Specific lysis of targets expressing varicella-zoster virus gpI or gpIV by CD4+ human T-cell clones.

Varicella-zoster virus (VZV)-specific CD4-positive T cells are known to lyse targets expressing VZV antigen, but little is known of the glycoprotein specificity or phenotype of these cells. To test the ability of T cells to distinguish between gpI and gpIV (which share an antibody-defined epitope), we prepared clones from blood from four healthy individuals by limiting dilution. Among 68 T-cell clones from four donors which were VZV specific in tests of proliferation, 30 lysed autologous Epstein-Barr virus-transformed lymphoblasts which had been superinfected with a recombinant vaccinia virus which included the whole VZV gpI sequence. These clones were characterized as major histocompatibility complex class II restricted by inhibition of their cytotoxicity with HLA-DR and CD4 monoclonal antibodies. Twenty-one clones lysed targets expressing gpIV. Fifteen of these clones lysed targets expressing gpI and gpIV. Four clones with gpI-gpIV specificity were examined in detail, and their dual specificity was confirmed by cold target inhibition. These four clones failed to kill target cells infected with a mutant gpIV recombinant vaccinia virus from which amino acid residues 212 to 354 had been deleted. This region includes one of the two gpIV decapeptides which have 50% homology with amino acids 111 to 121 of gpI. Our data confirm that T-cell-receptor-associated structures are required for specific lysis of VZV targets and indicate that (i) gpI-specific CD4 cytotoxic T cells outnumber gpIV-specific T cells in blood and (ii) 50% of gpI-specific T-cell clones also lyse gpIV-expressing targets.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

T-cell immune response to human herpesvirus-6 in healthy adults.

The proliferative response of peripheral blood mononuclear cells (PBMC) from healthy adults to human herpesvirus-6 (HHV-6) was examined. It was found that PBMC from 13 of 14 HHV-6-seropositive adults apparently proliferated in response to stimulation with HHV-6 antigen in contrast to the lack of response of cord blood mononuclear cells. In order to confirm the presence of HHV-6-specific memory T cells in the peripheral blood of healthy adults, we established HHV-6-specific T-cell clones from an HHV-6-seropositive individual. CD4+ T-cell clones generated from HHV-6-stimulated PBMC were found to proliferate upon stimulation with HHV-6 in the presence of autologous antigen-presenting cells, but not in response to herpes simplex virus type 1 antigen or mock-infected control antigen. These results indicate that a T-cell immune response against HHV-6 infection is generally present in healthy adult populations.     

Persistent high frequencies of varicella-zoster virus ORF4 protein-specific CD4+ T cells after primary infection

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-gamma) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-gamma cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.     

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.     

Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones.

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.     

Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones.

We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we identified seven CTL clones that recognized D4V capsid protein. Six of these CTL clones were cross-reactive between D2 and D4, and one clone was specific for D4. Using synthetic peptides, we found that the D4V-specific CTL clone recognized an epitope between amino acids (aa) 47 and 55 of the capsid protein, while the cross-reactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2V and D4V. This region of the capsid protein induced a variety of CD4+ T-cell responses, as indicated by the fact that six clones which recognized a peptide spanning this region showed heterogeneity in their recognition of truncations of this same peptide. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84 to 92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84 to 92. These results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level but in bulk culture responses as well. Since previous studies have indicated that the CTL responses to DV infection seem to be directed mainly against the envelope (E) and NS3 proteins, these results are the first to indicate that the DV capsid protein is also a target of the antiviral T-cell response.     

CD69 expression on alpha-gliadin-specific T cells in coeliac disease

Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is triggered in susceptible individuals by ingestion of gluten. The pathogenic mechanism of coeliac disease, and the role that alpha-gliadin specific T cells play in mucosal lesions and their involvement in peripheral blood is not yet explained at all. Previous studies have reported proliferative response to alpha-gliadin measured with the classic assay of 3HTdR incorporation. We analysed the activation antigen CD69 on T cells from CD patients and normal individuals following stimulation with alpha-gliadin and different antigens (tetanus toxoid, peptides unrelated to gliadin and PHA). CD69 coexpression with T cell CD3+ and proliferation marker Ki67 was evaluated with time. CD69 coexpression with T cell CD3+, CD4+ and CD8+ was also evaluated. It was found that peripheral blood mononuclear cells (PBMC) of coeliac patients increased their percentage of CD69 positive T cells when stimulated with alpha-gliadin, in comparison with cells from controls. Significant T cell activation was found only in subjects not treated with the gluten free diet; a positive response was found also in two coeliac patients with selective IgA deficiency, anti-endomisium negative, without circulating IgA anti alpha-gliadin or anti-tissue transglutaminase antibodies. The CD69 expression after stimulation was compared with the standard method of 3HTdR incorporation. Our data show that CD69 expression is useful to asses a specific T cell response to alpha-gliadin in coeliac disease. in a very short time. Moreover, the method allows to investigate T cell response at the lymphocyte subsets level, which represents a useful tool in the diagnosis of coeliac disease.     

Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells.

Epstein-Barr virus-transformed human B cells expressed cell surface varicella-zoster virus (VZV) antigens after superinfection with VZV although they did not form infectious centers in a plaque assay. The VZV-superinfected cells were lysed by autologous VZV-stimulated T-cell lines and their derivative clones. The effector cells were specific for VZV and an HLA DR antigen and were T4+. The specificity of lysis of Epstein-Barr virus-transformed, VZV-superinfected targets by prestimulated mononuclear cells in this system contrasted with the unrestricted lysis seen when the targets were VZV-infected fibroblasts.     

Lymphocyte clones from old subjects: growing rate and functional activity

Old subjects exhibit a decline in circulating T cells and an impaired proliferative response to mitogens, plus a relative increase in cells with NK phenotype not associated with a concomitant increase in their cytolitic activity.In the present study a limiting dilution assay was used to evaluate the phenotype, the functional activity and the proliferative capacity of clones obtained from peripheral blood lymphocytes of old and young subjects. CD5+ CD8+ clones from old people showed a significant impairment in their proliferative capacity and a decreased lytic activity against K562 and P815-IgG cell lines.     

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans.     

Helicobacter pylori has stimulatory effects on naive T cells

BACKGROUND: Despite an apparently active host response, Helicobacter pylori infection can persist for life. Unexpectedly, T cells from apparently uninfected individuals respond to H. pylori antigen by proliferating. Also, the T-cell proliferative response appears to be less in infected compared with uninfected individuals. MATERIALS AND METHODS: We have investigated the T-cell response of isolated human peripheral blood, naive, and memory CD4+ T cells to H. pylori antigen in infected and uninfected subjects. RESULTS: In agreement with previous findings, the peripheral blood proliferative response was higher in uninfected compared with infected subjects. Interestingly, there was a response in CD4+ CD45RO+ (memory) and CD4+CD45RA+ (naive) subsets. The RO/RA ratio of the response to H. pylori antigen was 0.8-2.1 in both H. pylori-positive and H. pylori-negative subjects, which was similar to that of a known superantigen (2.5 and 2.2 in Helicobacter-positive and -negative subjects, respectively) whereas the RO/RA response ratio to a recall antigen (tetanus toxoid) was 9.8 and 18.7 in Helicobacter-positive and -negative subjects, respectively. Mononuclear cells isolated from cord blood also responded to H. pylori antigen, whereas there was no response to tetanus toxoid. The cord blood response and CD4+ CD45RA+ cell response to H. pylori antigen were inhibited predominantly by anti-HLA-DR and to some extent by anti-HLA-DQ antibodies. Investigation of the response to five different recombinant H. pylori antigens identified two that produced a response in naive T cells. CONCLUSIONS: These data suggest that H. pylori possesses molecules that cause higher than expected proliferation of naive T cells.     

Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor following in vitro immunization

Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4(+) T-cell response. Therefore, understanding and characterizing HIV-specific CD4(+) T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4(+) T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4(+) T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4(+) T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4(+) T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.     

Anergic TH1 clones specific for hepatitis B virus (HBV) core peptides are inhibitory to other HBV core-specific CD4+ T cells in vitro.

A strong and transient hepatitis B virus core (HBc)-specific CD4+ T-cell response has been shown to be associated with viral elimination in acute self-limited hepatitis B but to be absent in chronic hepatitis B. So far, little is known about immunological mechanisms involved in the regulation of the HBc-specific CD4+ T-cell response. We studied 28 patients with acute hepatitis B, and frequently a sudden decrease in the HBc-specific CD4+ T-cell response was found between 4 and 8 weeks after disease onset. Thirty-two CD4+ T-cell clones specific for amino acids 50 to 69, 81 to 105, 117 to 131, or 141 to 165 of HBc were isolated from a patient shortly before the peripheral blood mononuclear cell response to most HBc-derived peptides abruptly disappeared. TH1 clones, but not TH0 clones, could be anergized in vitro by stimulation with specific peptides even in the presence of costimulatory cells. Moreover, when anergic cells were mixed with responsive cells, the proliferation of HBc-specific TH1 or TH0 clones was inhibited antigen specifically by anergic cells. The unusual susceptibility of HBc-specific TH1 clones to anergy induction in vitro as well as their potential to inhibit other HBc-specific TH1 and TH0 clones suggests that anergy induction may be involved in the downregulation of the virus-specific immune response during acute hepatitis B in vivo.     

