clearly significant T cell response to two I-E-restricted peptides. Our results suggest that expression of one isotype or allele of class II MHC molecules in the appropriate thymic environment enables the selection of a polyclonal repertoire restricted in part to non-selecting class II MHC molecules. Thus, although the requirement for interaction with the MHC by the TCR on developing thymocytes exists, the available adult repertoire need not be solely faithful to the specific MHC expressed on the thymic cortical epithelium: it can also be restricted to other isotypes and alleles of this same class of MHC molecule.

Murine suppressor T cell clones specific for minor histocompatibility antigens express CD4, CD8, and alpha beta T cell receptor molecules.

We have been able to establish stable, minor histocompatibility antigen-specific, non-cytolytic, MHC-class II-restricted suppressor T cell clones and lines. These suppressor T cells rearrange, transcribe, and express alpha beta T cell receptor. An unusual feature of all our clones and lines is the co-expression of CD4 and CD8 molecules on their cell surface, a characteristic which may distinguish them from conventional helper and cytolytic T cells in this system. The fact that multiple, phenotypically and functionally identical cell lines were reproducibly obtained from this system indicates that antigen-specific down-regulation of immune functions in certain circumstances is mediated by T cells that are neither conventional cytolytic nor helper T cells.

Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells.

It is now becoming accepted that one is not tolerant to all the determinants of self proteins: the T cell repertoire directed to some sequences in self proteins is intact and can be activated. When a self protein is exclusively expressed by tumour cells, the T cell repertoire directed to the particular self antigen can potentially be activated to attack the tumour: this would amount to induction of a beneficial autoimmune response. Prostate cancer offers a unique opportunity for activation of a tumour-specific immune response owing to the exclusive synthesis of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) by prostatic tissue and prostate tumour cells. In this study we examine the CD4 and CD8 T cell repertoires specific for peptides of PSA and PSM in normal human male individuals, using short-term, peptide antigen-driven CD4 and CD8 T cell lines. We show that short-term, CD4 T cell lines derived from six HLA-DR4 individuals showed strong proliferative responses to six of 10 tested peptides of PSA, selected as to contain a DR4 binding motif. Short-term, CD8 T cell lines from three HLA-A1 individuals showed specific cytolytic activity for autologous targets loaded with five of five tested peptides of PSA and PSM, selected to possess an HLA-A1 binding motif. One of the peptides chosen is termed a 'dual-motif' peptide, as it encodes determinants for both CD4 and CD8 T cells. These results, indicating the existence of CD4 and CD8 T cells against determinants of the self proteins, PSA and PSM, in healthy male individuals reveal the potential of the T cell repertoire from the typical prostate cancer patient to eradicate prostate tumours upon being appropriately activated.

Limitations in plasticity of the T-cell receptor repertoire.

How constrained is T-cell recognition? Is a truncated T-cell receptor (TCR) repertoire, missing half of its V beta components (where V indicates variable), still broad enough to produce an antigen-specific T-cell response to all determinants? These questions can be answered for certain T-cell antigenic determinants whose response in the wild type is limited to specific gene segments. Our results show that mice with such a deletion in their TCR V beta genes (V beta truncated haplotype, Va beta) are unable to respond to two antigen determinants (sperm whale myoglobin 111-121/I-Ed and myelin basic protein 1-11/I-Au) whose response in the wild type is restricted to the missing V beta (V beta 8.2 in the case of 111-121/I-Ed and V beta 8.2 and V beta 13 in the case of 1-11/I-Au) gene segments. Fundamentally, this restriction could have been attributed to another aspect of immunodominance--that a favored TCR with high affinity would dominate the response, but in its absence, a hierarchy of T cells with lesser efficiency and expressing alternate TCR V genes could take over. However, from our experiments it has become evident that there is an absolute limit to the flexibility inherent in the TCR repertoire. Since it is clear that mouse populations have many ambient deletion ligands (such as self-superantigens) that can result in the loss of multiple V beta gene segments during normal T-cell development, these deletions can have serious consequences, such as unresponsiveness to the antigen as a whole--a hole in the repertoire--if a dominant determinant of that antigen normally shows restricted TCR V beta gene usage.

Regulation of histamine receptor concentration on human PBMC by homologous hormone.

Human peripheral blood mononuclear cells (PBMC) preincubated with 10(-3)M histamine at 37 degrees C, washed and incubated with 3H-histamine showed a reduction in the binding of the labelled hormone as compared to cells which had not been pretreated with histamine. Using competitive binding assays it was further shown that the number of specific binding sites for histamine was reduced by 46-62%. However, the affinity of receptors as indicated by Kd values remained unaltered. It would thus appear that elevated levels of histamine lead to down-regulation of histamine receptors on human PBMC with a quantitative reduction in the number of binding sites without an alteration in their affinity characteristics. In view of the fact that histamine receptors have been shown to be present on suppressor lymphocytes, the down-regulation of these receptor sites may have relevance in physiological and disease states where perturbations in the levels of histamine are observed.

