Ability of T cells from patients with rheumatoid arthritis to respond to immunoglobulin G

The ability of T cells from rheumatoid factor (RF)-positive patients with rheumatoid arthritis (RA) to respond to immunoglobulin G (IgG) was assessed. Peripheral blood mononuclear cells (PBMC) from RA patients and normal individuals were cultured with and without human IgG or Mycobacterium tuberculosis-purified protein derivative (PPD) for 7 days and their proliferative response measured at intervals by their ability to take up tritiated thymidine. PBMC from 14/26 RA patients proliferated in response to IgG (taking a stimulation index of 3 or above as positive). The peak response varied between individuals but usually occurred on day 5, the same day, or 1 day later than the peak response to PPD. By contrast, PBMC from a significantly lower proportion (1/9) of normal individuals and patients with other arthritides (0/6) responded to IgG, although all responded to PPD. PBMC from 9/14 RA patients responded to Fab fragments of IgG but only 3/9 to the Fc fragment. Higher proliferative responses from RA PBMC were elicited by IgG aggregates than the original IgG preparation, but PMBC from 5/5 normal individuals and 5/6 patients with other arthritides failed to respond to the aggregates. The response to IgG was human leucocyte antigen (HLA)-DR restricted and mediated by CD4+ T cells. It is considered that these results advance the hypothesis that IgG-reactive T cells contribute to the initiation or perpetuation of RA.     

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring

The 65-kD hsp from Mycobacterium tuberculosis has been reported to induce an autopathogenic subset of T cells in at least two animal models of autoimmune disease. Reports of increased expression of human hsp60 in the inflamed synovial tissue of rheumatoid arthritis (RA) patients, increased proliferation of RA synovial fluid T cells to mycobacterial hsp65, and increased levels of anti-mycobacterial hsp65 antibody in synovial fluid, have suggested that the highly homologous human (hu) hsp60 may be recognized as an autoantigen in RA patients. In the present study, we have examined by ELISA the serum IgG antibody levels to mycobacterial hsp65 and hu hsp60, as well as to the Escherichia coli hsp60, groEL, in patients with RA, systemic lupus erythematosus (SLE), Reiter's syndrome, active tuberculosis, and normal controls. In all these groups, the levels of anti-groEL and anti-hu hsp60 were significantly higher than the anti-mycobacterial hsp65. Anti-hu hsp60 was positively correlated with anti-groEL, but not with anti-mycobacterial hsp65. Anti-hu hsp60 was competitively inhibited by either soluble groEL or hu hsp60, but little or none by mycobacterial hsp65. Reiter's sera were found to have somewhat higher levels of anti-groEL and anti-hu hsp60 than did normal controls. We conclude that IgG anti-hu hsp60 autoantibodies arise primarily as a consequence of the humoral immune response to E. coli groEL through the recognition of cross-reactive epitopes.     

Deficient interleukin 2 production in rheumatoid arthritis: association with active disease and systemic complications.

Interleukin 2 (IL-2) production and proliferative responses of peripheral blood mononuclear cells (PBMC) stimulated with three concentrations of PHA were measured in 75 patients with rheumatoid arthritis (RA) and 25 normal controls. All patients were on a standard therapeutic regime, and were assessed for disease activity by clinical and laboratory criteria. Rheumatoid cells showed significantly lower IL-2 production and proliferation than normal PBMC at all PHA doses. These differences were not attributable to different kinetics. Within the rheumatoid population, both IL-2 levels and proliferation were lower in patients with active disease than those with inactive RA. Patients with extra-articular disease showed the most pronounced defects. Proliferative responses showed an inverse correlation with clinical indices of disease activity but not with measures of the acute phase response. Rheumatoid patients had higher proportions of CD4+, TFR+ and Tac+ lymphocytes than controls. Both proliferative responses and IL2- levels showed a positive relationship with the proportion of CD4+ cells, and an inverse relationship with Tac+ lymphocytes. Monocyte depletion and partial reconstitution resulted in an increase of both proliferation and IL-2 production, which was more marked in RA patients, suggesting that depressed IL-2 production may relate in part to monocyte effects. However, this cannot completely explain the magnitude of the defects observed, because normal monocytes did not increase the responses of rheumatoid lymphocytes, neither did rheumatoid monocytes suppress the responses of normal lymphocytes.     

T-cell functional defects in rheumatoid arthritis: intrinsic or extrinsic?

