Human Mycobacterium tuberculosis-reactive CD4+ T-cell clones: heterogeneity in antigen recognition, cytokine production, and cytotoxicity for mononuclear phagocytes.

CD4+ T cells regulate the protective immune response which follows exposure to Mycobacterium tuberculosis by activating macrophages through the cytokines the CD4+ T cells secrete. In addition CD4+ T cells have been shown to be directly cytotoxic for antigen-pulsed mononuclear phagocytes (monocytes-macrophages). To explore the functional interaction between mycobacterial antigen-specific CD4+ T cells and mononuclear phagocytes further, CD4+ T-cell clones were derived from healthy purified protein derivative-positive individuals. Five T-cell clones were selected for detailed analysis. None responded to the purified recombinant or native mycobacterial antigens of 14, 19, 65, 71, and 30 (alpha-antigen/Ag6) kDa. However, the T-cell clones demonstrated heterogeneity in antigen recognition as measured by their Western blot (immunoblot) responses. Some T-cell clones made only interleukin 2, while others made only interleukin 4; all produced gamma interferon, although in differing amounts. Four of five T-cells clones were cytotoxic for purified protein derivative-pulsed monocytes at 1:1 and 10:1 effector-target cell ratios. When monocytes infected with live M. tuberculosis were used as targets, comparable levels of cytotoxicity were observed. The cytotoxicity was major histocompatibility complex class II restricted and inhibited by antibodies to ICAM-1 and LFA-1 and not by antibodies to tumor necrosis factor alpha, lymphotoxin, and gamma interferon. Cytotoxicity by CD4+ T cells for monocytes pulsed with mycobacterial antigens or infected with live M. tuberculosis is a common property of these cells and appears to be independent of the repertoire of lymphokines produced and not limited to recognition of defined mycobacterial heat shock proteins. Lysis of heavily infected mononuclear phagocytes may be one manner in which CD4+ T cells regulate host immune response to M. tuberculosis.     

T-cell receptor V beta repertoire of L3T4+ regulatory T cells in anti-L3T4 antibody-induced tolerant NOD mice.

In ongoing studies, we have found that short-term administration of anti-L3T4 monoclonal antibodies (mAb) prevents the development of overt diabetes in non-obese diabetic (NOD) mice. In the present work, we asked whether L3T4+ T cells or Lyt-2+ T cells can suppress the diabetes in these mice. L3T4+ T cells or Lyt-2+ T cells were sorted using a magnetic cell sorter, then were transferred into cyclophosphamide-induced male NOD mice. We obtained evidence that the L3T4+ but not Lyt-2+ T cells did inhibit the diabetes, thereby indicating that the former can regulate diabetes in anti-L3T4 mAb-induced tolerant NOD mice. Further analysis on T-cell receptor (TCR) V beta genes on splenic T cells from anti-L3T4 mAb-treated NOD mice revealed that V beta 4-positive T cells expanded predominantly, while L3T4+ T cells represented heterogeneity of the TCR V beta gene, hence, V beta 4-positive Lyt-2+ T cells generate predominantly. Our findings suggest that both L3T4+ and Lyt-2+ T cells renew and function as regulatory cells, through clonotypic interaction in tolerant NOD mice.     

Antigen-reactivity pattern of T-cell hybridomas from Mycobacterium bovis BCG-infected mice.

Lymph node cells from mice infected with live Mycobacterium bovis BCG were fused with BW5147 cells after short-term culturing in vitro. Both mycobacterium- and self-reactive T-cell hybridomas were identified. Some T-cell hybridoma clones displayed dual reactivity to self and to self plus mycobacterial antigen but did so to a different degree, indicating that infection with mycobacteria stimulates autoreactive immune responses.     

Investigation of antigen cross-reactivity of Mycobacterium leprae-reactive murine T-cell lines and clones.

Inguinal lymph node lymphocytes from BALB/c mice immunized intradermally with 10(8) 60Co-irradiated Mycobacterium leprae were cloned by limiting dilution culture. In general, cloned T-cell lines exhibited helper type activity producing interleukin-2, macrophage activation factor and gamma-interferon and lines were further characterized in terms of their cross-reactivities with other species of mycobacteria. M. leprae clones derived after a period of in vitro restimulation were found to cross-react with other species of mycobacteria probably recognizing non-specific or closely related common cell wall associated mycobacterial determinants. On the other hand, lines established by cloning directly from immune mice appeared more M. leprae-specific, exhibiting antigen-dependent lymphokine production and proliferation in vitro.     

