Regulation of delayed-type hypersensitivity. VII. The role of I-J subregion gene products in the inhibition of delayed-type hypersensitivity to major histocompatibility antigens by specific suppressor T cells

Suppressor T cells for delayed-type hypersensitivity (DTH) to alloantigens are induced by injecting mice intravenously with a high dose of X-irradiated allogeneic cells. Using the same protocol, suppression of DTH directed against H-2 subregion products could also be induced, provided that the H-2 incompatibility between the cells used for the induction of suppression and the recipients includes the I-J subregion genes. Thus, the I-J subregion difference is both necessary and sufficient for the induction of suppression of DTH to the whole or part of the H-2 gene products. The suppression appears to be mediated by antigen-specific suppressor T cells which recognize the allo-I-J molecules and are able to suppress the DTH response to other H-2 subregion gene products in an associative recognition manner. T cells from (B10 × BALB/c)F₁ mice suppressively primed against B10.A(5R) cells (directed against J^kE^k antigens), when adoptively transferred to normal syngeneic recipients, were capable of suppressing the hosts' DTH response to B10.A(4R) cells (K^kA^k antigens) when the recipients were challenged with [B10.A(4R) × B10.A(5R)]F₁ cells (K^kA^k × J^kE^k). The recipients express normal DTH reactivity to B10.A(4R) cells when challenged with a mixture of B10.A(4R) and B10.A(5R) cells (K^kA^k + J^kE^k). These results provide direct evidence that when functioning as alloantigens, the I-J determinants preferentially induce suppressor T cells which specifically impair the immune response to the I-J molecules as well as other H-2 gene products if they are physically associated with the I-J determinants. The role of I-J subregions as the suppressor genes is discussed in terms of the possible application in transplantation immunity and the host's defence against infectious diseases.     

Major histocompatibility complex (MHC) restricted antigen recognition: high frequency of human T-cell clones recognizing novel MHC class II determinants

We have used antigen-specific human T-cell clones to study the relationship between MHC and antigen recognition specificities expressed by T cells. Tetanus toxoid (TT)-specific T-lymphocyte clones were derived from a immunized HLA-DR2,7 heterozygous donor by limiting dilution from peripheral blood mononuclear cells (PBM) restimulated with TT in vitro. Clones were screened for MHC-restricted antigen recognition against antigen-presenting cells (APC) from a panel of HLA-typed donors, using an in vitro T-cell proliferation assay. Several distinct patterns of antigen recognition were identified. In addition to T cells that recognized TT in association with donor class II MHC antigens, we found clones that simultaneously expressed self-restricted antigen recognition and alloreactivity, and clones with specificity for antigen in the context of MHC antigens not expressed by the T-cell donor. This was confirmed in inhibition studies using well-characterized monoclonal antibodies against class II MHC antigens to block specific proliferative responses. We propose a possible structure for the determinant recognized by two of the clones. These results suggest that the T-cell antigen receptor undergoes random or antigen-dependent changes in vitro, and that this may be a mechanism for somatic diversification of the T-cell repertoire.     

Regulation of delayed-type hypersensitivity. II. Specific suppressor factor for delayed-type hypersensitivity to sheep erythrocytes in mice.

An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x 10(9) SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH to SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 56 degrees C for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.     

Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

Mice injected with 1 X 10(8) sheep red blood cells (SRBC) into the footpad showed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after the injection. In contrast, mice injected intravenously with 1 X 10(9) SRBC were unresponsive to DTH induction through 1 X 10(8) SRBC injected into the footpad. This suppression of DTH was maintained for at least 6 weeks and was transferable spleen, lymph node and thymus cells to normal syngeneic recipients. Bone marrow cells, on the other hand, did not contain the suppressor cells. The suppression of DTH was antigen-specific in that DTH to chicken red blood cells and contact sensitivity to 2,4-dinitrofluorobenzene was not affected. The suppressor cells were theta-positive and Ig-negative. They appeared in the spleen in optimum number 3-4 days after induction. The suppressor cells affected both the induction and manifestation of DTH. The presence of suppressor and effector cells for DTH inducible by different routes of antigenic presentation reflects the dynamic balance in the regulation of DTH.     

