人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

Generation of cytotoxicity with various MHC class I restrictions against autologous LCL by stimulating the lymphocytes with autologous and/or allogeneic LCLs sharing HLA alleles with the responder.

We exposed human blood lymphocytes to autologous and to allogeneic lymphoblastoid lines (LCLs), each alone or in combination, and analyzed the MHC Class I restriction pattern of the generated auto-LCL reactive cytotoxicity. In the cultures of two EBV-seropositive, HLA A11-positive individuals the majority of cytotoxic lymphocytes generated after repeated stimulation with autologous LCL were restricted by this molecule. One of the cultures was subjected to various stimulation strategies. A relatively low proportion of HLA A2- and HLA B7-restricted cytotoxic T cells could be detected in the autostimulated cultures. Such cells were enriched at the expense of A11-restricted ones by stimulating with allogeneic LCLs which lacked HLA A11 but expressed A2 or B7. Interestingly, stimulation of the lymphocytes with only allogeneic LCL also generated autoreactive CTLs. Thus, by including or using exclusively allogeneic LCL stimulators, the CTL fractions represented by few cells could be enriched.     

A permanent human cytotoxic T-cell line with high killing capacity against a lymphoblastoid B-cell line shows preference for HLA A,B target antigens and lacks spontaneous cytotoxic activity

The optimal conditions for the generation of highly cytotoxic human T lymphocytes (CTL) in vitro against a lymphoblastoid B-cell line (JY) in primary and secondary mixed lymphocyte cultures (MLC) were investigated. Variation of the stimulator:responder (S:R) cell ratio influenced both the specific cytotoxicity and the spontaneous cytotoxicity as well as the recovery of the responder cells. T lymphocytes of donor JR (HLA A23,29; B7,7; DRw5) were stimulated with JY (HLA A2,2; B7,7; DRw4,6) in primary and secondary MLC at S:R ratios of 1:50 and 1:10, respectively, since stimulations at these S:R ratios resulted in the highest specific cytotoxicity against JY, the lowest spontaneous cytotoxicity against K562 and Daudi, and in a good recovery of the responder cells. From these T-lymphocyte cultures an exponentially growing CTL line (JR-2) was obtained by weekly stimulations with irradiated JY cells at a S:R ratio of 1:1. After 2 months of culturing the growth rate of the JR-2 cells declined, but could be restored by the addition of conditioned medium, containing T-cell growth factor (TCGF). Irradiated JY cells or TCGF alone were insufficient to maintain proliferation. JR-2 cells were strongly cytotoxic for JY (50% lysis was obtained at an effector:target ratio of 1:2) but the cytotoxic activity against a classical target cell for spontaneous cytotoxicity (K562) was negligible. The cytotoxic activity of JR-2 cells against JY could be inhibited by a monoclonal antiserum W6/32, which recognizes all HLA A, B, and C specificities, and by a monoclonal antiserum directed against β2 microglobulin, whereas monoclonal anti-Ia antisera showed no inhibition. JR-2 cells lysed fresh HLA A2-homozygous lymphocytes more efficiently than HLA A2-heterozygous lymphocytes, whereas the latter were better killed than HLA A2-negative lymphocytes.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

Modifications of lymphocyte HL-A antigens as a consequence of therapy in patients with manic-depressive psychosis]

In 54 (= 46.96%) of 115 patients with maniacal-depressive psychosis a HLA modification could be identified. This modification turned out to be temporary and from a serological point of view it revealed a different character. In 29 cases a loss of HLA antigens could be observed, in 3 cases there was a decrease, in 14 cases a combination of both changes, twice a polyreactivity was observed and 6 times a change of the antigen HLA-A 2 in A 28 could be determined. These serological modifications appeared after therapy with lithium as well as with various antidepressive and neuroleptic medicaments. The connection between therapy and development of HLA modification could be ensured statistically. The modifications of HLA antigens A 10 and B 7 developed after administering neuroleptic medicaments, those of HLA antigens A 9, A 11, B 12, and B 13 after therapy with antidepressive medicaments. HLA antigens B 27 and B 40 showed a relative resistance towards therapy. The significance of these findings for the possibility of mistakes in HLA typing and from the standpoint of therapy efficiency in connection with the patient's HLA phaenotype is discussed.     

