Potent immune responses of Ag-specific Vgamma2Vdelta2+ T cells and CD8+ T cells associated with latent stage of Mycobacterium tuberculosis coinfection in HIV-1-infected humans.

OBJECTIVE: To investigate immune responses of peptide-specific CD4+ and CD8+ T cells, and nonpeptide-specific Vgamma2Vdelta2+ T cells during clinical quiescence of latent Mycobacterium tuberculosis coinfection in HIV-1-infected humans. METHODS: One hundred HIV-1-infected individuals who had HIV infection only [HIV+tuberculosis-(TB-)], latent Mycobacterium tuberculosis coinfection (HIV + LTB), or active tuberculosis (HIV + TB) were recruited to measure mycobacterium purified protein derivative (PPD)-specific IFNgamma+ CD4+ and CD8+ T cells, and phosphoantigen HMBPP-specific IFNgamma+ Vgamma2Vdelta2+ T cells using enzyme-linked immunospot and intracellular cytokine staining assays. RESULTS: Both HIV + TB and HIV + LTB groups had low levels of PPD-specific IFNgamma+ CD4+ T cells regardless of CD4+ peripheral blood lymphocytes counts. However, numbers of PPD-specific IFNgamma+ CD8+ T cells in the HIV + LTB group were significantly greater than those in the HIV + TB group. Surprisingly, numbers of phosphoantigen hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells in the HIV + LTB group were much greater than those in the HIV + TB group (P < 0.001). This difference was present in the subgroups of HIV + LTB whatever the levels of CD4+ T-cell counts more than 200/microl or less than 200/microl. Numbers of hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells were even five times greater than those of PPD-specific IFNgamma+ CD8 T cells within the HIV + LTB group. CONCLUSION: Potent immune responses of hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells and PPD-specific IFNgamma+ CD8+ T cells were detected in HIV + LTB persons but not HIV + TB patients. The robust immune responses of Vgamma2Vdelta2+ and CD8+ T effector cells were associated with the latent stage of Mycobacterium tuberculosis coinfection in HIV-1-infected humans.

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs.

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.

Conservation of nonpeptide antigen recognition by rhesus monkey V gamma 2V delta 2 T cells.

We have previously found that monkey Vgamma2Vdelta2(+) T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guérin infections. We have now analyzed rhesus monkey gammadelta T cell responses to nonpeptide Ags and superantigens. Like human Vgamma2Vdelta2(+) T cells, rhesus monkey gammadelta T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags. Responsiveness was limited to gammadelta T cells expressing Vgamma2Vdelta2 TCRs. Rhesus monkey Vgamma2Vdelta2(+) T cells also responded to the superantigen, staphyloccocal enterotoxin A. Sequencing of the rhesus monkey Vgamma2Vdelta2 TCR revealed a strong sequence homology to human Vgamma2Vdelta2 TCR that preserves important sequence motifs. Moreover, chimeric TCRs that pair human Vgamma2 with monkey Vdelta2 and monkey Vgamma2 with human Vdelta2 retain reactivity to nonpeptide Ags and B cell lymphomas. A molecular model of the rhesus monkey Vgamma2Vdelta2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vgamma2Vdelta2 TCR and preserves the topology of the complementarity-determining region loops. Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human gammadelta T cell Ag responses.

Both TCR alpha and TCR delta chain diversity are regulated during thymic ontogeny.

TCRalpha and TCRdelta chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCRalpha and TCRdelta chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCRalpha and TCRdelta transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCRdelta repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCRdelta chains, but also for TCRalpha chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.

Characterization of an avian (Gallus gallus domesticus) TCR alpha delta gene locus.

Mammalian TCR delta genes are located in the midst of the TCR alpha gene locus. In the chicken, one large V delta gene family, two D delta gene segments, two J delta gene segments, and one C delta gene have been identified. The TCR delta genes were deleted on both alleles in alpha beta T cell lines, thereby indicating conservation of the combined TCR alpha delta locus in birds. V alpha and V delta gene segments were found to rearrange with one, both or neither of the D delta segments and either of the two J delta segments. Exonuclease activity, P-addition, and N-addition during VDJ delta rearrangement contributed to TCR delta repertoire diversification in the first embryonic wave of T cells. An unbiased V delta 1 repertoire was observed at all ages, but an acquired J delta 1 usage bias occurred in the TCR delta repertoire. The unrestricted combinatorial diversity of relatively complex TCR gamma and delta loci may contribute to the remarkable abundance of gamma delta T cells in this avian representative.

