Lymphocyte subsets and mitogen induced proliferation in adjuvant arthritis. Part II. Changes in function and numbers of lymph nodes T lymphocytes.

To evaluate the effects of lymphocytes on the pathogenesis of adjuvant arthritis (AA), the lymphocytic responses to phytohemagglutinin (PHA) and concanavalin A (Con A) and the percentages of T cell subpopulations were investigated in the inguinal lymph nodes of Long-Evans (LE) rats and Sprague-Dawley (SD) rats after a single injection of complete Freund's adjuvant. The response of LE to PHA increased to its maximum on the 14th day and correlated with the severity of AA. The responses of LE and SD rats to Con A were decreased on the 7th, 11th, and 14th days. Additionally, percentage of suppressor T cells of LE rats was lower than that of SD rats. Significant increases of percentage of helper T cells and of helper/suppressor T cell ratio were observed in LE rats, indicating a decrease or defect in the suppressor T cells in LE rats. In LE, we postulated that helper T cells are involved in the induction of AA, and a defect in the suppressor T cell function or numbers would subject LE to the development of AA. SD rats, on the other hand, with higher suppressor T cell numbers, were less susceptible to AA.     

Lymphocyte subsets and mitogen induced proliferation in adjuvant arthritis. Part III. Numbers and functions of T lymphocytes in spleen.

The changes and the possible roles of splenic T lymphocyte subpopulations in high-responder Long-Evans (LE) rats and low-responder Sprague-Dawley (SD) rats with induced adjuvant arthritis (AA) were investigated. Total splenic T lymphocytes of both strains did not change significantly after the adjuvant injection. However, LE rats exhibited an increase in percentage of helper T cells and helper/suppressor T cell ratio, whereas SD rats expressed a decrease in the ratio with concomitant increase of suppressor T cells. This observation suggested that an imbalance of T lymphocyte ratio not only existed in the peripheral blood as reported by other investigators, but also existed in the spleen of rats with AA. In vitro measurement of phytohemagglutinin (PHA) responses of splenic T lymphocytes revealed an increased PHA response in LE but a markedly decreased PHA response in SD. Both LE and SD rats showed decreased response to concanavalin A (Con A) stimulation. We concluded that PHA response, as an index of helper T cell function, coincided with the development of AA and may be responsible for the immunoregulation of the disease. The increased proliferation of suppressor T cell in SD rats may also be significant in regulating the immune response to AA.     

Suppressor T cell numbers and function in atopic patients with high serum IgE levels

Suppressor T cell function and the sensitivity of lymphocyte transformation to histamine has been studied in 9 atopic patients with high serum IgE levels and in 14 controls. The effect of concanavalin A (Con A) induced suppressor T cells on the proliferative response of fresh lymphocytes to mitogens and antigens was measured; in atopic subjects the median suppression to 5.0 micrograms/ml Con A was 80% to 1.0 microgram/ml Phytohaemagglutinin (PHA) was 73%, to Dermatophagoides pteronyssinus extract was 45% and to streptokinase--streptodornase was 24%. Indomethacin increased lymphocyte proliferation to mitogens, but to a variable degree, in both groups. Histamine suppressed lymphocyte transformation to PHA and Con A (median suppression in normal subjects 38 and 46%, and in atopics 51 and 60%) and to D. Pteronyssinus extract. There was no significant difference between normal subjects and atopics in any of these functional assays. The relative and absolute numbers of total T cells, helper T cells and suppressor T cells measured by monoclonal antibodies and the helper to suppressor T cell ratio were normal in the atopic group. These results show that the activity of Con A induced suppressor T cells and the effect of histamine and indomethacin on lymphocyte proliferation is normal in highly atopic subjects. No suppressor T cell defect has been identified using these assays.     

Mitogen induced lymphoproliferative responses and lymphocyte sub-populations in patients with sickle cell disease

Mitogen induced lymphoproliferative responses and lymphocyte sub-populations were studied in a group of sickle cell disease (SCD) patients with homozygous SS hemoglobinopathy. Even though the response to a sub-optimal dose of Con A (0.5 microgram/ml of culture) was significantly decreased in patients with SCD, the proliferative responses to optimal doses of Con A, to PHA and to PWM were preserved in the patients. Addition of indomethacin to the cultures increased to a more significant degree the response to Con A of lymphocytes from patients than from the normal controls. Study of the mononuclear cell subsets indicated that the relative and absolute numbers of B lymphocytes as well as those of monocytes were significantly increased in the patients' group. The percentage of T3+ lymphocytes was found decreased in SCD. However, a rise in the number of T11+ and T4+ lymphocytes as well as in the helper/suppressor cell ratio was observed in the patients as compared to controls.     