This study investigated two mechanisms which may underlie abnormal T-cell function [lymphocyte proliferation and interleukin-2 (IL-2) production] in rheumatoid arthritis (RA). These were: (a) a possible lack of the IL-2-producing CD4+2H4+ lymphocytes and (b) the possible inhibitory role of monocytes and neutrophils. Numbers of CD4+2H4+ cells did not differ between normal controls and patients with RA, although IL-2 produced by the peripheral blood mononuclear cells (PBMC) of the same individuals was markedly reduced in the patient group (P less than 0.001). Many rheumatoid peripheral blood mononuclear cell preparations, but very few control, were contaminated with neutrophils (P less than 0.001). This was more marked in patients with active RA than in those with inactive disease (P less than 0.001). Numbers of monocytes were similar in all groups. Monocyte depletion, or addition of indomethacin and/or catalase in PBMC, caused a significantly greater increase of responses in RA patients than in controls. This effect was significantly higher in patients with active disease than in the inactive group. These findings suggest that activated monocytes and neutrophils found in the rheumatoid PBMC preparations exert inhibitory effects mediated, in part, by the production of prostaglandins and reactive oxygen intermediates. Monocyte depletion and partial reconstitution resulted in significant increase of lymphocyte proliferation and IL-2 production in both controls and patients. None of the manipulations performed succeeded in normalizing the deficient rheumatoid T-cell responses. These data support the hypothesis that non-lymphoid cell populations play an important role in the T-cell dysfunction characteristic of RA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.     

Enhanced interleukin 1 generation by monocytes in vitro is temporally linked to an early event in the onset or exacerbation of rheumatoid arthritis.

Twenty-one patients with rheumatoid arthritis (RA) and 12 age and sex matched healthy controls were examined for the ability of their monocytes (adherent cells, AC) to spontaneously secrete interleukin 1 (IL-1) and for their peripheral blood mononuclear cells (PBMC) to secrete interleukin 2 (IL-2) induced by Staphylococcal Protein A (SPA). All RA patients had PBMC which secreted normal amounts of mitogen induced IL-2 regardless of disease activity or disease history. However, AC from RA patients who had a recent (less than 6 months) onset of their disease, or exacerbation of existing RA, had enhanced spontaneous IL-1 secretion. AC from patients with equally active RA but with historically stable disease generated normal amounts of IL-1. Enhanced in vitro IL-1 generation by circulating monocytes is temporally linked to an early event in the onset of exacerbation of RA.     

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.     

Elevated IgG Antibody Levels to the Mycobacterial 65-kDa Heat Shock Protein Are Characteristic of Patients with Rheumatoid Arthritis

We have previously demonstrated raised levels of IgG and IgA antibody to the mycobacterial 65-kDa heat shock protein (hsp) in the sera of patients with rheumatoid arthritis (RA). We have now attempted to determine whether this phenomenon is specific for RA, and whether it is seen only with the mycobacterial homologue of this particular hsp gene family. We therefore screened antibody levels to the mycobacterial and Escherichia coli hsp 65, and the mycobacterial, E. coli, and human hsp70, in sera from RA, systemic lupus erythematosus (SLE), tuberculosis (TB), ankylosing spondylitis (AS), Crohn's disease, and control donors. RA sera show the greatest increase in IgA binding to the mycobacterial hsp65, but no increase in IgA binding to the E. coli homologue. Similarly, only RA and TB sera show increased IgG binding to the mycobacterial hsp65, and we have shown previously that the titre is greater in RA. In contrast, the use of mycobacterial and E. coli hsp70 preparations as control bacterial hsp gene products has shown that RA patients do not differ from TB or SLE patients in their antibody binding to these proteins. Moreover, neither IgA nor IgG antibody to the human hsp70 in RA sera were higher than in TB, and the IgA binding was not higher than in SLE. These findings suggest that elevated IgG antibody levels to the mycobacterial hsp65 shows some disease specificity, and further studies with the human homologue and at the T-cell level are required.     

