Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs II

We compared five cytotoxic T lymphocytes raised by primary mixed lymphocyte cultures of HLA-A, -B and -C serologically identical Bw35-positive responder-stimulator combinations. When tested on a panel of third-party target cells, the reactivity pattern of these cytotoxic T lymphocytes allowed the distinction of three subtypes of HLA-Bw35. Cold-target inhibition experiments and analysis of CTL activity at the clonal level showed the existence of subsets of CTLs directed against distinct antigenic determinants associated with HLA-Bw35.     

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs I

Cytotoxic T lymphocytes were activated in primary one-way mixed lymphocyte cultures of cells matched for serologically defined HLA-A, -B and -C antigens. In 16 out of the 29 combinations mismatched for the HLA-D/DR antigens, cell-mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA-Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T-target cells enriched by rosetting with sheep red blood cells, whereas target cells from the 'non-T' fraction were strongly lysed, indicating that antigenic determinants associated with Class-II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA-typed third-party target cells. One cytotoxic T-lymphocyte population preferentially lysed HLA-DR2-positive target cells. Family studies, including a family with a recombination between HLA-B and -D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T-lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA-A-, -B- and -C-identical, D/DR-different combinations, cell-mediated lympholysis of stimulator target cells could not be detected, not even on enriched 'non-T' target cells. Thus, after primary mixed lymphocyte culture of HLA-A-, -B- and C-identical, HLA-D/DR-non-identical cells, cytotoxic T lymphocytes directed against sensitizing Class-II molecules can be detected in some combinations, but not in others.     

Alloantigen pretreatment induces a down-regulation of the in vivo subsequent development of cytotoxic T lymphocytes directed against linked alloantigens

In the present study, we describe a new regulatory system that influences the in vivo development of cytotoxic T lymphocytes (CTL) and that could be related to epitopic suppression. Epitopic suppression has been previously shown to occur when carrier-primed mice are subsequently immunized with a "new" epitope coupled to the priming carrier. The suppression specifically inhibited the antibody response to the "new" epitope without affecting the secondary antibody response to the carrier. In this report, using a carrier/hapten-carrier type of immunization protocol, we have demonstrated that a similar regulatory system could also affect the induction of CTL directed against allogeneic cells. Priming mice with an alloantigen 1 (carrier) inhibits the induction of alloantigen 2 (hapten)-specific cytotoxic responses when the alloantigen 2 is presented in association with the alloantigen 1 on an F1 stimulator cell (hapten-carrier conjugate). This has been demonstrated by the specific decrease of anti-H-2b or anti-H-2d CTL responses generated in C3H/He mice (H-2k) previously primed with, respectively, H-2d or H-2b spleen cells before immunization with F1 (H-2d x b) spleen cells. This suppression of the CTL responses against the second immunizing alloantigen is associated with a strong CTL response against the first priming alloantigen. The induction of the suppression is dependent on the dose of H-2d spleen cells administered before immunization with F1 spleen cells and is not related to antigen elimination since a strong suppression of the CTL response against H-2b antigens is shown following immunization with a mixture of F1 cells and H-2b-bearing cells of H-2d-primed animals.     

Permissible and immunogenic HLA-A mismatches: cytotoxic T-cell precursor frequencies reflect graft survival data.

Analysis of the in vivo immunogenicity of single HLA mismatches, in the context of a patient's own human leukocyte antigen (HLA) phenotype, has been used to define permissible and immunogenic HLA mismatches. Kidney graft survival in the case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. The present study tested whether such permissible and immunogenic HLA mismatches are reflected in the in vitro cytotoxic T-lymphocyte (CTL) allorepertoire. Limiting dilution experiments were performed to analyze the number of precursor CTL directed against individual HLA class I antigens. In general, the frequency of CTLp directed against permissible HLA-A antigens (n = 70, mean frequency 27 CTLp per million peripheral blood lymphocytes [PBL]) was found to be significantly lower compared with the CTLp directed against immunogenic HLA-A antigens (n = 73, mean frequency 59 CTLp per million PBL). The difference was found both in healthy individuals and a population of renal transplant candidates. These results were confirmed by a retrospective analysis of CTLp frequencies performed between partly mismatched unrelated bone marrow donors and their potential recipients. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a particular patient should be determined on an individual basis.     

Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.     

HLA-A, -B and -DR allele and haplotype frequencies in Malays

One thousand four hundreds and forty-five Malays registered with the Malaysian Marrow Donor Registry were typed for HLA-A, HLA-B and HLA-DR. Fifteen HLA-A, twenty nine HLA-B and fourteen HLA-DR alleles were detected. The most common HLA-A alleles and their frequencies were HLA-A24 (0.35), HLA-A11 (0.21) and HLA-A2 (0.15). The most common HLA-B alleles were HLA-B15 (0.26), HLA-B35 (0.11) and HLA-B18 (0.10) while the most common HLA-DR alleles were HLA-DR15 (0.28), HLA-DR12 (0.27) and HLA-DR7 (0.10). A24-B15-DR12 (0.047), A24-B15-DR15 (0.03) and the A24-B35-DR12 (0.03) were the most frequent haplotypes. This data may be useful in determining the probability of finding a matched donor and for estimating the incidence of HLA associated diseases.     

HLA-Dw/LD directed cytotoxic T cell clones

T lymphocyte clones were derived by micromanipulation from an MLC between a stimulator and responder matched for class I but mismatched for class II HLA antigens; among the possible stimulating antigens were DR4 and Dw4. Two of the clones, when tested for cytotoxicity on a panel of DR4 positive and negative targets, appeared to recognize a determinant closely associated with Dw4, but did not lyse, with one exception, targets expressing other DR4 associated Dw specificities or DR4 negative targets. Blocking studies, using monoclonal antibodies directed against monomorphic epitopes on class I or class II molecules, revealed that the cytotoxic activity of these clones was strongly inhibited by an anti-class II (L-243) but not by an anti-class I (w6/32) monoclonal antibody. Both clones were T3⁺, T4⁺, T8^?. These findings show that cytotoxic T cell clones, (directed against class II antigens), may have specificity that correlates with a T lymphocyte-defined LD/Dw determinant. The blocking experiments using monoclonal antibodies give further support to the idea that these clones recognize determinant(s) on a class II molecule. The cell surface phenotyping results are in agreement with previous reports that most anti-class II cytotoxic clones have a T4⁺ T8^? phenotype.     

HLA-A, -B and -DRB1 allele frequencies in the Bangladeshi population

Population genetic studies have become an invaluable tool because of the extreme polymorphism found at some of the loci of the human leukocyte antigen (HLA) system. In this study, we are reporting for the first time the genetic polymorphism of 141 healthy unrelated Bangladeshi Bangalees living in central region of Dhaka. We studied the HLA-A, -B and -DRB1 loci using polymerase chain reaction with sequence-specific primers. The allelic frequencies, two and three locus haplotype frequencies were statistically analyzed. A total of 16 HLA-A alleles, 26 HLA-B alleles and 14 HLA-DRB1 alleles were detected. A*33-B*44 (8.15%) was the most common two loci class 1 haplotype, whereas A*33-B*44-DRB1*07 (6.38%) was the most frequent three loci haplotype. The most common HLA-A, HLA-B and HLA-DRB1 alleles were A*33 (17.02%), B*15 (19.5%) and DRB1*15 (29.07%), respectively. Construction of phylogenetic tree using average linkage between groups and correspondence analysis showed close associations with Indian non-tribal random Dravidians, north Indian Hindus and some relations with Mongolian and Pakistani populations. We believe this data will provide useful information for bone marrow registry, legal medicine, disease association and anthropological studies.     

Inhibition of proliferative and cytotoxic activities of human T lymphocytes with rabbit antibodies directed against leucoagglutinin-reactive T cell surface components