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation.

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.

Circulating TCR gammadelta cells in the patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell-mediated responses against several self-antigens of affected cells. The last studies demonstrate that the role of gammadelta T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR gammadelta T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR gammadelta cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing gammadelta TCR in most SLE blood specimens were significantly lower than in the control group (P<0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage gammadelta T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vdelta2+ and Vgamma9+ subtypes of pan gammadelta T cells, Vdelta3+ T cells number was higher in SLE patients (20 x 10 cells/microl) than in healthy control group (2 x 2 cells/microl) (P=0.001). However, we found no differences between the numbers of pan gammadelta T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vdelta3+ subtype of gammadelta T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.

Interleukin-15 may be responsible for early activation of intestinal intraepithelial lymphocytes after oral infection with Listeria monocytogenes in rats.

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-gamma) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor kappaB (NF-kappaB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) gamma delta+ T cells, NKR.P1(+) cells, and CD3(+) CD8(+) alpha alpha cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-gamma upon stimulation with immobilized anti-TCR gamma delta or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-gamma at an early phase of oral infection with L. monocytogenes.

Isolation and N-terminal sequence determination of a novel gamma/delta T cell surface antigen.

An antigen complex unique for porcine gamma/delta T cells has previously been identified using the monoclonal antibodies MAC319 and MAC320. Here we use digestion with the proteolytic enzyme bromelain to selectively release the MAC319 antigen as a soluble fragment, for further characterisation. A cytofluorometric inhibition assay was developed to follow the purification of this fragment, as the conformation sensitivity of the MAC319 epitope prevented the use of immunoblotting techniques. The antigen has been purified using a combination of anion-exchange and hydrophobic-interaction columns, followed by separation on a size-exclusion column. Fractions from the size-exclusion column containing the antigen consisted of one major band at Mr 95,000. This species was shown to be specifically absorbed onto MAC319-coupled Sepharose, thereby identifying the MAC319 antigen. N-terminal amino acid sequencing of this band has revealed a previously unidentified sequence. This fragment was also shown to be glycosylated, most likely with a single sugar moiety. Enzymatic removal of the sugars showed that they did not appear to be necessary for binding of the polypeptide to the MAC319 antibody.

The canonical T cell receptor of dendritic epidermal gamma delta T cells is highly conserved between rats and mice.

Two monoclonal antibodies with specificity for rat gammadelta T cell receptor (TCR) were generated. One, called V65, reacts with all CD3+ alphabeta TCR- rat Tcells and thus recognizes a constant determinant of the rat gammadelta TCR (Kühnlein et al., Journal of Immunology 1994, 153: 979). The other, called V45, reacts with approximately 80% of gammadelta T cells in peripheral lymphoid organs. In rat epidermis, V65 but not V45 detects a dense network of the dendritic epidermal Tcells (DETC). Analysis of epidermal RNA by polymerase chain reaction (PCR) indicated that Vgamma3 and Vdelta1 are the predominant, if not exclusive TCR V transcripts present at this site. Sequence analysis of cDNA clones obtained by reverse transcription-PCR with Vgamma3- and Vdelta1-specific primers revealed that the variable domains of rat DETC gamma and delta chains are very homologous to those described in mice (92% and 95% identity at the protein level). The complete conservation between the two species of the amino acid sequences at the V-(D)-J transitions of this monomorphic receptor indicates that the interaction of the DETC TCR with its as yet unknown ligand must be of central importance for DETC function.

The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum.