Interesting relations in the HLA system

There exists no absolute binding between the antigens HLA-Cw 2, Cw 3 and Cw 4, on the one hand, and HLA-B 27, HLA-B 15 and HLA-Bw 35, on the other hand. Even if 91% of human beings with HLA Cw 4 will simultaneously have the antigen HLA-Bw 35, another antigen as HLA-B 27 or HLA-B 15 can be identified in approximately 55% of individuals with HLA-Cw 2 and Cw 3. In this connection, the joint presence of some pairs of cross-reacting HLA antigens (A 2 and A 28, B 5 and Bw 35, B 7 and B 27, B 8 and B 14, B 12 and Bw 2) could be proved and their frequency be determined. 2 cases of a simultaneous presence of two subtypes of HLA-A 10 antigen, A 25 and A 26, could be found in family examinations. Moreover, two atypical bindings of anti-HLA-Bw 4 and anti-HLA-Bw 6 cytotoxins with HLA antigens could be identified: 7,49% of HLA-Bw 35 positive lymphocytes no positive response with anti-HLA-B 4 and 1,69% of HLA-B 12 with anti-HLA Bw 6. The importance of the findings for determining HLA in practice is discussed.     

Degenerate immunogenicity of an HLA-A2-restricted hepatitis B virus nucleocapsid cytotoxic T-lymphocyte epitope that is also presented by HLA-B51

The recent identification of hepatitis B virus (HBV) epitopes restricted by multiple HLA alleles has greatly expanded the epitope repertoire available for T-cell-mediated therapeutic vaccine development. The HLA-B51-restricted peptide HBc19-27 is particularly interesting because it is located entirely within the HLA-A2-restricted HBc18-27 epitope. Here we show that HLA-B51-restricted cytotoxic T lymphocytes specific for HBc19-27 from a patient with acute HBV infection were also able to lyse HLA-B51-positive target cells pulsed with HBc18-27 and to produce gamma interferon when stimulated by that peptide, implying that HBc18-27 can be presented by HLA-B51 as well as by HLA-A2. These results demonstrate the concept of degenerate immunogenicity across HLA class supertype boundaries in a human viral disease setting. In addition, they could facilitate the development of an epitope-based therapeutic vaccine to terminate chronic HBV infection that could provide a broad and diverse population coverage with a single peptide.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

HLA system antigens in persons with differing susceptibility to the causative agents of acute respiratory diseases

The relationship between the susceptibility of the body to infections caused by influenza A and B viruses, parainfluenza viruses, adenoviruses, Mycoplasma pneumoniae and antigens of the HLA system was studied on a group of 400 adolescents placed under clinico-epidemiological surveillance for two years. The relationship between histocompatibility antigens and acute respiratory diseases was manifested in a decrease or increase in the occurrence of recurrent diseases and infections or in the probability of the development of the diseases in infected persons. HLA B40 was associated with resistance to influenza A, B18 and B21 were associated with resistance to parainfluenza, B15 and B35 were associated with resistance to M. pneumoniae infection; susceptibility to influenza B was registered in persons with HLA B12 and to M. pneumoniae infection, in persons with HLA B16 and B18. With respect to different infective agents, the relative risk of infection varied within 1.7 and 5.0.     

In vivo and in vitro modulation of HLA-DM and HLA-DO is induced by B lymphocyte activation.