Three rabbit antisera (870, 872 and 873) were raised against leucoagglutinin-reactive components from the surface of human T cells. The antibodies reacted with two major glycoproteins of 175 kDa and 105 kDa. None of the antibodies triggered peripheral blood lymphocytes or T cells to proliferation when tested under different culture conditions. All antibodies inhibited the proliferative response to concanavalin A or to allogeneic lymphocytes in mixed lymphocyte culture when whole IgG fractions were used. Complete inhibition of cytotoxic activity was obtained in cell-mediated lympholysis and in natural killer cell cytotoxicity (NK) when fresh peripheral blood lymphocytes were used as effector cells. Weak inhibition was also obtained in NK when mixed lymphocyte culture-activated effector cells were used. The inhibition was stronger, when NK activity was determined against MOLT4 target cells as compared to K562. Whereas F(ab')2 fragments of 873 IgG inhibited cytotoxic T lymphocyte activity completely, Fab fragments of 873 IgG neither inhibited proliferation nor cytotoxic T lymphocyte activity, but gave some inhibition of NK against MOLT4 targets. The results indicate that antibodies against these leucoagglutinin-reactive structures reacted with polypeptides similar to or identical with the human "leukocyte function-associated antigen-1" (LFA-1) considered to be an important mediator of cell-cell interactions and nonspecific adherence.     

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8(+) activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8(+) T lymphocytes and CD8(+) NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on the activity of EBV-specific CD8(+) cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I-negative cells transfected with one gene encoding either classical or non-classical HLA class I antigens. Both classical and non-classical sHLA class I molecules trigger apoptosis in CD8(+) T lymphocytes and in CD8(+) NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8(+) cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class I molecules inhibit the cytotoxic activity of EBV-specific CD8(+) CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.     

Synergistic cytotoxic effects of antibodies directed against different cell surface determinants.

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L2C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognized by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L2C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.     

HLA-A2限制性Survivin点突变肽诱导特异性抗肝癌CTL反应

目的 探讨HLA-A2限制性Survivin点突变抗原肽体外诱导CTLs的抗肝癌作用.方法 生物信息学软件BIMAS和SYFPEITHI用来鉴定点突变的HLA-A2限制性Survivin抗原九肽;流式细胞术检测突变肽与HLA-A2分子结合效率;肽体外刺激肝癌患者腹水来源的肿瘤浸润淋巴细胞(TILs)诱导反应性CTLs,用流式细胞术及ELISA检测反应性CTLs释放IFN-γ情况;肽诱导的CTLs与肝癌细胞系HepG2及BEL-7402共孵育,CytoTox 96(R)非放射性细胞毒性检测法检测对肿瘤细胞的裂解情况,倒置相差显微镜观察肝癌细胞形态学变化.结果 生物信息学分析筛选出与MHC分子结合效率评分显著提高的点突变Survivin抗原九肽Sur79M2 (KMSSGCAFL),流式细胞术检测证实负载Sur79M2的T2细胞,HLA-A2表达率显著提高,经Sur79M2体外刺激诱导的CTLs与靶细胞共孵育后能释放较高水平的IFN-γ,Sur79M2诱导的CTLs能通过HLA-A2限制性机制有效杀伤肝癌细胞系HepG2,对HLA-A2阴性的BEL-7402细胞无明显杀伤作用.结论 点突变肽Sur79M2能在体外诱导反应性CTLs产生,该CTLs能以HLA-A2限制性方式有效杀伤肝癌细胞系.     

Subtypes of HLA-A1 defined on the basis of CTL precursor frequencies

Recently we have shown that limiting dilution analysis can be used to detect cytotoxic T-cell precursor frequencies directed against individual HLA class I antigens. Using the same protocol, we have been able to define two subtypes of HLA-A1, which are indistinguishable by conventional typing sera as well as by cell-mediated lympholysis. One-dimensional isoelectric focusing analysis of the variants did not show any overall charge differences. However, family studies indicated that these HLA-A1 subtypes are genetically determined and can be distinguished on the bases of T-cell precursor frequencies in HLA-A1-negative blood donors.     

A cytotoxic monoclonal IgM antibody (Tü 101) directed against an antigenic determinant shared between the HLA-A allospecificities A2 and A28

A complement-fixing murine monoclonal IgM antibody (TÜ 101) strictly directed against a supertypic determinant present on the HLA-A locus antigens A2 and A28 was defined from a fusion experiment employing T cell blasts as immunizing cells. The specificity of this antibody was established in the microcytoxicity assay on 91 normal Caucasian blood donors, as well as in the SpA-Ig assay on a panel of lympho- and hematopoietic cell lines. TÜ 101 segregates only with its defined HLA allotypes in families. This reagent may be of particular value as a probe for analyzing a molecular relationship of different antigenic determinants on the HLA-A2 and A28 specificities in comparison with two recently defined anti-HLA-A2/A28 monoclonal antibodies and may help to characterize structural variations of these HLA-molecules on a serological and immunochemical basis.     