Stable expression of Lyt-2 homodimers on L3T4+ T cell clones

The murine T lymphocyte antigen Lyt-2 is considered to act as an accessory molecule to the class I-restricted T cell receptor during antigen recognition. We have previously described two unusual Lyt-2+L3T4+ class II-restricted T cell clones whose activation by antigen is inhibited by antibodies to L3T4 but not to Lyt-2 (B. Fazekas de St. Groth et al., Proc. Natl. Acad. Sci. USA 1986. 83: 2594). The Lyt-2 immunoprecipitated from one of these clones was indistinguishable from the molecule found on splenic T cells, as analyzed under reducing conditions on polyacrylamide gels, in two-dimensional charge/size separations and in peptide mapping. The molecule from the second clone showed slightly more extensive glycosylation but was within the range described for functional Lyt-2 on cytotoxic T cell lines. Lyt-2 mRNA from both clones showed no abnormalities on Northern analysis. Lyt-2 is normally expressed on thymocytes and peripheral T cells as a heterodimer disulfide bonded to the Lyt-3 glycopeptide, yet Lyt-3 could not be detected on the cell membranes of our clones; Lyt-2 existed as stable homodimers without Lyt-3. Thus Lyt-3 is not required structurally for the spontaneous expression of Lyt-2 on lymphoid cells.     

T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae.

The 18-kilodalton (kDa) antigen of Mycobacterium leprae was expressed as a fusion protein with a 2-kDa leader peptide and used in proliferation assays with peripheral blood cells. Fifty percent of untreated tuberculoid leprosy patients and 93% of long-term leprosy contacts responded to the recombinant protein in lymphocyte transformation tests. Comparison of the stimulation indices in the two groups showed that the contacts responded more strongly than the tuberculoid leprosy patients. Seventy percent of Mycobacterium bovis BCG-vaccinated European donors responded, although with low stimulation indices. The isolation of 18-kDa antigen-responsive T-cell lines from a BCG-vaccinated British donor confirmed that the 18-kDa antigen contains at least one cross-reactive epitope. These results indicate that the 18-kDa protein is an important antigen in the immune response to leprosy.     

A role of L3T4+ antigen in the Con A response of regenerating splenic L3T4+ T cells after Cy treatment.

The role of L3T4 antigens in the concanavalin A (Con A) response of regenerating spleen cells (Cy-SCs) after cyclophosphamide (Cy) treatment was studied. Anti-L3T4 monoclonal antibodies (Mabs) markedly inhibited the Con A response of the regenerating Cy-SCs, which do not require Ia molecules expressed on accessory cells (ACs) for Con A activation. However, the Con A response of normal spleen cells (N-SCs), which do require Ia molecules on ACs, was not inhibited by the same Mabs, although the Con A response of N-SCs, as well as that of Cy-SCs, was demonstrated to be mediated by L3T4+ T cells. The optimal times for the inhibitory effect of anti-L3T4 Mab was 7 days after Cy treatment, when the number of spleen cells increased to a maximum following a regenerative phase. Its inhibitory effect was reduced by high concentrations of Con A, and was restricted to the early phase of the Con A response. A short time exposure of the Cy-SCs to the anti-L3T4 Mabs was sufficient to decrease the response to Con A. Our results cannot explain the hypothesis that the L3T4 molecule functions solely by interacting with non-polymorphic parts of Ia molecules on ACs. Taken together, these results and those of other groups of investigators suggest that Con A-induced T-cell activation may be mediated by at least two or more interaction mechanisms involving either Ia or L3T4 molecules. Firstly, normal L3T4+ T cells may mainly interact with Con A involving self Ia molecules on the ACs. The extent of this interaction is sufficient to induce T-cell activation, and then does not need another L3T4 molecule. Secondly, the regenerating L3T4+ T cells may usually interact with the cell surface antigens of other T cells, including L3T4+, by the binding of both cell surface molecules to Con A in the absence of ACs, and then transmit a signal for T-cell activation. Anti-L3T4 Mabs may exert inhibitory effects somewhere in this process.     

Diversity in antigen recognition by Mycobacterium tuberculosis-reactive T cell clones from the synovial fluid of rheumatoid arthritis patients

In a previous study we have shown that synovial fluid mononuclear cells from many rheumatoid arthritis (RA) patients exhibit an enhanced response to M. tuberculosis antigens as compared to peripheral blood mononuclear cells. The 65-kDa heat-shock protein of M. tuberculosis was shown not to play an important role in this response, therefore other mycobacterial proteins must be involved. In this study we have investigated the possibility that synovial fluid T cells from RA patients predominantly recognize a limited number of M. tuberculosis antigens, as a result of a lesion-specific activation of only those M. tuberculosis-reactive T cells that have cross-reacted with joint-related autoantigens. From the synovial fluid of four RA patients M. tuberculosis-reactive T cell clones were isolated and analyzed for their phenotype, HLA-DR restriction and proliferation to immunoblot fractions containing sodium dodecyl sulfate-polyacrylamide gel-separated M. tuberculosis proteins of known molecular weight range. The overall M. tuberculosis immunoblot recognition pattern of the clones was strikingly heterogeneous. Within a panel of 15 clones 12 different antigenic specificities could be distinguished. In other words, we did not observe a dominant recognition of a few M. tuberculosis antigens by synovial fluid T cells. This argues against the hypothesis that the elevated synovial T cell reactivity against M. tuberculosis is a reflection of an in vivo expansion of a limited number of different types of M. tuberculosis-reactive T cells as a result of a cross-reaction with putative joint autoantigens.     