Ag presentation via HLA class II molecules in B lymphocytes depends on the coordinated action of HLA-DM, the catalyst of class II-peptide loading, and HLA-DO, a pH-dependent modulator of DM, the expression of which is almost completely restricted to B lymphocytes. The relative expression levels of both class II modulators are critical for the composition of the HLA class II peptide repertoire. The data in this work demonstrate that DO and DM expression are both dependent on the cellular activation status in primary human B lymphocytes. In vivo low-density activated primary human B lymphocytes show a prominent reduction in DO and DM expression when compared with high-density resting primary B lymphocytes. In vitro, reduction of DO and DM expression can be induced by B lymphocyte activation via the B cell receptor or by use of the phorbol ester, PMA. Specific inhibition of protein kinase C resulted in a significant reduction of HLA-DO and is potentially due to protein degradation in lysosomal compartments as the phenomenon is reversed by chloroquine. Thus, the expression of the dedicated HLA class II chaperone DM and its pH-dependent modulator DO is regulated and tightly controlled by the activation status of the B lymphocyte.     

Anti-group A streptococcal vaccine epitope: structure, stability, and its ability to interact with HLA class II molecules

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue α-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 α-helix residues, whereas the B cell epitope is in the second microdomain and showed no α-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCor-overlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.     

Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus

Epstein-Barr virus is ubiquitous and is causally implicated in lymphoid and epithelial malignancies. Virus invades oropharyngeal mucosa and establishes latency in B lymphocytes. Reactivating lymphocytes shed virus into saliva for spread to new hosts. A complex of three virus glycoproteins, gH, gL and gp42, is essential for entry. B-cell entry requires binding of gp42 to human leukocyte antigen (HLA) class II whereas entry into epithelial cells lacking HLA class II requires complexes without gp42. To accommodate infection of each, the virus carries both three-part and two-part complexes. We show here that HLA class II in the virus-producing cell alters the ratio of three-part to two-part complexes. As a consequence, virus originating in epithelial cells efficiently infects B cells whereas B-cell derived virus better infects epithelial cells. This molecular switch is a novel strategy that could alter tropism of virus from epithelium to B cells and then back to epithelium in a new host.     

Effectiveness of immunizing donors with adsorbed tetanus anatoxin in relation to their genetic markers

Blood groups, including rhesus grouping, have been determined in 218 donors and HLA of loci A, B, C in 121 donors immunized with adsorbed tetanus toxoid (40 binding units). The presence of HLA Aw 32, B 8; HLA Bw 16, haplotypes A3 Bw 16 and A1, blank antigens of locus C is linked with the production of, respectively, high or low (less than or equal to 16 I.U./ml) antibody titers. The effectiveness of immunization is influenced by agglutinogen B, which is confirmed by its associations (in a complex with HLA Bw 16 or Bw 35) with low immune response.     

Antibody responses following rubella immunization analyzed by HLA and ABO types

HLA typing was performed on 232 rubella seronegative (HAI antibody titer <8) children immunized with RA 27/3 vaccine. High convalescentphase geometric mean antibody titers were noted in children one to five years of age, in girls one to 12 years of age, in subjects with AB blood type and in vaccinees with HLA types B14 and BW22. Of nine children with rubella titers512, 44 percent had HLA type A28; in contrast, only eight percent of the total group had A28 HLA type. HLA-A2 was present in four, and HLA types B12 and BW17 were present in three of the six children in whom seroconversion did not occur. Fifteen of 37 sibling pairs had identical antibody responses; in contrast, only 18 percent of randomly paired children had titers identical to each other. Vaccine-related symptomatology could not be associated with any HLA type. The results of this study suggest that infection with and antibody response to RA 27/3 rubella vaccine is influenced by genetic factors, which include cellular membrane determinants.     

A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.     

Human cytotrophoblast populations studied by monoclonal antibodies using single and double biotin-avidin-peroxidase immunocytochemistry