IL-1基因多态性对类风湿关节炎病情及IL-1蛋白表达影响

目的:探讨白细胞介素-1α(IL-1α)和白细胞介素-1β(IL-1β)基因多态性对类风湿关节炎(RA)病情及对IL-1β产生的影响。方法:检测136例RA病人及102例正常人的IL-1等位基因分布,其中72例病人系已明确患病2年内出现关节破坏者,IL-1基因多态性检测采用PCR-RFLP法,同时分析不同类型基因型病人外周血单个核细胞受刺激后分泌IL-1β水平的不同。结果:IL-1α等位基因1、2的频率和携带率在RA组及正常人组无显著差异。IL-1β纯合子等位基因2频率在2年内出现骨侵蚀组明显高于2年内未出现骨侵蚀组,IL-1β纯合子等位基因2携带病人组具有较高的血沉及关节肿胀、压痛指数,从携带IL-1β纯合子等位基因2病人中分离出的外周血单个核细胞经脂多糖刺激后分泌的IL-1β量明显高于其它组病人。结论:RA与IL-1β基因多态显著相关,IL-1β等位基因2促进IL-1β分泌,IL-1β等位基因2的检测可作为预测RA严重性的一个有用指标。     

Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis

Heat shock proteins (hsp) are highly conserved, immune-dominant microbial proteins, whose expression is increased at sites of inflammation. In the experimental model of adjuvant arthritis (AA) immune responses to hsp determine the outcome of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response directed against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic arthritis. Next to hsp60, other hsp have similar protective effects in arthritis, while other conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in suppressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candidates for immune therapy in chronic arthritis.     

Immune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans.

The course of human infection with Leishmania braziliensis is variable, ranging from self-healing infection to chronic disease. It is therefore a useful system in which to study immunoregulatory aspects of leishmaniasis, including the effects of parasite antigens on host responses. In the present study, we report on the cloning of, expression of, and comparative analyses of patient immune response to two different L. braziliensis genes homologous to the genes for the eukaryotic 83- and 70-kDa heat shock proteins. rLbhsp83 contains a potent T-cell epitope(s) which stimulated peripheral blood mononuclear cells (PBMC) from all L. braziliensis-infected individuals to proliferate and to produce interleukin-2 (IL-2) gamma interferon, and tumor necrosis factor alpha. The elicitation of IL-4 and IL-10 mRNAs was found to differ depending on the portion of the rLbhsp83 used to stimulate PBMC. rLbhsp83a, which represents the nearly full-length protein, stimulated IL-10 but not IL-4 mRNA. In contrast, a approximately 43-kDa protein representing the C-terminal region of Lbhsp83 stimulated the production of IL-4 but not IL-10 mRNA. rLbhsp70 stimulated PBMC proliferation from patients with mucosal disease but, unlike rLbhsp83, did not stimulate PBMC from self-healing individuals. PBMC from mucosal patients were not stimulated by rHuhsp70 to either proliferate or produce cytokines. This suggests that the hyperresponsiveness of mucosal patient PBMC to Leishmania heat shock proteins does not involve an auto-immune phenomenon resulting from cross-reactivity with self hsp70. In general, although the cytokine profile of patient PBMC in response to both of these Leishmania heat shock proteins represents a mixed Th1-Th2 pattern, the levels of gamma interferon and IL-2 were significantly higher than those of the Th2 cytokines IL-4 and IL-10. Patients with active mucosal and cutaneous disease but not self-healing individuals had significant anti-immunoglobulin G antibody titers to both rLbhsp83 and rLbhsp70 but not to the homologous rHuhsp70. It therefore appears that differential patient immune responses to Leishmania hsp83 and hsp70 may be of particular significance in the induction of protective immune responses as well as in the development of tissue damage in cases with particularly strong hypersensitive reactions.     

Cells with dendritic morphology and bright interleukin-1 alpha staining circulate in the blood of patients with rheumatoid arthritis.

Freshly isolated peripheral blood mononuclear cells (PBMC) from 10 healthy volunteers, 28 patients with rheumatoid arthritis (RA), eight patients with osteoarthritis, and five patients with ankylosing spondylitis were examined for interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) production using monoclonal antibodies and an indirect immunofluorescent method. In freshly isolated PBMC from healthy controls very few cells were stained for either IL-1 type. All 20 RA patients who were not receiving parenteral gold therapy had PBMC staining for IL-1 alpha. In these patients, up to 7.5% of PBMC showed bright IL-1 alpha staining (range 1.2-7.5%). No IL-1 beta staining was seen. These IL-1 alpha-staining cells had a dendritic morphology and the percentage of cells staining correlated well with levels of C-reactive protein, an index of disease activity in these RA patients. Significantly fewer IL-1 alpha-staining cells were present in the peripheral blood of RA patients receiving gold therapy and in the blood of patients with osteoarthritis and ankylosing spondylitis. These IL-1 alpha-containing cells, circulating in the blood of RA patients and correlating with disease activity have not been previously described. These results support the idea that IL-1 alpha plays an important role in the pathogenesis of rheumatoid inflammation.     

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-gamma delta+ lymphocytes.