Mycobacterium bovis BCG-induced human T-cell clones from BCG-vaccinated healthy subjects: antigen specificity and lymphokine production.

A total of 121 human T-cell clones were raised from nine Mycobacterium bovis BCG-vaccinated healthy individuals. Three clones were autoreactive, 74 responded to BCG in the presence of antigen-presenting cells, and the others required in addition exogenous interleukin 2. Only one clone was CD8+ CD4-, and the rest were CD4+ CD8-. Testing with a panel of mycobacteria suggested that the clones were recognizing epitopes of varied specificity. Out of 44 clones tested, 15 were specific to BCG and Mycobacterium tuberculosis, 22 showed limited cross-reactivity, and 8 were broadly cross-reactive. None of the 22 BCG responder clones could differentiate between Danish, French, Prague, and Moreau strains of BCG. BCG and M. tuberculosis H37Rv also paralleled very closely; however, 6 of 18 BCG- and M. tuberculosis H37Rv-responding clones did not proliferate to Mycobacterium africanum. BCG- and M. tuberculosis H37Rv-specific as well as cross-reactive T-cell clones could be induced to produce interleukin 2, gamma interferon, and granulocyte-macrophage colony-stimulating factor activity.     

The effect of lipoylation on CD4 T-cell recognition of the 19,000 MW Mycobacterium tuberculosis antigen.

The mechanisms contributing to the dominant and degenerate recognition of the N-terminal region of the Mycobacterium tuberculosis 19,000 MW protein have been investigated. Using polyclonal and cloned T cells it was found that the apparently promiscuous response to the N-terminal peptide was due to the presence of multiple epitopes recognized in the context of different HLA determinants. This finding did not, however, explain the concentration of T-cell recognition on this part of the molecule. The 19,000 MW antigen is a lipoprotein, which raised the possibility that presence of a lipidation motif preceded by a signal peptide influenced the processing and presentation of the protein. Modified peptides covalently attached to lipid moieties were, therefore, tested on both polyclonal and cloned T cells. It was found that whilst lipoylation enhanced polyclonal T-cell recognition, the effect on cloned T cells was variable and depended on their epitope specificity and restricting HLA determinants. This suggested that whilst lipoylation may enhance some aspect of signalling for polyclonal T cells it does not affect the presentation of peptide to T-cell clones. The explanation for the immunodominance of the N-terminal region may, therefore, lie in some aspect of its processing.     

Influenza virus-specific cytotoxic T-cell recognition: stimulation of nucleoprotein-specific clones with intact antigen.

The present study was undertaken to investigate the role of 'antigen processing' in influenza virus-specific cytotoxic T (Tc)-cell recognition. H-2 Db-restricted Tc-cell clones specific for the 1934 influenza nucleoprotein (NP) were tested in an in vitro proliferation assay for the recognition of intact virus, purified protein or peptide antigen. Inactivated virus required further 'processing', which was inhibited by either NH4Cl treatment or paraformaldehyde fixation of antigen-presenting cells (APC). Purified NP, by contrast, was readily presented by both normal peritoneal exudate cells and H-2 Db gene 'transfected' L cells. The response was not inhibited by either NH4Cl or prior paraformaldehyde treatment of APC. Peptone-induced peritoneal exudate cells presented ineffectively unless treated with NH4Cl or prefixed with paraformaldehyde. Comparison of the responses to either purified protein or a synthetic peptide implies that the epitope recognized by the three NP specific clones is not 'cryptic' and, therefore, that the purified protein, in this case, does not require 'processing'.     

Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells.

Immunity to infection with Eimeria vermiformis was transferred in NIH mice by both the nylon wool-adherent (B-cell-enriched) and nonadherent (T-cell-enriched) fractions of lymphocytes (spleen and mesenteric lymph node) taken from infected donors. Transfer was more variable with the adherent fraction, and when contaminating T cells were removed by treatment with anti-Thy1 monoclonal antibody (MAb) and complement, this fraction lost all protective activity. The protective effect of T-cell-enriched populations of mesenteric lymphocytes was abrogated by treatment with anti-L3T4 MAb and complement in vitro before transfer or by opsonization with this MAb in vitro before intravenous inoculation into recipients. Similar treatments of cells with anti-Lyt2 MAb did not have this effect, confirming that Thy1+ L3T4+ cells mediate the adoptive transfer of immunity to E. vermiformis. Thy1+ L3T4+ cells were also shown to limit the replication of E. vermiformis in primary infections: mice depleted of this subset (by thymectomy followed by intravenous injection of anti-L3T4 MAb) passed greater numbers of oocysts over a longer period of time than did mice similarly depleted of Lyt2+ cells.     