Antigen specific suppressor T cells from chronic hepatitis B virus (HBV) carriers inhibit the responsiveness to HBsAg of allogeneic high-responder lymphocytes

Immunoregulatory mechanisms in chronic HBsAg carriers have been investigated through the study of in vitro proliferative responses to HBsAg by allogeneic coculture experiments between T lymphocytes from HBsAg + chronic active hepatitis (CAH) patients (HBsAg no responder) and PBMC from subjects boosted with anti-hepatitis B vaccine (high responder). When high-responder PBMC have been challenged with the hepatitis B surface antigen (HBsAg) in the presence of HBsAg no-responder T lymphocytes, HBsAg no-responder T lymphocytes caused an antigen specific, dose-dependent, suppression of the responsiveness of high-responder PBMC. On the other hand, T cells from patients with autoimmune CAH did not exert any suppressor effect in our system. The suppressor T lymphocytes were mitomycin C resistant and were positive for OKT8, but were negative for OKT4. When T8 + cells were depleted from HBsAg no-responder PBMC, the in vitro immunoproliferative response to HBsAg in chronically HBV infected patients was markedly improved. Out data clearly demonstrate the existence of T8 + suppressor T lymphocytes that can control low responsiveness to HBsAg in chronic HBV patients.     

Immune dysfunction in diabetes-prone BB rats. Interleukin 2 production and other mitogen-induced responses are suppressed by activated macrophages

Spleen cells of diabetes-prone BB Wistar rats were found to generate excessively low proliferative responses, and interleukin 2 (IL-2) levels in response to T-dependent mitogens. This abnormality was not due solely to abnormal T cell numbers since: (a) addition of BB spleen cells of BB splenic macrophages to normal major histocompatibility complex (MHC)-matched Wistar Furth (WF) spleen cells resulted in severe suppression of concanavalin A (Con A)-, phytohemagglutinin (PHA)-, and pokeweed mitogen (PWM)-mediated proliferation, and IL-2 production; (b) macrophage depletion from BB spleen cells, but not B cell or T cell depletion, removed completely the suppressive effects of BB cells on WF cells; (c) macrophage depletion greatly enhanced the response of BB lymphocytes to T-dependent mitogens. Although suppressor macrophages could also be found in the spleen of WF control rats they were present in much smaller numbers than in the spleen of BB rats. The suppressive effect of BB macrophages was partially reduced by addition of the prostaglandin synthetase inhibitor indomethacin to cultures. Furthermore, indomethacin (but not catalase or PMA) considerably augmented IL-2 secretion of Con A-stimulated BB spleen cells, but had little effect on WF spleen cells. In contrast, prostaglandins E1 and E2 (PGE1 and PGE2) suppressed IL-2 production. While IL-2 secretion was severely depressed in BB rats unstimulated and lipopolysaccharide (LPS)- stimulated IL-1 secretion by splenic macrophages was normal. BB macrophages did not inactivate IL-2. Low IL-2 production and macrophage- mediated suppression were features of all BB rats tested.     

Arthritis induced in rats by cloned T lymphocytes responsive to mycobacteria but not to collagen type II.

We have been studying the pathogenesis of adjuvant arthritis in rats using a long-term cell line of T lymphocytes, the A2 line, which can induce polyarthritis and can also be used to vaccinate rats against adjuvant arthritis. Although line A2 was selected for its proliferative response to mycobacteria, it also responded to collagen type II. To elucidate its role of responsiveness to collagen type II and the relationship between arthritogenicity and vaccination, we cloned A2 and selected a subline A2b. We now report that subline A2b, which bore a marker of helper/delayed hypersensitivity T lymphocytes, was strongly arthritogenic, but could not vaccinate against arthritis. Moreover, A2b showed no response to collagen type II. Therefore, reactivity to collagen type II is not a requisite for arthritogenicity, and mediation of arthritis and vaccination can be distinct properties of different populations of T lymphocytes.     