Major histocompatibility complex (MHC)-linked microsatellite markers in a founder population

The Finnish population is genetically relatively homogeneous and has a narrow gene pool as a result of founder effect followed by rapid population growth. We here demonstrate that microsatellite markers are highly informative tools for major histocompatibility complex (MHC) analysis in this population. First, no variation in 12 MHC-linked microsatellites could be observed in certain CYP21-deficient chromosomes, which as a result of founder effect most likely derived from common ancestors. Second, amongst 131 Finnish chromosomes, some, but not all, apparently HLA-identical chromosomes also carried identical microsatellites, suggesting that these loci could be applied for identification of haplotypes which have a relatively recent shared origins. Finally, when the microsatellites were studied between ethnically more distant individuals (Finnish vs. non-Finnish), who were matched for the HLA alleles, much more differences were observed. This showed that the similarity in microsatellites was population specific. The microsatellite typing can therefore be informative in fine mapping MHC-linked susceptibility genes and can help in matching bone marrow transplants in isolated populations. Linkage disequilibrium was found to be much higher in the MHC than in another region (5q31) of similar size, indicating that there may be particular mechanisms keeping the MHC haplotypes conserved.     

Regulation of delayed-type hypersensitivity. III. Effect of cyclophosphamide on the suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

A previous study (Eur. J. Immunol. 1977. 7: 714) has shown that mice injected intravenously (i.v.) with 4 x10(9) sheep red blood cells (SRBC) produce cells which suppress delayed-type hypersensitivity (DTH). These suppressor cells are theta-positive, antigen-specific and act via a soluble factor which does not bear immunoglobulin determinants (Eur. J. Immunol. 1978. 8: 168). The present paper demonstrates that these suppressor cells are inhibitable by cyclophosphamide (CY). Mice injected with graded amounts of CY two days prior to SRBC injection, showed maximum augmentation of DTH at 200 mg/kg body weight, a dose which completely suppressed the appearance of splenic plaque-forming cells (PFC) to SRBC. In contrast, lower doses of CY enhanced both DTH and PFC responses. Time course studies showed that CY inhibited the precursors of suppressor cells and had little or no effect on suppressor cells which have already encountered antigens. This was further confirmed by passive transfer studies which showed tha- suppressor cells were inhibited if CY was administered at the same time or 2 days before SRBC injection, but were not affected if CY was given after antigen stimulation. Direct evidence for the effect of CY on suppressor cells was obtained by cell fractination with a Ficoll density gradient. The denser suppressor cell population was absent from the spleens of mice treated with 200 mg/kg of CY 2 days before i.v. injection with 1 x 10(9) SRBC.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Major histocompatibility complex (MHC) factors predisposing to and protecting against Graves' eye disease

We investigated the influence of gene mapping within the major histocompatibility complex on the susceptibility to Graves' eye disease. We studied 133 randomly selected patients with Graves' disease, many of whom had eye disease. HLA B8 and DR3 carried the greatest risk for disease but the difference between the two patient groups was not statistically significant. An earlier finding that Hungarian patients with a subset of B8, (B8 + DR7 +) had a greatly enhanced risk for eye disease was confirmed in Newfoundland patients. HLA B8 and DR7 are probably carried on different homologous chromosomes and their interaction enhances eye disease. HLA-DR4 was negatively correlated with eye disease. In particular, a subset of DR4 (B35 + DR4 +) appears to protect against eye disease. We have also derived the haplotypes of 22 probands, half of whom had eye disease. The haplotype data emphasized the high frequency of HLA A1 B8 DR3 C4A*QO and C4B*1 in both patient groups, 15% of the haplotypes in the group with eye disease and 25% in that without eye disease. Forty-one percent of haplotypes in the eye disease group and 32% in the no eye disease group were either C4A*QO or C4B*QO. In one proband with eye disease B8 and DR7 were carried on separate chromosomes. The phenotype DR4, C4A*3 C4B*1 was found in 3/20 haplotypes of patients without eye disease but in 0/20 of patients with eye disease. This finding is in keeping with the increased frequency of the DR4 C4A*3 C4B*1 in the patient group with no eye disease when 94 patients were phenotyped.(ABSTRACT TRUNCATED AT 250 WORDS)     

The surface phenotype of a suppressor cell of delayed-type hypersensitivity in the mouse.

Delayed-type hypersensitivity (DTH) to horse red blood cells was induced in cyclophosphamide-treated CBA/H mice. The DTH reaction, represented by an increase (0 x 8--1 x 0 mm) in footpad thickness 24 h after secondary challenge, could be suppressed by the adoptive transfer of 10(7) splenic lymphocytes from syngeneic mice primed with 10(9) HRBC. The surface antigenic phenotype of the suppressor cell was determined by the formation of EA, EAC, or Ig rosettes followed by depleting the rosetted populations on Isopaque-Ficoll. The suppressor cell was found to be Ig-, FcR- and CR-, although some suppression was observed with FcR+ cells. Cell depletions with cytotoxic alloantisera and rabbit complement further characterized the suppressor cell as being Thy-1+, Ly-1+, 2-, 3-, 4-, 5+, 6+, 7-, Ia- and IJ-. This cell surface phenotype if unique and differs from the Ly-1-, 2+, 3+, I-J+ suppressor cell of antibody formation and from the recently described Ly-+, 2+, 3+ feedback suppressor T cell.     