Single and double biotin-avidin-peroxidase immunocytochemical methods in conjunction with an anti-trophoblast monoclonal antibody 18B/A5 and an anti-HLA-A,B,C monoclonal antibody W6/32 were used to study various human trophoblast populations. Several combinations of peroxidase substrates were tried in the double-labeling procedure. It was concluded that the use of 4-chloro-1 naphthol to develop the primary sequence peroxidase and of 3-amino-9-ethyl carbazole for the second sequence peroxidase was the most suitable. The significant findings were: Monoclonal antibody 18B/A5 proved to be a useful marker for villous as well as nonvillous trophoblast, which facilitated the identification of these cells particularly in the placental bed. The expression of MHC Class I antigens was not confined to extravillous trophoblast but these antigens were also demonstrable on the villous cytotrophoblast proliferating to form new primary villi. Double labeling revealed that many of these cells, particularly those furthest away from the mesenchymal core, expressed both trophoblast and HLA antigens as shown by a mixing of the colors produced by the two reaction products. A large number of these HLA-A,B,C, positive trophoblast cells were found to infiltrate deep into the uterine myometrium. The hypothesis was put forward that these fetal cells could be the ones that are responsible for maternal sensitization.     

Host genetic factors in susceptibility to herpes simplex type 1 virus infection: contribution of polymorphic genes at the interface of innate and adaptive immunity

HSV-1 establishes life-long latency that can result in clinical relapses or in asymptomatic virus shedding. Although virtually all adults have been exposed to HSV-1, the clinical course varies remarkably. Genetic host variability could be related to this clinical diversity. In this study, we analyzed the contribution of gene families in chromosomes 1, 6, 12, and 19, which encode key regulators of the innate and adaptive immunity, in a cohort of 302 individuals. Class I and class II alleles of the HLA system, the copy-number variation of NK cell receptor genes (KIR and NKG2C), the combinations of killer cell Ig-like receptor and their HLA ligands, and CD16A and CD32A allotypes of variable affinity for IgG subclasses were all studied. Although no major susceptibility locus for HSV-1 was identified, our results show that the risk of suffering clinical HSV-1 infection is modified by MHC class I allotypes (B*18, C*15, and the group of alleles encoding A19), the high-affinity receptor/ligand pair KIR2DL2/HLA-C1, and the CD16A-158V/F dimorphism. Conversely, HLA class II and CD32A polymorphisms and NKG2C deletion did not seem to influence the clinical course of herpetic infection. Collectively, these findings support an important role in host defense against herpetic infection for several polymorphic genes implicated in adaptive immunity and in surveillance of its subversion. They confirm the crucial role of cytotoxic cells (CTL and NK) and the contribution of genetic diversity to the clinical course of HSV-1 infection.     

Detection of common determinants of HLA antigens A2 and B17 by absorption using human HLA serum

Attempts of cross absorption where sera of anti-HLA A2 + B17, anti-HLA A2 and anti-HLA B17 with thrombocytes were absorbed from donors of HLA A2 positive, B17 negative and HLA A2 negative, B17 positive, revealed that anti-HLA A2 and anti-HLA B17 could be eliminated from the sera of both HLA types on the platelets. Thus, the findings allow the existence of a common determinant of HLA A2 and B17-antigens to be assumed. This is the first case where the evidence of a cross reaction between antigens of two different HLA loci with human sera could be established.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

HLA class I polymorphism in the Albanian population

The HLA class I polymorphism was studied in a sample of the Albanian population. Ninety-three unrelated healthy Albanians were typed for HLA-A, -B and -Cw antigens by standard microlyphocytotoxicity test. The antigens with the highest frequencies were: HLA-A2 (34.4%), A3 (14.5%) and A1 (12.4%); B51 (19.3%), B35 (12.4%) and B18 (10.2%); Cw4 (16.2%), Cw7 (16.2%) and Cw6 (10.8%). The HLA haplotypes with high frequency in Albanians included A2-B51 (4.3%), A2-B18 (2.4%), A2-B35 (2.4%), Cw4-B35 (7.6%), and Cw7-B18 (6.5%), which are not significantly different from the other neighboring populations. Low frequency of HLA-A1-B8 haplotype (1.1%) is noted in the Albanian population. The frequency of HLA-B27 antigen (1.1%) is one of the lowest frequencies observed in Caucasians. Such results are important in studies of HLA-A1-B8, HLA-B27 and disease associations. These findings should be also useful in understanding the origin of Albanians, representing a base for future studies about HLA polymorphism in the Albanian population.