Cellular requirements for lipopolysaccharide adjuvanticity. A role for both T lymphocytes and macrophages for in vitro responses to particulate antigens

By employing primary cultures of purified spleen cells from lipopolysaccharide (LPS) responder (C3H/HeN or C57BL/10Sn) or nonresponder (C3H/HeJ or C57BL/10ScN) mice incubated with particulate antigen and LPS prepared by phenol-water extraction (Ph), we have presented evidence that both T cells and macrophages (MO) are required for LPS-induced adjuvanticity. First, MO derived from C3H/HeN spleen cells, when mixed with responder, C3H/HeN lymphocytes and Ph-LPS, elicited enhanced antibody responses to sheep erythrocytes (SRC) antigen, whereas lymphocytes from the nonresponder, C3H/HeJ mouse strain did not evoke this response. Similarly, purified T cells from C3H/HeN spleens, when cultured with responder, nu/nu spleen cells, and Ph-LPS yielded enhanced anti-TNP PFC responses; whereas, C3H/HeJ T cells did not potentiate immune responses when mixed with optimal concentrations of Ph-LPS. LPS prepared by butanol-water extraction elicited significant adjuvant effects with all cell combinations. Finally, purified responder T cells and MO enabled either responder or nonresponder B cells to elicit LPS potentiation. These data indicate that T cells and MO are controlling LPS-induced augmentation of B-cell responses.     

Effects of cytotoxic immunosuppressants on tuberculin-sensitive lymphocytes in guinea pigs.

The immunosuppressive activities of two phase-specific drugs, 6-mercaptopurine (6-MP) and methotrexate, and a cycle-specific agent, cyclophosphamide, were evaluated on the lymphocytic component of established tuberculin hypersensitivity in guinea pigs. In these animals, purified protein derivative (PPD)-sensitive lymphocytes are in an intermitotic phase of their proliferative cycle. Neither phase-specific drug significantly altered either the number or functional activities of these lymphocytes. By two in vitro criteria, PPD-induced lymphoproliferation and elaboration of migration inhibition factor (MIF), the responses of lymph node cells were equivalent to sensitized controls. In addition, these agents did not deplete pools of T lymphocytes, impair responses to phytohemagglutinin (PHA), nor inhibit cutaneous reactivity if employed before sensitization. In contrast, cyclophosphamide showed broader immunosuppressive effects including significant toxicities for intermitotic lymphocytes. This drug depleted pools of T cells and markedly impaired the in vitro proliferative responses of residual lymphocytes. The latter occurred with both PHA and PPD. Suppression of PHA reactivity was a dose-dependent phenomenon but was evident even with small quantities of this alkylating agent. The suppression of antigen-induced responses was independent of the proliferative status of target lymphocytes in vivo, after a single large dose, it persisted for more than 3 wk. In total, these results indicate that the effective use of cytotoxic drugs as immunosuppressants must include consideration of both the cycle specificities of the agent and the proliferative activities of the target lymphoid population.     

Deficient concanavalin-A-induced suppressor cell activity in women with untreated breast cancer

Peripheral blood mononuclear cells from 14 women with untreated breast cancer and 5 patients with non-malignant breast disease were studied for concanavalin-A (Con A) inducible suppressor activity against proliferative response of lymphocytes to phytohemagglutinin (PHA). Six of 14 patients with breast cancer demonstrated deficiency of suppressor cell activity against proliferation of autologous lymphocytes to PHA. This is in contrast to only 1 of 5 patients with benign breast disease who demonstrated deficiency of inducible suppressor activity. No correlation was observed between the deficiency of suppressor cells and the presence or absence of metastasis in the regional lymph nodes. The significance of these observations is discussed.     

A study of the initial suppressor function of lymphocytes]

The new method for analysis of initial suppressor lymphocyte function in newly isolated monocultures is based on quantitative cytofluorometric analysis of early mitogen-induced activation of B lymphocytes in the presence of prednisolone that switches off a T lymphocyte immunoregulatory effect. Suppressor lymphocytes monitor B lymphocyte exit into proliferative pool and early processes of nuclear chromatin activation. The levels of initial suppressor lymphocyte function and lymphocytic proliferative activity are in inverse correlation. Suppressor lymphocyte function has been essentially reduced in rheumatoid arthritis patients as against normal subjects. A reduction as low as 70 percent of the normal value (recorded in 1/3 of the patients) is compensated with unchanged regulatory control over B cells, and reduction as low as 40 percent of the normal value (in 2/3 of the patients) is characterized by loss of regulatory control and B cell hyperactivation.     