Development of autoreactive L3T4+ T cells from double-negative (L3T4-/Ly-2-) Thy-1+ spleen cells of normal mice

Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.     

A single amino acid substitution in influenza hemagglutinin abrogates recognition by monoclonal antibody and a spectrum of subtype-specific L3T4+ T cell clones

A fine specificity analysis of influenza hemagglutinin-specific IAk-restricted T cell clones using natural virus variants of the H3N2 subtype, monoclonal antibody-selected variants and a synthetic peptide corresponding to a variable region of the HA1 polypeptide has provided insight on the structural basis for T cell recognition. A glycine to arginine substitution at HA1 135 abrogates recognition by a panel of T cell clones which, according to their reactivity for natural virus variants, have different antigenic specificities: three clones recognize a synthetic peptide (HA1 residues 118-138) but fail to recognize the monoclonal antibody-selected mutant (Gly135/Arg). There is no correlation, however, between differences in T cell specificity for the natural virus variants and HA1 amino acid sequences in this region. Two further clones have a reduced proliferative response to mutant recognize a completely different spectrum of natural variants, and only one of these clones recognizes the synthetic peptide. We speculate that influenza hemagglutinin employs a common strategy during antigenic drift to evade antibody recognition and effective processing/presentation to subtype-specific T cell clones.     

Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells.

The resistance of mice against Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells was studied. Thymectomized mice were treated with rat monoclonal antibodies against the L3T4 or Lyt-2 molecule to selectively eliminate the respective T-cell subset. In both L3T4+ and Lyt-2+ T-cell-depleted mice, resistance against subsequent infection with M. tuberculosis was markedly impaired compared with that in untreated controls, with L3T4+ T-cell-depleted mice showing more pronounced effects. Simultaneous depletion of L3T4+ and Lyt-2+ T cells did not further exacerbate infection. These findings suggest that both L3T4+ and Lyt-2+ T cells are involved in the acquisition of resistance against tuberculosis.     

Mitogenic activity of soluble preparations from Plasmodium berghei-infected erythrocytes to L3T4+, Lyt2- and L3T4-, Lyt2+ T-cell subsets

The mitogenic activity of soluble preparations of Plasmodium berghei-infected erythrocytes (PBEP) in cultures of T or non-T cells isolated from spleens of naive mice was investigated. The proportion of cells at each phase in the cell cycle was examined by flow cytometry after incubation with various concentrations of PBEP; the proliferation index (PI-cell numbers at S, G2 and M phases/cell numbers at all phases x 100) was calculated. An addition of PBEP led to significant increases in the PI levels of T cells, but not in those of non-T cells. Moreover, when PBEP were added to L3T4+, Lyt2- (helper) or L3T4-, Lyt2+ (suppressor) T-cell fractions separated from rosette-forming T cells in spleens, significant increases in PI levels were detectable in both helper and suppressor T-cell cultures. These results indicate that PBEP have a mitogenic activity directed to both helper and suppressor T-cell fractions isolated from spleens of naive mice and that PBEP or Plasmodium parasites may in this way induce complicated immune responses during malaria infection.     

Tumor-specific L3T4+ and Lyt-2+ lymphocytes in mice primed to mutagenized cell variants.

We have investigated the tumor-specific reactivity of different T-cell subsets from mice primed with clonal variants of L5178Y and P815 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both tumor systems, anti-parental tumor immunity and protection against non-immunogenic clones were only induced by vaccinating the hosts with highly immunogenic cell variants, and the effect correlated with the detection of TATA-specific delayed-type hypersensitivity (DTH) reactions. The footpad reaction was transferable with spleen cell populations from immunized mice, and enrichment of splenic lymphocytes in L3T4+ but not Lyt-2+ lymphocytes increased the footpad swelling. Unfractionated spleen cell populations from immunized mice released high amounts of IL-2 and IFN-gamma in vitro in response to parental antigens. Purified L3T4+ and Lyt-2+ lymphocytes also produced IFN-gamma when incubated in vitro with the parental tumors and accessory cells. It is suggested that the mechanisms of anti-parental tumor immunity induced by MNNG-treated variants may be similar to those described previously for triazene-xenogenized L5178Y/DTIC cells, and may involve induction of a tumor-specific DTH reaction and IFN-gamma-mediated stimulation of non-specific tumoricidal effects.