Assembly of the multisubunit T cell antigen receptor (TCR) complex is an intricate process requiring coordinated regulation of at least six different gene products (alpha, beta, gamma, delta, epsilon, and zeta) and the ordered pairing of partner chains within the endoplasmic reticulum (ER). To date, two proteins have been implicated as functioning as molecular chaperones in the assembly of nascent TCR proteins: calnexin, a resident ER transmembrane protein, which associates with all TCR components except zeta, and T cell receptor-associated protein, which selectively associates with CD3gammaepsilon pairs. In this study, we examined the association of calreticulin, a soluble protein with significant sequence homology to calnexin, with newly synthesized TCR proteins. Analogous to calnexin, processing of glycan chains by glucosidase enzymes was required for initial association of TCRalpha and -beta proteins with calreticulin; however, several major differences were noted regarding interaction of calnexin and calreticulin chaperones with TCR proteins. First, TCRalpha and -beta proteins showed prolonged association with calnexin molecules compared with calreticulin; interaction of TCRalpha proteins with calreticulin was particularly transient, with most calreticulin-TCRalpha protein complexes dissociating within 15 min of their initial assembly. Second, we found that, unlike calnexin, which associated with clonotypic TCRalpha and -beta proteins and invariant CD3delta and -epsilon polypeptides, calreticulin associated specifically with clonotypic TCRalpha and -beta proteins. These studies identify calreticulin as a molecular chaperone for nascent clonotypic TCRalpha and -beta proteins and demonstrate that calreticulin and calnexin differentially associate with newly synthesized TCR proteins within the ER.

Selective gamma-chain T-cell receptor gene rearrangements in a patient with Omenn's syndrome: absence of V-II subgroup (V gamma 9) transcripts.

Only gamma-chain T-cell receptor transcripts utilizing V-1 subgroup gene segments were found in peripheral blood lymphocytes from a patient with Omenn's syndrome. gamma-Chain T-cell receptor transcripts utilizing the V gamma 9 (V-II subgroup) gene segment were absent in peripheral blood lymphocytes from this patient. V gamma 9 J gamma 1.2 C gamma 1 rearrangements are those primarily found in peripheral blood lymphocytes (70 to 85%) from normal donors.

Phenotypic and functional properties of murine gamma delta T cell clones derived from malaria immunized, alpha beta T cell-deficient mice.

Six murine T cell clones expressing gamma delta TCR were generated from malaria immunized, alpha beta T cell-deficient mice. Phenotypic characterization of these clones has revealed that, in contrast to conventional alpha beta T cells, there is a considerable degree of heterogeneity among these gamma delta clones with regard to their surface markers and their lymphokine profile. One clone was found to display significant anti-parasite activity in vivo upon adoptive transfer. We attempted to determine whether the protective clone differs in one or more key characteristics from the non-protective clones. Although no obvious pattern peculiar to the protective gamma delta clone was observed, it appears that more than one parameter may, in combination, define a distinct protective phenotype, and thus explain the functional difference between the protective and non-protective gamma delta clones.

Isolation of T cell receptor gamma/delta chain constant genes from minke whale.

The polymerase chain reaction using oligonucleotides based on consensus sequences from other species allowed us to amplify minke whale (Balaenoptera acutorostrata) T cell receptor (TcR) gamma and delta constant genes. Two types of Cgamma clones were obtained which only differ by one mismatch. These minke whale Cgamma clones showed 83% nucleotide and 70% amino acid sequence similarity to the corresponding region of the human TRGC genes. The minke whale Cdelta sequences were all identical, and showed 86% nucleotide and 77.8% amino acid sequence similarity to the human TRDC gene. The whale Cgamma and Cdelta clones were used as probes for Southern blot analysis. The data confirmed that there is a unique Cdelta gene in minke whale. Two different Cgamma genes were detected, one of them also cross-hybridizing with a human Cgamma probe, suggesting that the Cgamma locus in minke whale consists of at least two different constant genes.