佐剂关节炎大鼠心功能变化与细胞因子、调节T细胞和Foxp3表达的相关性

目的:观察佐剂性关节炎(adjuvant arthritis,AA)大鼠足跖肿胀度(E)、关节炎指数(AI)、心功能、细胞因子、调节T细胞及Foxp3的表达,探讨细胞因子、转录因子Foxp3与调节T细胞(Treg)在AA大鼠心功能改变中的作用机制.方法:24只Wistar雄性大鼠随机分为正常组和模型组(Model组)各...     

In vivo and vitro effects of interferon-alpha 2b on functional activity of T-lymphocytes from patients with rheumatoid arthritis

AIM: To investigate the effect of recombinant alpha 2b-interferon (r alpha 2b-IFN) on functional capacity of peripheral blood (PB) T cells in rheumatoid arthritis (RA) patients and the relationship between functional characteristics of T lymphocytes and the disease activity. MATERIALS AND METHODS: PB mononuclear cells (PBMC) were separated by Ficoll-Verografine++ gradient centrifugation from 24 healthy donors (HD) and 75 RA patients 19 of which were treated with r alpha 2b-IFN (realdiron, Biofa, Lithuania) in the dosage 1 million IU i.m. each other day for 20 days, 10 injections a course. Cell surface markers (CD3, CD4, CD8) and adhesion molecules (CD18, CD54, CD2) were analyzed using specific monoclonal antibodies (MoAbs) and flow cytometry on the PBMC, freshly isolated and treated for 72 hours with medium alone, PHA, r alpha 2b-IFN and their combination. The proliferative response of PBMC to MoAbs for CD3, PHA and r alpha 2b-IFN were assessed by 3H-thymidine incorporation. The percentage of spontaneous and inducing apoptosis was quantified by flow cytometry using propidium iodide staining. RESULTS: The expression of CD18 was lower on RA PB lymphocytes compared to HD PB lymphocytes (p < 0.05). After stimulation of PBMC in both RA patients and HD with PHA, percentages of CD2+, CD3+, CD4+, CD18+ cells significantly diminished (p < 0.05), whereas the percentages of CD54+ and CD18+ (p < 0.05) cells increased. We have found three types of RA PB lymphocytes response to complex factors in vitro: 1) the presence of the proliferative response to T-mitogens but not to r alpha 2b-IFN (56% of the patients); 2) the presence of the increased proliferative response to T-mitogens and r alpha 2b-IFN (17% of the patients); 3) the absence of the proliferative response to T-mitogens and r alpha 2b-IFN (27% of the patients). PBMC of HD demonstrate only the first type of the response. R2 alpha b-IFN demonstrated own mitogenic effect and increased mitogen-induced proliferation in PBMC cultures with a high proliferative response to T-mitogens. The levels of spontaneous and inducing apoptosis were increased in RA PB lymphocytes compared to HD. After stimulation with PHA, RA PB lymphocytes preferentially underwent apoptosis whereas cells of HD proliferated. High disease activity correlated positively with an increase of a proliferative response to mitogens and apoptosis and a decrease in the percentage of lymphocytes, expressed adhesion molecules. The treatment with r alpha 2b-IFN induces changes in T-cell response to mitogens similarly to those after incubation with r alpha 2b-IFN in vitro before treatment. CONCLUSION: Functional capacity of RA PB lymphocytes relates to the disease activity. Inhibitory or stimulatory effects of r alpha 2b-IFN depend on functional activity of RA lymphocytes. Using the test with alpha 2b-IFN incubation, we may predict changes of apoptosis and proliferation levels caused by different agents in RA lymphocytes after treatment with r alpha 2b-IFN.     

Regulatory mechanisms in cell-mediated immune responses. VI. Interaction of H-2 and non-H-2 genes in elaboration of mixed leukocyte reaction suppressor factor