Limiting dilution analysis of alloreactive T helper cells: precursor frequencies similar to that of alloreactive cytotoxic T cells

Precursor frequencies for alloreactive T helper cells involved in the generation of primary cytotoxic responses from thymocytes were determined in splenic T cells and selected Lyt-1 lymphocytes by limiting dilution analysis. T helper precursors at frequencies ranging from 1/5000 to 1/13,500 were found in individual experiments in unsensitized selected Lyt-1 populations reacting to H-2 alloantigens. After preactivation of Lyt-1 lymphocytes with antigen in limiting dilution, the frequencies of T helper cells were increased 2-3 fold when cultured in the absence and 10-50-fold when cultured in the presence of T cell growth factor. The frequencies for T helper precursors found in Lyt-1 cells were comparable to those of unselected T cells, indicating that a significant portion of T helper cells resides in the Lyt-123 population. Activation of T helper precursors with H-2 antigens or with H-2 and non-MHC (plus MLs) antigens resulted in similar frequencies, suggesting that the same T cell can respond to H-2 and non-MHC determinants. The data suggest that alloreactive T helper precursors exist at frequencies similar to that of CTL precursors. In addition, the results indicate that the induction of CTL by T helper cells is subject to regulation presumably by suppressive cells and that Lyt-1 inducer cells may be involved in the development of suppression for CTL responses.     

Expression of Qat-4 and Qat-5 alloantigens on cytotoxic precursor and effector cells: different surface phenotypes of alloreactive and H-2-restricted cytotoxic T Cells

Monoclonal anti-Qat-4 and anti-Qat-5 antibodies, which define antigens expressed on peripheral T cell subsets, have been used to study the phenotypes of alloreactive and H-2-restricted cytotoxic effector cells and their precursors. Depletion of Qat-4⁺ or Qat-5⁺ cells from the T cell pool prior to their sensitization in bulk cultures prevented the development of alloreactive and H-2-restricted cytotoxic activities in the selected populations. No reconstitution of cytolytic activities to normal levels was obtained when mixtures of Qat-4⁺ and Oat-5⁺ cells were sensitized in bulk cultures to H-2 or non-H-2 antigens. Sensitization of limiting numbers of Qat-4^? or Qat-5^? lymphocytes under optimal conditions for help (interleukin 2), with the appropriate antigens (H-2, H-Y) did not result in the generation of cytotoxic T cells, indicating that the majority of all cytotoxic T lymphocyte (CTL) precursors are Qat-4⁺, Qat-5⁺. When CTL effector populations were treated with the antisera and complement (C) at their maximum CTL activity, it was found that H-2-restricted CTL were totally eliminated by anti-Qat-4 and considerably reduced by anti-Qat-5 antisera and C. In contrast, alloreactive CTL effector cells were insensitive to anti-Qat-4 and to anti-Qat-5 plus c. Although alloreactive CTL effector populations regained some Qat-4 antigens during further in vitro culture, it was shown that H-2-restricted CTL were at all times more sensitive to anti-Qat-4 than were alloreactive CTL. The findings suggest that during maturation of alloreactive and H-2-restricted CTL from their precursors, both alloantigens undergo differential quantitative variations in their expression that lead to different Qat-4,5 phenotypes of alloreactive and H-2-restricted CTL.     

Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein

To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.     

Suppression of the cytotoxic T cell response to minor alloantigens in vivo. Linked recognition by suppressor T cells

This report describes the activity of transferable suppressor T cells (Ts) generated in vivo in response to minor alloantigens. These Ts cells are antigen specific in both primary and secondary in vivo cytotoxic T lymphocyte responses to minor alloantigens and are the result of a host response rather than of a graft-vs.-host reaction. The Ts cells are produced soon after immunization and their activity is transient. They act via "linked recognition", since they can suppress the cytotoxic T lymphocyte response to noncross-reactive minor antigens, but only if these are presented on the same antigenic cell. A model for dominant low responsiveness in (high X low responder)F1 animals is proposed, whereby Ts cells, activated via the low responder allele, work by linked recognition to suppress helper cells activated via the high responder allele.