Human T lymphocyte clones specific for malaria (Plasmodium falciparum) antigens.

Peripheral blood mononuclear cells (PBM) from a patient who had lived in a malarial-endemic area were cultured in the presence of malarial antigens (a lysate of Plasmodium-infected erythrocytes). Responding cells were grown in IL-2-containing medium and then cloned, and subsequently subcloned, in the presence of phytohemagglutinin and allogeneic irradiated PBM. Ten clones were specific for malarial antigens. They proliferated in response to P. falciparum extract, but not to a lysate of uninfected erythrocytes. The response was HLA-restricted. All the clones tested responded to lysates of cells infected with parasites of either African or Asian origin. Six clones had the T4+/T8- phenotype and four the T4-/T8+ phenotype. Two of the T4+ clones recognised a parasite antigen of apparent mol. wt. approximately 50 000. All of the clones tested produced gamma-interferon following antigen stimulation.     

Vaccinia virus-specific human CD4+ cytotoxic T-lymphocyte clones.

Vaccinia virus-specific cytotoxic T-lymphocyte (CTL) clones were established from a healthy donor, who had been immunized with vaccinia virus vaccine, by stimulation of peripheral blood lymphocytes with UV-inactivated vaccinia virus antigen. The phenotype of all of the clones established was CD3+ CD4+ CD8- Leu11-. We used a panel of allogenic vaccinia virus-infected B-lymphoblastoid cell lines and demonstrated that some of the clones recognized vaccinia virus epitopes presented by human leukocyte antigen (HLA) class II molecules. Monoclonal antibodies specific for either HLA-DP or HLA-DR determinant reduced the cytotoxicity of specific clones. The HLA-restricted cytotoxicity of the clones is vaccinia virus specific, because vaccinia virus-infected but not influenza virus-infected autologous target cells were lysed. Using vaccinia virus deletion mutants, we found that some of the CTL clones recognize an epitope(s) that lies within the HindIII KF regions of the vaccinia virus genome. These results indicate that heterogeneous CD4+ CTL clones specific for vaccinia virus are induced in response to infection and may be important in recovery from and protection against poxvirus infections.     

Promiscuous malaria peptide epitope stimulates CD45Ra T cells from peripheral blood of nonexposed donors.

PBL from individuals with no history of malaria exposure, as well as cord blood lymphocytes, were tested for proliferation to T cell epitopes from the malaria circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax. Cells from many individuals proliferated in response to these peptides, but for two peptides (P. vivax317-336 and P. falciparum CS331-350) the response rate ranged from 64 to 93%, with the specific stimulation indices reaching as high as 38. The phenotype of the cells responding to PfCS331-350 was predominantly CD4+,CD8-,CD45Ra+,CD45Ro-, which was the inverse of the phenotype of the cells responding to tetanus toxoid with respect to CD45 isoforms. T cell clones from different individuals specific for PfCS331-350 were restricted by at least four different HLA-DR molecules and there was no evidence that the peptide was a "superantigen." Overlapping peptides were used to demonstrate that clones had different fine specificities although the peptide specificities of the DR4-restricted and DR11-restricted clones were similar. Although the individuals tested here have had no history of malaria exposure, these data demonstrate that they have T cells specific for malaria sequences present in high frequency that proliferate as intensely as some memory responses. Although one clone from an individual with a history of BCG vaccination did react strongly with PPD, the phenotype of these cells suggests that they are not classical memory cells for a cross-reactive recall Ag. Such cells may affect the induction or expression of malaria immunity.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.     

Interleukin-2 reverses T cell unresponsiveness to Plasmodium falciparum-antigen in malaria immune subjects

Peripheral blood mononuclear cells (PBMC) from a large proportion of 34 healthy adult native residents in a malaria endemic area showed null or marginal proliferative response (low-responders) to schizont-enriched Plasmodium falciparum malaria antigen (M.Ag) but good response to pokeweed mitogen. In contrast, substantial proliferative response to M.Ag was observed in 8/8 adult temporary residents with a history of one to three acute malaria episodes. Purified CD4+ T cells preferentially responded to M.Ag, however in low-responders CD4+ T cell proliferation was poor. Moreover, no inhibition of CD4+ T cell proliferation was observed when graded numbers of CD8+ T cells were added in culture. The addition of recombinant interleukin 2 (rIL-2) to M.Ag restored the proliferative response of low-responders' PBMC. This response was M.Ag-specific when CD4+ T cells grown in M.Ag plus rIL-2, but not in rIL-2 alone, were tested in secondary cultures.