The gamma delta T cell repertoire in Graves' disease and multinodular goitre.

gamma delta T cells are a subset of T cells with unknown function, and restriction of the gamma delta T cell receptor (TCR) repertoire has been described in rheumatoid arthritis and multiple sclerosis. Elevated numbers of gamma delta T cells have been reported in the peripheral blood and thyroids of patients with Graves' disease. We have carried out flow cytometric analysis on peripheral blood mononuclear cells (PBMC) and intrathyroidal lymphocytes (ITL) from 12 patients with Graves' disease and nine patients with multinodular goitre (MNG), a thyroid disease of unknown etiology. There was no significant difference between the proportion of gamma delta T cells in the PBMC of Graves' and MNG patients, nor between the PBMC and ITL populations in either patient group. We have also carried out polymerase chain reaction amplification on RNA prepared from matched PBMC, ITL and the activated (CD25+) subset of ITL using six TCR V delta-family specific primers. Although there were differences in the amounts of each V delta transcript amplified from PBMC and ITL, there was no difference between the two patient groups. No consistent differences were therefore found between the gamma delta T cell populations in Graves' and MNG patients, arguing against the direct involvement of this T cell subset in the pathogenesis of Graves' disease.

The gamma chain of the high-affinity receptor for IgE is a major functional subunit of the T-cell antigen receptor complex in gamma delta T lymphocytes.

T-cell activation is a consequence of the clonotypic T-cell antigen receptor (TCR) binding to an antigen followed by signal transduction via the invariant subunits of the TCR/CD3 complex. gamma delta TCR cells are a small subset of T cells that populate both the epithelial and lymphoid tissues and have unique antigen specificity and function. However, the composition of invariant chains within the gamma delta TCR/CD3 complex has not been well characterized. Here we report that, unlike the majority of alpha beta T cell, gamma delta T cells isolated from spleen and intestinal epithelial tissue express high levels of the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) as one invariant subunit of their TCR/CD3 complex. Fc epsilon RI gamma exists as both a homodimer and a heterodimer associated with the TCR zeta chain. Moreover, stimulation of the gamma delta TCR results in rapid tyrosine phosphorylation of Fc epsilon RI gamma. Our results suggest that utilization of distinct receptor signaling components may enable the coupling of antigen stimulation to the activation of different signal transduction pathways in alpha beta and gamma delta T cells.

The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation.

Recent work indicates that signaling events resulting from stimulation of the T cell antigen receptor (TCR) can be initiated by the CD3 complex (gamma, delta, epsilon) as well as the zeta chains of the receptor. To help characterize the signaling function of CD3 we examined its associated tyrosine kinase activity since induction of tyrosine phosphorylation is one of the earliest signaling events. Our results indicate that at least two kinases, lck and ZAP-70, contribute to the CD3-associated kinase activity. A likely target of this activity is the CD3 complex itself since we observed that TCR stimulation resulted in rapid tyrosine phosphorylation of the CD3 epsilon and delta chains. To examine the function of the CD3 epsilon chain in particular, we constructed a chimera that fused the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of CD3 epsilon. This chimera demonstrated that CD3 epsilon was independently capable of associating with proteins having tyrosine kinase activity, including ZAP-70. Our results show that the kinase activity that associates with the CD3 complex has characteristics that are quite similar to the previously characterized zeta-associated kinase activity. This finding suggests that both these components of the TCR initiate signaling events using a common mechanism. However, differences in their signaling function could result from recognition of distinct substrates.

Secretion of disulfide-linked human T-cell receptor gamma delta heterodimers.

The availability of soluble forms of T-cell antigen receptors (sTCR) should be of great use in the detailed characterization of their interactions with ligands, for the generation of anti-TCR monoclonal antibodies (mAb), and for the eventual determination of their three-dimensional structures by x-ray crystallography. Here, we show that efficient secretion of nonchimeric disulfide-linked human gamma delta TCR could be achieved by simply introducing translational termination codons upstream from the sequences encoding TCR chain transmembrane regions. This recombinant protein appeared to be correctly folded, as judged by its reactivity with a panel of anti-gamma and anti-delta mAbs, and proved to be a powerful immunogen, allowing generation of mAb that are able to recognize both soluble- and membrane-bound gamma delta TCR. While variable and constant domains of gamma delta sTCR seem to be folded into compact conformations, the extreme sensitivity of its interchain disulfide bridge to digestion with papain suggests that sTCR C-terminal portions are in a more extended configuration than the corresponding region in immunoglobulins. Finally, the gamma delta heterodimer remains stable even after removal of the interchain disulfide link, suggesting the existence of strong noncovalent forces holding the two chains together.