Induction of suppressor cells and cells producing antigen-specific suppressor factors by haptens bound to self carriers

Injection of TNP-, DNP- or oxazolone-substituted syngeneic cells into mice causes the development of hapten-specific T suppressor cells which prevent the animals from being activity sensitized with homologous hapten. These cells injected together with immunized cells abrogate the latter's ability to transfer passively the contact sensitivity (CS) reaction into normal recipients. T lymphocytes from animals made unresponsive and sensitized with homologous hapten synthesize in vitro antigen-specific suppressor factors (SF) which when incubated with immune lymphocytes prevent them transferring adoptively the CS reaction. The type of cell used to induce suppression or production of suppressor factor (haptenated erythrocytes, thymocytes or macrophages) is not critical suggesting that a hapten-substituted common membrane structure is recognized as a tolerogen. The present work demonstrates that while the specific unresponsiveness induced by cell-bound hapten in vivo is long lasting, cells from tolerized animals are able to suppress the immunized cells in passive transfer or produce in vitro antigen-specific suppressor factors only when tested several days after tolerization.     

Schwann cells co-cultured with stimulated T cells and antigen express major histocompatibility complex (MHC) class II determinants without interferon-gamma pretreatment: synergistic effects of interferon-gamma and tumor necrosis factor on MHC class II induction

Schwann cells (SC) do not express major histocompatibility complex (MHC) class II antigens under normal culture conditions. SC can, however, be induced in vitro to express MHC class II molecules by exposure to high concentrations of interferon-gamma (IFN-gamma) and can present antigens to antigen-specific T cell lines. In the present study immunohistochemical labeling showed that most SC (greater than 90%) prepared from rat neonatal sciatic nerves expressed MHC class II molecules when cultured together with mycobacterial antigen and T cells, and as a consequence were able to function as antigen-presenting cells in lymphoproliferation assays, without requiring pretreatment with IFN-gamma. Antigen or T cells alone were ineffective in stimulating MHC class II expression and induction of class II molecules was MHC restricted, requiring the presence of syngeneic T cells. Addition of monoclonal antibody DB1, directed against IFN-gamma to co-cultures of SC and T lymphocytes stimulated with antigen, prevented the induction of MHC class II antigen on SC. When SC were incubated with recombinant (r)IFN-gamma alone, up to 50% of SC showed positive labeling for MHC class II antigen. This level of expression was enhanced to greater than 80% when recombinant tumor necrosis factor (rTNF) was also added. rTNF alone had no effect, and addition of DBI antibody inhibited the synergistic effects of rTNF on MHC class II expression. The effects of rIL 4 were also investigated but neither rIL 4 alone nor rIL 4 in combination with rIFN-gamma induced MHC class II expression by SC. These results show that in the presence of sensitized T lymphocytes and antigen, SC do not require pretreatment with exogenous rIFN-gamma to express MHC class II antigens and function as antigen-presenting cells. T cell-derived TNF and IFN-gamma appear to act as mediators of the T cell-induced expression of MHC class II by SC.     

Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.     

Junin virus-induced suppressor cells are effective only on the efferent limb of the delayed-type hypersensitivity response

Junin virus (JV), the etiological agent of Argentinian hemorrhagic fever, induces high mortality in the suckling BALB/c mouse, which correlates with delayed-type hypersensitivity (DTH)-like immune response. In contrast, the adult mouse is resistant to infection, and no DTH response can be detected. An antigen-nonspecific DTH suppressor T-cell activity induced by JV has been described that may be related to adult mouse survival. In this report, we present evidence supporting the inability of such cells, identified here as bearing the L3T4 marker, to affect the normal establishment of a DTH reaction. In the same form, virus-induced suppressor cells were unable to alter the expression of DTH effector cells in the absence of antigen-specific DTH suppressor cells. Besides, we found that a single high dose (200 mg/kg) of cyclophosphamide before or after JV inoculation was unable to alter the activity of virus-induced suppressor cells.     