Previous studies have shown that alloantigen-activated spleen T cells produce a soluble factor which suppresses mixed lymphocyte reaction proliferative responses, and that the interaction between suppressor and responder cells is controlled by genes of the H-2 complex. However a defect in the expression of suppressor activity was identified in the mouse strain C57BL/6J. Factor prepared from alloactivated B6 spleen cells failed to suppress MLR responses of syngeneic or H-2 compatible responder cells. Unimpaired suppressor factor production by other H-2 (b) strains and failure of suppressor factor production by a B6 congenic strain, B6.C-H-2(d) isolated the defective gene to the non-H-2 portion of the genome. In addition, the defect appeared to be related specifically to inability to produce an active factor, while the capacity to respond to suppressor molecules was unimpaired. The genetic character of the non-H-2 gene action was identified in F1 hybrid studies. Initially F(1) hybrids of the nondefective histoincompatible strains were studied. Suppressor factor from F1 cells suppressed the responses of both parental strains, and parental factors each suppressed the response of F(1) cells. Adsorption of F(1) factor with Con A-activated thymocytes of either parental strain removed suppressor activity specific for that strain, leaving activity against the other parental strain intact. The data support cedominant expression and production of distinct, parental H-2 haplotype-specific suppressor molecules by F(1) suppressor cells. An F(1) hybrid of the defective B6 strain with nondefective BALB/c produced suppressor factor which was also capable of suppressing both parental strains. Production of a suppressive B6-reactive factor by F(1) cells was verified by adsorption studies. Thus it appears that non-H-2 genes of the BALB/c parent acted in a genetically dominant fashion to provide the function required for expression of B6 suppressor molecules. We conclude that multiple genes control the expression of alloactivated suppressor cell activity, with at least one gene mapped to the I-C subregion of the murine major histocompatibility complex and one or more genes mapped to the non-H-2 gene complement.     

Genetic control of immune response. The dose of antigen given in aqueous solution is critical in determining which mouse strain is high responder to poly(LTyr, LGlu)-poly(LPro)--poly(LLys)

Antibody response to different doses of (T,G)-Pro--L, given in aqueous solution, was investigated in the high responder SJL and low responder DBA/1 strains by measuring hemolytic plaque-forming cells (PFC) in the spleens as well as hemagglutination titers in the sera. The gene responsible for the difference between the two strains in the response to this antigen, given in complete Freund's adjuvant, has been previously denoted Ir-3. This gene is not linked to the major histocompatibility locus. In the response to the optimal dose (1 mug) of antigen, no difference could be shown between the strains. The peak of the response and the numbers of direct and indirect PFC were similar in both strains in the primary and secondary response. After injection of higher doses (10-100 mug) of antigen, both the direct and indirect PFC responses were lower in the low responder than in the high responder strain. Moreover, the peak of the response occurred earlier in the high responder strain in the primary response to the 10 mu dose of antigen. After administration of a suboptimal dose (0.02 mug) of antigen, the low responder strain produced in the primary response 4-20 times more indirect plaques than the high responder strain. Also the number of direct plaques was higher in the low responder than in the high responder strain. The serum antibody responses to the optimal and higher doses of antigen were parallel to the PFC responses. From inhibition of PFC with free antigen, it was concluded that a similar proportion of cells was producing high and low affinity antibodies to (T,G)-Pro--L in both strains. High and low zone tolerance could be induced in the two strains with (T,G)-Pro--L, but no difference could be shown between the strains. It is suggested that the Ir-3 gene plays a role in the regulation of the balance stimulation and suppression according to the dose of antigen given.     

Deficiency of the autologous mixed lymphocyte reaction in patients with primary biliary cirrhosis.

In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.     

Immunoregulatory abnormalities in chronic lymphocytic leukemia. I. Correlations of immunoregulatory subpopulations with stage of disease

Previous studies have demonstrated multiple immunologic abnormalities in Chronic Lymphocytic Leukemia (CLL). We, therefore, investigated the relationship of immunoregulatory mononuclear cell subpopulations and disease activity in inactive and active CLL. The absolute number of T cells is increased in both groups compared to controls and a significant increase (p less than 0.001) in the number of monocytes was found in active patients. When the number of Fc gamma bearing T cells was compared to the number of B cells, active patients revealed a 993% decrease and inactive patients a 93% decrease compared to controls. Inactive patients also revealed increased proliferation when stimulated (PHA) after 48 hours in media alone compared to fresh cells. The active group revealed no increase in preincubated (PHA) stimulated cultures. As this suggested the possibility of immunoregulatory differences, suppressor cells were studied. Con A induced suppressor cells decreased proliferation of PHA stimulated cultures in inactive and control groups but had no effect in active patients. Isolated fresh autochthonous T cells (1:1) decreased PHA-induced proliferation 86% in the inactive group, 50% in the control group but had no effect in active patients. Pre-incubation (48 hr) followed by T cell separation revealed decreased Fc gamma T cells and abrogation of this suppressive effect in both inactive and control groups. Finally, isolated adherent cells decreased PHA stimulation by 86% in active patients but had insignificant effects on preincubated PHA stimulated cultures in the other groups. These data suggest that inactive CLL is characterized by a population of T suppressor cells that are more active than similar numbers of this population in control cultures. This population is short-lived and correlated with the Fc gamma bearing T cell population. This population appears inactive or non-functional in active CLL where adherent suppressor cells are increased.     