A unique pattern of lymphokine synthesis is a characteristic of certain antigen-specific suppressor T cell clones.

We report that the lymphokines (IFN-gamma) and IL-10 are co-synthesized by previously described CD3+ TCR alpha beta+, minor antigen-specific suppressor T cell clones. IFN-gamma and IL-10 are known to (i) be characteristically produced by different helper T cell types, Th1 and Th2 respectively, and (ii) inhibit the function of the reciprocal subset of T cells: IFN-gamma inhibits the function of Th2 and IL-10 that of Th1 cells. Although Th0 cells are also known to synthesize cytokines of both the Th1- and Th2-type T cells, the suppressor T cells described in this report are different from Th0 cells in that they produce (i) neither IL-2 nor IL-4 molecules and (ii) stimulation via their CD3-TCR system seems independent of both IL-2 and IL-4, the typical autocrine molecules for T cell proliferation. The lymphokine profile of these suppressor T (Ts) cell clones, as well as those of human antigen-specific Ts cells reported earlier, suggests that co-synthesis of some Th1-like and some Th2-like cytokines may be a characteristic of antigen-specific Ts cells as opposed to the type of reciprocal inhibition mediated through IFN-gamma or IL-10, which is antigen non-specific.

Antigen processing and T cell repertoires as crucial aleatory features in induction of autoimmunity.

Induction of self-reactive T cell responses leading eventually to autoimmune pathology involves several key events: (1) availability of a determinant cross-reactive with the pathogenic self or foreign determinant upon processing of native antigen; the foreign molecule bearing the related determinant may have additional processing sites flanking the determinant, or at least different ones (the same determinant may only be available on the native self molecule under inflammatory conditions) (2) a T cell bearing T cell receptor (TCR) capable of response to the autoantigen; (3) ability of the processed self determinant to bind efficiently to the appropriate major histocompatibility complex (MHC) molecule as well as to interact with the appropriate TCR, coordinated with the absence of competitively dominant determinants; and (4) the lack of regulation. At any step of this cascade of interactions, the conditions could either favour induction of an autoreactive T cell response or the process may be truncated/stalled at any step without any adverse effect. A major component determining the outcome of the above interactions is the aleatory nature of the antigen processing events. Experiments highlighting these aleatory events are the focus of this report.

Anaemia in acute, Plasmodium falciparum malaria in children from Orissa state, India.

The severity of anaemia associated with acute, Plasmodium falciparum malaria and the extent to which haemolysis, bone-marrow suppression, and pre-existent iron deficiency contribute to the anaemia were assessed in 102 Indian children aged 2-12 years. Blood haemoglobin (Hb), plasma unconjugated bilirubin and haptoglobin, serum iron and transferrin concentrations and transferrin saturation, red cell morphology and reticulocyte response were investigated in the patients and in 50 control children. Twenty-three patients with severe anaemia (< 70 g Hb/litre) were investigated further, by bone-marrow biopsy followed by iron staining of sections or touch smears of the biopsy material. There was evidence of haemolysis in the malaria cases: in the peripheral blood smears and the significantly higher plasma concentrations of unconjugated bilirubin, lower plasma concentrations of haptoglobin and lower blood concentrations of Hb than those seen in the controls. Haemoglobin concentration correlated directly with haptoglobin (r = 0.489; P < 0.001) and inversely with unconjugated bilirubin in malaria patients (r = -0.526; P < 0.001) but not in controls (r = -0.140 and -0.061, respectively). Parasitaemia (parasites/microliter) was not significantly correlated with Hb, haptoglobin or unconjugated bilirubin. Compared with the earlier samples, follow-up samples from the patients, collected 2 weeks after discharge from hospital and antimalarial therapy, showed significant increase in Hb, haematocrit, haptoglobin and decreases in both total and unconjugated bilirubin. There was evidence of hypercellularity and mild-moderate erythroid hyperplasia, mainly of normoblastic maturation with adequate reticulocyte response, in the bone-marrow samples from the cases of severe anaemia; dyserythropoiesis was only noticed in one case and no stainable iron was detectable in 17 of the 23 cases. These observations indicate that haemolysis is the prime cause of the anaemia seen in acute falciparum malaria, although destruction of parasitised erythrocytes is not the sole cause of the haemolytic process. Bone-marrow suppression appears to have an insignificant role but pre-existent iron deficiency aggravates the severity of the anaemia.