Differential sensitivity to cyclophosphamide of helper T cells for humoral responses and suppressor T cells for delayed-type hypersensitivity

The relative sensitivity to cyclophosphamide (CY) of helper T cells in antibody responses and of suppressor T cells in delayed-type hypersensitivity (DTH) responses against sheep red blood cells (SRBC) was measured by pretreating cell suspensions in vitro with the microsomally activated form of CY and evaluating their subsequent function in transfer experiments. It was found that suppressor T cells involved in DTH responses were sensitive to much lower concentrations of activated CY than were helper T cells for anti-SRBC antibody responses. Since the cytotoxic activity of CY is thought to be dependent on its ability to alkylate nuclear DNA, the differential sensitivity observed may be more easily explicable in terms of additional secondary enzyme-mediated degradation or DNA repair mechanisms.     

The basis for major histocompatibility complex (MHC) and immunoglobulin gene control of helper T cell idiotopes

BALB/c helper T cells, prepared against 2,4,6-trinitrophenyl (TNP)-derivatized syngeneic spleen cells, fail to recognize other hapten modifications [(4-hydroxy-3-nitrophenyl)acetyl, fluorescein isothiocyanate] of syngeneic cells, as well as TNP-derivatized cells from major histocompatibility complex-congenic donors. T helper cell interactions with "presenting" cells and "target" B lymphocytes are inhibited by monoclonal antibodies directed either to the hapten or to I-A molecules on target cells. Helper activity is also inhibited by monoclonal anti-idiotypic antibodies directed to an idiotope expressed by the TNP-binding myeloma protein MOPC460, but not by soluble TNP-protein conjugates. The study of congenic mouse strains revealed that while the fine specificity of TNP recognition and the quantitative levels of M 460 idiotope expression are I-A linked, the expression of the M 460 idiotope by helper cells is controlled by IgCh-linked genes. Absence of anti-idiotope inhibition of helper cells prepared in anti-mu-suppressed mice suggests, however, that immunoglobulin idiotype expression by T cells results from network interactions selecting available lymphocyte repertoires which operate before antigenic stimulation.     

Tumour-induced suppression of delayed-type hypersensitivity in the mouse: independence of cyclophosphamide-sensitive suppressor cells

Delayed-type hypersensitivity (DTH) responses to sheep red blood cells and ovalbumin were markedly reduced in mice receiving viable Landschütz ascites tumour cells at the time of immunization. Similar inhibitory effects were observed in control immunized animals when cell-free ascitic fluid or tumour bearer serum was administered together with antigen at the challenge site. Normal mouse serum exhibited much less pronounced inhibitory activity over the range of concentrations tested. High-dose cyclophosphamide (Cy) prior to immunization enhanced DTH responses to both antigens in normal mice. However, in animals also given tumour or soluble tumour-associated material, the immunosuppressive effect which had been observed in non-Cy-treated controls was preserved. It is concluded that the observed suppression of DTH is not dependent on Cy-sensitive T suppressor cells or on the presence of immunosuppressive factors during induction of the immune response.     

Major histocompatibility complex (MHC2+) perivascular macrophages in the axotomized facial motor nucleus are regulated by receptors for interferon-gamma (IFNgamma) and tumor necrosis factor (TNF)

The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.     

Major histocompatibility complex (MHC) antigens polymorphism and alloimmunization study in thalassemia patients with febrile non-hemolytic transfusion reaction (FNHTR)

ObjectivesHLA alloimmunization is one of the most troublesome consequence of regular transfusion which is itself a mainstay measure to provide longevity to the thalassemia patients. Febrile non-hemolytic transfusion reaction (FNHTR) is one of the most common complication which might be related to the HLA alloimmunization. Here, we studied the HLA antigenic system and alloimmunization rate in the Iranian β-thalassemia patients who suffered from FNHTR compare to the β-thalassemia patients without FNHTR.Materials & methodsTotal of 60 β-thalassemia patients with FNHTR (case group) and 20 β-thalassemia patients without FNHTR (control group) randomly have been selected and enrolled in the study. All were tested for HLA-A and -B loci by PCR-SSP method and also for the presence of anti-lymphocyte antibodies by LIFT method. Comparisons between two groups were performed by Pearsonʼs χ2 test.ResultsTotally, a significant predominance was noted for two HLA alleles, HLA-A*24 (P = 0.029) and B*55 (P = 0.034) which have higher prevalence in control group. Although no significant association was found between the presence of anti-leukocyte antibodies and the development of FNHTR, the HLA-A*32 (P = 0.047) allele was considered as possible genetic markers in the susceptibility to the development of anti-leukocyte antibodies.ConclusionHere some evidences about the possible role of HLA polymorphism in susceptibility to FNHTR are provided. Those results indicated that HLA-A*24 and HLA-B*55 might play protective role on inducing FNHTR in β-thalassemia patients. Further studies which investigate the allele level of HLA-I alongside with specific reactivity of HLA-I antibodies might reveal more deep data about these phenomena.