Role of the thymus in induction and transfer of vaccination against adjuvant arthritis with a T lymphocyte line in rats.

Adjuvant arthritis is an experimental disease of rats induced by immunization to antigens of Mycobacterium tuberculosis. Our observation that arthritis could be induced in irradiated rats by the A2 line of T lymphocytes in the absence of mycobacterial antigens suggested that adjuvant arthritis is an autoimmune disease. Moreover, the A2 line could be used to vaccinate unirradiated rats against the subsequent induction of adjuvant arthritis by active immunization to Mycobacteria. In the present study we found that thymus cells obtained from A2 vaccinated rats could transfer resistance to adjuvant arthritis to naive rats. This indicates that the mechanism of resistance induced by A2 vaccination is probably immunological and involves thymus-derived lymphocytes.     

佐剂性关节炎大鼠载脂蛋白高密度脂蛋白的变化及意义

目的观察佐剂关节炎(AA)大鼠载脂蛋白AI(ApoAI)、高密度脂蛋白(HDL)和血管内皮生长因子(VEGF)、E选择素(E-selection)的变化及相关性研究。方法将27只雄性大鼠随机分为正常组、模型组,分别为模型组15只,正常组12只,用弗氏完全佐剂(CFA)向模型组大鼠右后足跖皮内注射0.1 ml诱发大鼠产生关节炎,观察各组大鼠ApoAI、HDL和VEGF、E选择素的变化,并作相关性分析。结果(1)ApoAI模型组与正常组升高率分别为93.33%和50.00%,HDL模型组与正常组升高率分别为86.67%和41.67%,模型组升高率均显著高于正常组(P<0.05)。与正常组相比,模型组VEGF、E选择素、IL-1β、CRP显著升高(P<0.01或P<0.05);IL-10显著降低(P<0.01)。(2)模型组ApoAI、HDL与VEGF、E选择素、IL-1β呈显著负相关(P<0.05或P<0.01),与足跖肿胀度、关节炎指数、CRP呈显著正相关(P<0.05或P<0.01),与IL-10无明显相关。结论AA大鼠在足跖肿胀的同时出现ApoAI、HDL的升高及血管内皮细胞的损伤,提示免疫应激导致AA大鼠ApoAI、HDL的改变,并与疾病的活动度、细胞因子的紊乱、血管内皮的损伤密切相关。     

A Suppressor T Cell of the Mixed Lymphocyte Reaction Specific for the HLA-D Region in Man

The mixed lymphocyte reaction (MLR) is the proliferative response of one individual's lymphocytes cultured in the presence of another individual's lymphocytes. In man, the MLR is elicited by cell surface antigens coded for by the HLA-D gene locus. This locus is among a cluster of genes which are located on the sixth chromosome and which include genes coding for the major histocompatibility antigens HLA-A, B, and C as well as HLA-D. If the stimulator cell possesses D locus antigens not present in the responder, the lymphocytes of the latter will undergo blast transformation resulting in DNA synthesis which can be measured. A vigorous response in the MLR to allogeneic cells is the rule among healthy individuals.We describe studies of a 23-yr-old man whose lymphocytes respond normally to mitogens and soluble antigens but fail to respond to allogeneic cells in the MLR. His medical history is unremarkable except that he received thymic irradiation as an infant. HLA typing revealed that he is homozygous for HLA-A2, B12, and Cw5 as well as for the D locus antigen Dw4. When his lymphocytes were added to the responder lymphocytes of other persons homozygous for the same HLA antigens, their responses to allogeneic cells but not mitogens were suppressed by 50-95%. Their responses to a soluble antigen, tetanus toxoid, were suppressed to a lesser degree. These inhibitory effects were mediated by a relatively radioresistant thymus-derived (T) lymphocyte.Further studies of the requirements for MLR suppression revealed that only persons heterozygous or homozygous for the Dw4 antigen were inhibited by the suppressor T cell. This effect was not altered by differences in the HLA-A, B, or C antigens between the suppressor and responder. It is concluded that genes in or near the HLA-D locus code not only for antigens (primarily on bone marrow-derived (B) cells), that elicit the MLR, but also for structures on T cells, or possibly macrophages, which are recognized by MLR suppressor T cells.