T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocolitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-gamma delta+ lymphocytes in the 65-kD hsp-stimulated SF-derived lines. This expansion of TCR-gamma delta+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-gamma delta+ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-gamma delta+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-gamma delta+ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-alpha beta+ TLC responded vigorously to the human 65-kD hsp and in some cases also cross-recognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-gamma delta+ cells in response to 65-kD hsp or live bacteria.     

Humoral immune function in severe, active rheumatoid arthritis

Peripheral blood mononuclear cells (PBMC) from 30 patients with definite or classic active rheumatoid arthritis who were on no remittive drugs were studied for spontaneous and pokeweed mitogen (PWM)-stimulated immunoglobulin plaque-forming cell frequency (IgPFC), spontaneous IgM-rheumatoid factor (IgM-RF) secretion, and in vitro proliferative responses to soluble recall antigens. Rheumatoid spontaneous total (IgG + IgM + IgA) IgPFCs were higher than those of normal controls when assayed after 7 days in culture. Spontaneous and PWM-stimulated IgM-PFCs, in contrast, were significantly less than normal regardless of when assayed. Spontaneous synthesis of IgM-RF was observed in 56% of the RA patients, but absolute amounts produced were widely heterogeneous. Spontaneous IgM-RF production by RA PBMC was associated with low or absent spontaneous IgM-PFC production. Moreover, a strong association was found between the median amount of IgM-RF secreted and depressed proliferative responses to soluble recall antigens. Our results define several abnormalities of immunoglobulin production in a clinically homogeneous and highly active rheumatoid population and delineate methodologic variations that can complicate the interpretation of similar data in the literature. In addition, our findings suggest that subgroups of rheumatoid patients that show distinct cellular and humoral immune abnormalities can be identified.     

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.     

Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells. Introduction of an amplification step using ionomycin and phorbol myristate acetate.

The study of T cell responses in parasitic disease and allergy in humans has been limited by difficulties in the measurement of interleukin-4 (IL-4) in supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC). To obtain measurable amounts of IL-4 in vitro, we have added an amplification step to the antigen-specific response. Human PBMC were stimulated by tetanus toxoid (TT) or tuberculin (PPD) for 6 days and then pulsed with ionomycin and phorbol myristate acetate (PMA) for 24 h. TT-stimulated cells from nine revaccinated donors but not from seven unvaccinated donors and four that had only received childhood vaccinations against tetanus produced high levels of IL-4 (median (range) 1500 (300-3800), 316 (0-1600), and 270 (100-410) pg/ml, respectively, as measured by an enzyme linked immunosorbent assay, P = 0.005). PPD did not increase IL-4 production above the background level, although the majority of PPD-stimulated PBMC proliferated and produced interferon-gamma (IFN-gamma) in cultures without ionomycin and PMA. TT-induced IL-4 production correlated positively with proliferation. Culture supernatants did not interfere with IL-4 immunoreactivity and failed to affect ionomycin and PMA induced IL-4 production. The findings suggest that proliferating antigen-specific T cells were the source of IL-4 in these experiments. The method should prove useful for comparing the IL-4 producing ability of antigen-specific T cells from different individuals.     

Modulation of the cellular immune response during Plasmodium falciparum infections in sickle cell trait individuals.

Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P. falciparum-infected individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. HbAA and HbAS patients were compared for levels of plasma soluble IL-2 receptors (IL-2R) and the in vitro cellular reactivity to affinity-purified soluble P. falciparum antigens (SPAg), PPD and phytohaemagglutinin (PHA). At diagnosis, HbAS patients with clinical disease had lower plasma-soluble IL-2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients with asymptomatic infections had comparable soluble IL-2R levels and parasite counts. PBMC from HbAS patients had higher proliferation and IFN-gamma production in response to SPAg than PBMC from HbAA patients. The difference in the lymphoproliferative responses to SPAg between the two groups was evident in patients with asymptomatic infections. In all patients, the clinical severity, the soluble IL-2R levels and the parasite counts were directly related. The former two were inversely related to the proliferative responses to SPAg. After treatment, all the studied parameters were comparable in the two groups. The study indicates that during P. falciparum infection, HbAS compared with HbAA patients had lower in vivo cellular activation and higher in vitro cellular reactivity in response to soluble malaria antigens.     

Analysis of Human Peripheral Blood T Lymphocytes Proliferating in Response to Sensitizing Antigens: Effect of Interleukin-2 (Il-2)-Responsive Bystander Cells

The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (TT) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4⁺T8^- (helper)-enriched population, or T4⁺T8^- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures.