Microcytotoxicity Assay Research Articles

CTLA4Ig ENHANCES THE INCIDENCE OF XENOGENEIC BONE MARROW ENGRAFTMENT AND ALTERS THE CLINICAL PRESENTATION OF GRAFT-VERSUS-HOST DISEASE

534 Bone marrow transplantation (BMTx) has been shown to increase heart and liver graft survivals across both allogeneic and xenogeneic barriers. However, lower rate of engraftment and graft-versus-host disease (GVHD) are the limiting factors in the widespread clinical utility of this approach. CTLA4Ig is a fusion protein that has been used to prevent allogeneic GVHD via CD28-B7 costimulatory signal blockade. Given this observation, the role of CTLA4Ig in xenogeneic BMTx was examined in a hamster-to-rat model. Lewis rat recipients were prepared by splenectomy and total body irradiation (10.5 Gy) prior to receiving 3×108 unfractionated hamster BM cells. BMTx recipients were randomly assigned to either study (n=8) or control (n=6) groups. The study group received CTLA4Ig (0.5 mg/d IP days 0-7) while the control group received no treatment. Animals were then observed for clinical signs of GVHD. Engraftment was determined by FACS using rat-anti-hamster polyclonal antibodies as well as PCR using hamster-specific primers. On d15 after BMTx, animal sera were collected for microcytotoxicity assay against either rat or hamster lymphocytes. We also performed one-way mixed lymphocyte reactions in which rat and hamster lymphocytes were used as either responder or stimulators in order to assess T cell proliferation in the presence and absence of CTLA4Ig, MLR data showed that CTLA4Ig at the given dose suppressed both rat and hamster T cell proliferation significantly. When BMTx recipients were treated with CTLA4Ig, five (63%) rats engrafted, as compared to only two (33%) from the control group. Microcytoxicity assay showed that there was low titer of anti-rat antibody developed in one of the control animals, but this was not seen in any of the CTLA4Ig-treated animals. When examined histopathologically, animals in the study group showed extensive lymphocyte infiltration in skin while lymphocytes infiltration in lungs, kidneys and livers was variable. Of the two survivors from the control group, one had hepatocyte necrosis with minimal cellular infiltration around the bile ducts while other organs appeared normal; the other animal had cellular infiltrates in skin, bile ducts and renal collecting tubules as well as hepatocyte necrosis with polymorphonuclear cell infiltration, resembling a combined cellular and humoral injury. In this experiment, we showed that CTLA4Ig enhanced the rate of engraftment of xenogeneic BM. While CTLA4Ig did not prevent GVHD altogether, it altered its clinical presentation. The presentation of GVHD in the control animals was heterogeneous in that we observed two histologically different types of organ injuries. On the other hand, GVHD among the CTLA4Ig-treated animals was more homogenous with predominantly cellular involvement.

Analysis of HLA antigens in Japanese patients with chronic sinusitis.

Genetic factors likely play a role in the etiology of chronic sinusitis and this disease is often associated with diffuse panbronchiolitis, which is strongly associated with HLA B54 antigen. The purpose of this study is to examine whether genetic factors are involved in the pathogenesis of chronic sinusitis. Eighty-two Japanese patients with intractable chronic sinusitis were selected on the basis of the following criteria: 1) persistent mucous or mucopurulent nasal discharge and/or postnasal dripping for longer than 3 years and 2) opacification in bilateral maxillary sinuses and ethmoid cells in plain X-ray films. Both class I and class II HLA antigens were analyzed by conventional microcytotoxicity assays in these patients and 176 healthy control subjects. In class I antigens, B54 antigen significantly increased in the patient group (antigen frequency = 29.3%, relative risk = 3.23, corrected P value = .037) compared with normal control group (antigen frequency = 11.4%). For class II antigens, no antigens were significantly increased. These data indicate that certain genetic factors play a role in the etiology of chronic sinusitis.

A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of transplanted pig organs. The purpose of this study was to establish a suitable in vitro method that would allow for screening and comparison of various agents and methods potentially useful in the prevention of hyperacute rejection. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG08472), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytotoxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conventional 51 Chromium ( 51 Cr )-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotoxicity assay performed in Terasaki trays. Using the microcytotoxicity assay, a 1-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studied, PK15 cells were the most sensitive to cytotoxic injury, followed by AG cells and the primary endothelial culture. Good correlation between the 51 Cr -release and the two-color fluorescence method was documented. There was good agreement between the results obtained using the 2-chambered slide method and the microcytotoxicity assay, as there was between the 1- and the 2-step assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluorescence assay is suitable for the rapid and inexpensive screening of therapeutic interventions that might be useful in the prevention of hyperacute xenograft rejection, and that PK15 cells are suitable for use in this assay.

The influence of ABO blood groups on sensitization of potential kidney transplant recipients

In this study 50 chronic renal failurepatients were tested for blood ABO groups and forthe presence of lymphocytotoxic antibodies against apanel of 20 donor lymphocytes (of known HLAtypes) using microcytotoxicity assay. The influenceof other factors affecting sensitization, such asnumber of blood transfusions, pregnancies andprevious graft rejections were analyzed too. Theresults showed that41.2 % of blood group 0 patients,61.1 % of group A I, 90% of group B, and 80% ofgroup A IB are sensitized (PRA> 10%).These results pointed to higher incidence ofsensitization in patients with blood groups B andA IB as compared to groups A I and 0.Our data suggest an impact of the ABO system on thesensitization phenomenon.

Identification of a 25-aminoacid sequence from the major African swine fever virus structural protein VP72 recognised by porcine cytotoxic T lymphocytes using a lipoprotein based expression system

Identification of African swine fever virus (ASFV) proteins recognised by cytotoxic T lymphocytes (CTL) from swine surviving ASFV/NH/P68 infection was assessed using expression vectors based on the Pseudomonas aeruginosa outer membrane lipoprotein I gene ( oprI). Viral antigens expressed as fusion lipoproteins were shown to be taken efficiently by porcine blood-derived macrophages incubated with outer membrane protein preparations from transformed E. coli. To assess recognition by CTL the fusion lipoprotein-treated macrophages were used as targets in 51Cr release microcytotoxicity assays. Using this approach it was shown that the aminoacid sequence HKPHQSKPILTDENDTQRTCSHTNP from the major structural ASFV protein (VP72), encoded by a recombinant clone (pVUB72) is presented by macrophages, which are lysed under restriction of SLA class I antigens. Overall, the results demonstrate that the oprI based vectors are valuable tools to study ASFV-specific CTL activity.

THE USE OF MHC CLASS II-DEFICIENT MICE TO DETERMINE THE ROLE OF DIRECT VS INDIRECT ANTIGEN PRESENTATION IN THE PATHOGENESIS OF CHRONIC REJECTION

14 The relative contributions of direct vs indirect donor alloantigen presentation to recipients' T cells on the development of chronic rejection (CR) is as yet ambiguous. It has been argued that while direct allorecognition may play an important role in initiating acute cellular rejection (ACR); CR on the contrary, is predominantly an outcome of donor allopeptides being presented in the context of recipient MHC. Given that CR is one of the two most common causes for late organ allograft failure, it is somewhat imperative that the prevailing conundrum concerning its pathogenesis be resolved. To address unequivocally this issue, we transplanted aortas (AO) from C57B1/6 Aβb (H2b, MHC class II-1-)→naive C3H (H2k) recipients (Group V; n=6). It was reasoned that due to lack of MHC class II expression in the donor tissue, the antigen (Ag) presentation would be primarily indirect allowing us to study its role in the development of CR. Additionally, the role of direct Ag presentation in the pathogenesis of this lesion was also studied by transplanting AO from naive C3H→C57Bl/6 Aβb recipients (Group VI; n=6). Controls (n=6/group) included Tx of AO from C3H → C3H (Group 1), C57Bl/6 Aβb→C57Bl/6 Aβb (Group II), C3H→C57Bl/6 WT (Group III) and C57Bl/6 WT→C3H (Group IV). As published previously, these aortic allografts undergo self-resolving ACR with spontaneous acceptance, eventually developing changes pathognomonic of CR by d30 post-Tx. At the time of sacrifice (d30-40 post-Tx), the grafts were harvested for immunohistochemical analysis and the sera and splenocytes were also recovered for the determination of α-donor antibody and proliferative responses by complement-dependent microcytotoxicity and MLR assays respectively. Unlike that from Groups I and II, AO harvested from animals in Groups III and IV had marked circumferential intimal thickening due largely to proliferation of α-smooth muscle actin✦ (α-smA✦) cells. Additionally, both the α-donor and α-third party proliferative responses and the titers of cytotoxic antibodies remained comparable in animals in Groups III and IV. On the contrary, AO obtained from the majority of Group V animals exhibited marked reduction in intimal thickening which was largely eccentric and patchy with preservation of elastic membranes; this finding was associated with unmitigated α-donor proliferative and humoral responses. Interestingly, despite the lack of α-donor proliferative and cytotoxic antibody responses, circumferential intimal thickening of moderate severity was witnessed in the majority of aortic allografts harvested from Group VI recipients. Taken together, these data suggest that despite preservation of α-donor immune responses, presentation of Ag by indirect pathway alone does not precipitate the fulminant development of CR indicating that perhaps both pathways of Ag presentation are required for optimal immune activation. More importantly, the presence of moderate CR in recipients which lack α-donor cellular and humoral responses underscore the importance of numerous immune and non-immune pathways which operate in concert towards the development of this lesion.

T CELL ANERGY BUT NOT DELETION IS THE PREDOMINANT MECHANISM FOR INDEFINITE ISLET ALLOGRAFT SURVIVAL IN RECIPIENTS TREATED WITH rhCTLA4-Ig AND ??-gp39 ANTIBODY

48 We had previously reported that in mice, combined simultaneous blockade of costimulatory pathways by short perioperative treatment with rhCTLA4-Ig fusion protein and α-gp39 mAb results in indefinite islet allograft survival with reversal of streptozotocin (STZ)-induced diabetes. Furthermore, islet allograft survival was shown to be comparable in controls and in animals treated with either rhCTLA4-Ig or α-gp39 mAb alone, suggesting that combined simultaneous but not distinct blockade of CD28/B7 and CD40/gp39 costimulatory pathways is imperative for prolonging allograft survival. To ascertain the mechanism underlying these observations, we proceeded to transplant islets isolated from the pancreata of DBA/2 (H2d, Mlsd, Vβ6-CD4+) animals under the kidney capsule of STZ-treated diabetic MHC and MIs-disparate C3H (H2k, Mlsc, Vβ6+CD4+) recipients. The rationale for selecting this strain combination was triggered by the observation that in the donor but not the recipient, Vβ6+CD4+ were deleted during ontogeny thus prompting us to suggest that perhaps this could be used as a surrogate marker to determine the mechanism of T cell tolerance in these animals. In addition to MLR and complement-dependent microcytotoxicity assays, the proliferative status and the presence or absence of Vβ6+CD4+ T cells in the peripheral blood and splenocytes of the recipients was also determined by antibody-mediated TcR crosslinking and flow cytometric analysis respectively. The recipient treatment was as follows: Group I (n=4), isotype control mAb; Group II (n=6), anti-gp39 mAb (250µg/d on d0,2 and 4 post-Tx, i.m); Group III (n=5), rhCTLA4-Ig (200µg on d0,2,4 and 6 post-Tx, i.p) and Group IV (n=6), rhCTLA4-Ig + anti-gp39 mAb. All in vitro analysis were performed using samples obtained from the recipients either after islet allograft rejection (Groups I, II and III) or else at d81 post-Tx (Group IV). While donor-specific proliferative responses and cytotoxic antibody titers remained unchanged in Groups I, II and III, they were markedly reduced in animals in Group IV. These findings were corroborated by TcR crosslinking assays, in which the use of α-Vβ6 mAb resulted in marked proliferation of cells obtained from animals in Groups I, II and III; markedly reduced proliferation was witnessed in cells obtained from Group IV animals. Interestingly, flow cytometric analysis of T cell repertoire both in the PBMC and splenocytes of islet allograft recipients in all four groups revealed the presence of an equivalent number (∼8-9%) of donor-reactive Vβ6+CD4+ T cells arguing against deletion being the mechanism of the observed T cell tolerance. Take together, these preliminary data suggest that T cell anergy is perhaps the predominant mechanism for the indefinite islet allograft survival witnessed in animals subjected to combined simultaneous blockade of CD28/B7 and CD40/gp39 costimulatory pathways.

HLA antibodies present in the sera of sensitized patients awaiting renal transplant are also reactive to swine leukocyte antigens.

To determine whether preformed HLA alloantibodies present in the sera of patients awaiting kidney transplantation will be detrimental to a potential porcine xenograft, we tested their cross-reactivity to swine leukocyte antigens (SLA). Sera obtained from patients with varying levels of HLA sensitization (high panel-reactive antibodies > 70%, n= 7; moderate panel-reactive antibodies 30-40%, n=2) were analyzed. Pooled normal human AB sera and sera from nonsensitized patients (n=3) served as negative control. IgG was purified by protein-G chromatography, and xenoreactive natural antibodies (XNA) were depleted by passing the IgG through a series of melibiose and thyroglobulin-agarose columns. The elimination of XNA from HLA IgG preparations was confirmed by GS-IB4 lectin blocking assay and by an ELISA. IgG isolated from normal AB serum and three nonsensitized patients, which was depleted of XNA (HLA-IgG), did not react to human or porcine lymphocytes (peripheral blood mononuclear cells; PBMC) either by flow cytometry or by complement-dependent microcytotoxicity assays. However, HLA-IgG isolated from nine sensitized patients were reactive to a panel of porcine peripheral blood lymphocytes (n=6) by flow cytometry (>50 mean channel shift) and in complement-dependent microcytotoxicity assays in addition to their reactivity to human PBMC. The binding of HLA-IgG to porcine PBMC was significantly reduced by preabsorption with pooled human platelet concentrate. Further, the HLA IgG showed recognition of 45-kDa affinity-purified SLA class I on Western blots. This study demonstrates that HLA antibodies present in the sera of sensitized individuals can cross-react with SLA. Thus, xenotransplantation of porcine organs into HLA-sensitized patients has the potential to be rejected by humoral mechanisms. Testing to avoid such cross-reactive antibodies should be considered.

Higher Frequency of Selective Losses of HLA-A and -B Allospecificities in Metastasis Than in Primary Melanoma Lesions

Expression of HLA class I molecules is essential for the recognition of tumor cells by CD8+ T cells. In this study, 48 bioptic samples of 42 patients in all stages of melanoma were investigated after short-time cultivation of tumor cells. To confirm melanocytic origin of cultured cells, samples were screened for mRNA expression of melanoma markers gp100, tyrosinase, MAGE-3, MelanA, and MUC18 by reverse transcriptase-polymerase chain reaction. Surface expression of specific HLA-A and -B allospecificities on melanoma cells were analyzed with a standard microcytotoxicity assay after stimulation with interferon (IFN)-alpha and compared with the background found in peripheral blood mononuclear cells from the corresponding patients. Immunohistochemistry and flow cytometry confirmed specific losses in cases where the appropriate monoclonal antibodies were available. The level of expression of HLA-I, HLA-II, and intercellular adhesion molecule 1 antigens on melanoma cells cultured in the presence or absence of IFN-alpha and IFN-gamma was determined cytofluorometrically. All cell cultures tested were found to be positive for one or more melanocytic markers by reverse transcriptase-polymerase chain reaction. The specific HLA-I alleles on the cultured cells were detectable in 45 of 48 samples. In 11 cases a specific loss of one HLA-I allele was observed (2 x A2, B7, B8, B18, 4XB44, B47, B49). Ten of these samples were derived from locoregional lymphnode metastases or from distant metastatic tumors. Only one sample from a primary melanoma showed a specific loss of HLA-I (B47). IFN-alpha upregulated expression of HLA-I up to 4-fold. IFN-gamma enhanced the appearance of HLA-II up to 35-fold and the expression of intercellular adhesion molecule 1 up to 40-fold. Selective loss of HLA-I allospecificities might be a major step in tumor progression.

CREATION OF MIXED BONE MARROW CHIMERA BY SUBLETHAL IRRADIATION AND COMBINED BLOCKADE OF CD28/B7 AND CD40/CD40L COSTIMULATORY PATHWAYS

316 The chronic use of nonspecific immunosuppression to prevent allograft rejection is invariably associated with undesirable complications which include opportunistic infection, a high incidence of malignancies and chronic rejection. However, it has been unequivocally demonstrated that establishment of mixed chimerism by prior bone marrow transplantation (BMTx) confers to the recipient a state of donor-specific tolerance allowing for subsequent acceptance of allografts from donor but not third-party strain animals. Despite the obvious benefit, no ideal preparative regimen currently exists which would result in widespread clinical utility of this strategy. We therefore, proceeded to address this issue by transplanting 2×107 unmodified BM cells harvested from the tibia and femur of CBA/J(H-2k, Mlsd) donors into BALB/c (H-2d, Mlsc) recipients. Depending on the conditioning regimen employed in the recipients prior to Tx, the animals were divided into the following groups: Group 1 (n=5) total body irradiation (TBI; 3cGy); Group II (n=4) CTLA4-Ig fusion protein (250µg/d on d0, 2, 4 & 6 post-Tx; i.p.) + anti-CD40L mAb(250µg on d0, 2, & 4 post-Tx; i.m.); Group III (n=5) TBI + CTLA4-Ig + anti-CD40L mAb. Animals in all three groups remained healthy throughout the duration of their follow-u without any clinical evidence of GVHD suggesting that this conditioning regimen was well tolerated. Using monoclonal antibodies specific for donor MHC (H-2k), no chimeric cells were detectable by three-color flow cytometry in the lymphoid organs and in the peripheral blood of animals in Groups I and II. Additionally, animals in these two groups also rejected subsequently transplanted donor and third-party skin allografts. On the contrary, 2/5 (40%) animals in Group III developed sustained macrochimerism with donor cell levels of 36% and 31% in their peripheral blood at d28 and 45 respectively, post-Tx. This was associated with acceptance in these animals of skin allografts from donor but not third-party strain animals. Furthermore, the absence of donor-reactive Vβ6+ CD4+ T cells in these recipients was suggestive that clonal deletion was perhaps the predominant mechanism underlying the induction of persistent donor-specific tolerance. The lack of anti-donor humoral responses in these animals as determined by complement-dependent microcytotoxicity assays was as yet another evidence for the lack of T cell-mediated B cell help in the generation of an effective antibody response. In conclusion, these preliminary data suggest that mixed chimerism along with robust donor-specific tolerance can be reliably achieved by sublethal TBI and transient blockade of costimulatory pathways. This relatively benign preconditioning regimen which was free of any apparent toxicity is deemed to have widespread clinical application in organ transplantation.

HLA-DP Epitope Typing Using Monoclonal Antibodies

We have made a panel of murine anti-DP monoclonal antibodies for serological typing of HLA-DP polymorphisms; they can be used in microcytotoxicity (for 7 epitopes) and binding assays (for 8 epitopes). The antibodies detect polymorphic differences in both alpha and beta chains. As immunogens we sometimes used B-lymphoblastoid lines or purified DP molecules but mostly used mouse fibroblast transfectants expressing DP molecules. The DP beta genes were made from a cloned DPB1∗0201 gene by replacing its major area of polymorphism with matching stretches of DNA amplified from other alleles; cloned DPA1∗01 and DPA1∗02 genes were used for transfection along with the beta chain genes. The monoclonal antibodies showed reaction patterns that correlated with the presence of particular amino-acid sequence motifs; thus none of the antibodies is allele-specific. They bind instead to epitopes which are found on a number of different HLA-DP types. We have constructed frequency tables so that the epitope (motif) data can be interpreted as the most likely genotype in each case. The basic assumption to justify this work is that HLA-DP matching or mismatching will likely influence transplant outcome, particularly in bone marrow transplantation. The present challenge is to define permissive and non-permissive combinations of HLA-DP; it may be that matching for epitopes, rather than for full alleles, will help to resolve this issue.

Synergistic effect of donor-specific blood transfusion and a short course of deoxyspergualin in rat kidney transplantation

Abstract Abstract Deoxyspergualin (DSG), an analogue of spergualin produced by B. larerosporus, has a strong im-munosuppressive effect in various transplantation models. We have in vestigated the mechanism of donor-specific prolongation of survival time in rat kidney grafting by donor-specific blood transfusion (DST) and a short course of DSG. Lewis (LEW) kidney allografts were transplanted into fully allogeneic BN rats. Fresh, whole LEW blood 1.0 ml, was injected iv. into BN rats 2 days prior to transplantation. Then, DSG, 6 mg/kg per day, was administered by in. injection on days 0,1, and 2 after transplantation. The recipients were divided into five groups: group 1 (n = 6) no treatment: group 2 (n = 6) DST only; group 3 (n= 7) DSG only: group 4 (n = 7) DST and DSG: and group 5 (n= 6), third party (ACI rats) blood transfusion and DSG. Lymphocytes (cervical lymph nodes) and serum were harvested from BN recipients on day 7 postgrafting. For suppressor cell assays, lymphocytes from BN recipients in each group were added as a third cell to the mixed lymphocyte reaction (MLC) between nontransplanted BN lympho cytes (responder) and LEW or other third party (PVGC, ACI, WKA rats) lymphocytes (stimulator). An-tidonor lymphocytotoxic antibody (ADLA) was checked by microcy-totoxicity assays. Median survival times (MST) for each group were: group 1, 10 days; group 2, 10 days; group 3,13 days; group 4, 75 days; and group 5,13 days. Remarkable prolongation of MST was only noted in group 4. In the suppressor cell as say, group 4 showed significant sup pression (40 %: P < 0.05); the other groups did not show any suppression. This suppressive activity in group 4 was effective only during the MLC between BN and LEW, not during the MLC of third party-BN combinations. Thus, suppressor cells from DST/DSG-treated BN recipients appear to be donor-specific. In the microcytotoxicity assay, the only group that showed any ADLA was group 2, which was not treated with DSG. These results clearly show that both induction of donor-specific suppressor cells and inhibition of ADLA production are associated with the remarkable donor-specific prolongation of kidney allograft survival in DST/DSG-treated recipients.

Strong association of dermatomyositis-specific Mi-2 autoantibodies with a tryptophan at position 9 of the HLA-DR beta chain.

To characterize the clinical and immunogenetic features of patients with Mi-2 autoantibodies. Eighteen adult white patients with Mi-2 antibodies were clinically characterized and compared with 41 Mi-2-negative dermatomyositis (DM) patients. HLA class I and class II typing for DRB alleles was done by microcytotoxicity assay and for DQA and DQB alleles by polymerase chain reaction-based oligotyping. Seventeen of the 18 Mi-2-positive patients had DM. Symptoms of scleroderma, lung involvement, and arthritis were less common in this group than in the Mi-2-negative DM patients; the V-sign rash and nailfold involvement were found more frequently. Mi-2 antibodies were strongly associated with HLA-DR7 (88% versus 24% in healthy controls), HLA-DQA1*0201 (86% versus 23%), and DR7 "homozygosity" (31% versus 0%). A tryptophan residue at position 9 of the HLA-DR beta chain was present in all Mi-2-positive patients (100% versus 62%; homozygous in 81% versus 15%). Our results reemphasize the specificity of Mi-2 antibodies for DM, and extend previous reports that Mi-2 antibody production is associated with certain HLA class II antigens. We propose beta 9-Trp as a candidate epitope on the HLA-DR beta chain as a prerequisite for this type of autoimmune response.

Vogt‐Koyanagi‐Harada syndrome in patients of Vietnamese ancestry

Vogt-Koyanagi-Harada syndrome (VKH), is an idiopathic, multisystem inflammatory disorder primarily involving the eye. HLA typing has shown a strong association between the HLA-DR4 antigen and people of Japanese, Han Chinese and Hispanic ancestry with VKH. This study reviewed the clinical features and HLA typing of Vietnamese patients with VKH. A retrospective review of four unrelated Vietnamese patients with VKH seen in private practice and hospital clinic. The American Uveitis Society (1978) criteria for VKH diagnosis were satisfied. Standard microcytotoxic assays for Class I antigens and HLA-DNA typing of Class II DR antigens (DRB1 genotyping) by the PCR-SSO method were performed. The clinical features of VKH in Vietnamese were comparable to those seen in other Asian races. HLA-DR4 was present in three of the four VKH patients. Two of these patients also expressed the allele DRB1*0405. The strong association between HLA-DR4 and the DRB1*0405 allele and VKH seen in Japanese people, may well also exist in Vietnamese people. The HLA association suggests an immunogenetic predisposition to VKH.

The Humoral Response to Vascular and Nonvascular Allografts of Bone

The cytotoxic donor specific antibody response after vascularized and nonvascularized bone allograft implantation was assessed in rats and dogs. Nonvascularized segmental femoral grafts were studied in rats; nonvascularized fresh and cryopreserved massive osteochondral allografts were studied in dogs; and vascularized and nonvascularized fibular allografts were studied in dogs. The major histocompatibility complex antigens of all animals were defined. All grafts were stabilized by internal fixation and the antibody response was measured in a 51chromium release microcytotoxicity assay using donor lymphocytes as target cells. In all cases, donor specific antibody responses were elicited by major histocompatibility complex mismatched grafts. The response was directed primarily at Class I specificities although there was likely and antiClass II response as well. Among fully mismatched grafts, antidonor antibody was detectable earlier in animals receiving vascularized grafts (1 week after surgery) than in animals receiving nonvascularized grafts (3 weeks after surgery). Massive grafts elicited a sustained response whereas relatively smaller grafts, such as the fibula did not. The antidonor antigen antibody response was transient and less frequent in animals receiving frozen grafts. The clinical implications of these data are unclear. Although some improvement of clinical outcome has been observed with grafts matched for major histocompatibility complex antigens, the potential benefits of tissue antigen matching or modulation of the host immune response remain unresolved.

CD13-specific autoimmunity in cytomegalovirus-infected immunocompromised patients.

Cytomegalovirus (CMV) infection has been suggested to be associated with various autoimmune manifestations, such as hemolytic anemia, granulocytopenia, and the formation of autoantibodies. Earlier we found that CMV is associated with a human protein, CD13 (aminopeptidase N), emanating from CMV-infected cells and serving an important function during CMV infection of susceptible cells. We hypothesized that CD13 might become immunogenic if presented to the immune system as a part of the CMV virion. The presence of CD13-specific antibodies was tested using a microcytotoxicity assay against CD13-positive human monocytes, or by flow cytometric assays against mouse cells transfected with human CD13; specificity was assessed by specific blocking with monoclonal antibodies. CD13 reactivity was also demonstrated in immunoprecipitation experiments. CD13-specific antibodies were identified in 15 of 33 bone marrow transplant patients, but exclusively in patients who had experienced either CMV disease (9/10) or CMV viremia (6/9), and appeared at the time of CMV detection. None of the remaining 14 patients without signs of CMV infection were positive for CD13 antibodies (P<0.0001). No antibody of this specificity was found in any of the control individuals (0/24), including patients with various autoimmune diseases, CMV-seropositive or -seronegative healthy individuals, and patients with acute EBV or HSV-1 infections. Thus, the CMV-associated autoantigen CD13 is immunogenic during CMV infection in bone marrow transplant patients. A specific response against autoantigens associated with infectious virus particles is suggested as a new and general mechanism to explain virus-induced autoimmune manifestations in man.

An assay for the identification of antigens recognized by cytotoxic T cells, based on transient transfection of COS cells

The studies reported here describe methodology permitting the direct identification of antigens recognized by cytotoxic T lymphocytes. We demonstrated that bovine alloreactive CTL can detect a bovine MHC molecule transiently expressed in a COS cell population in a standard microcytotoxicity assay. We then showed that alloreactive CTL can detect cells expressing the bovine class I MHC molecule in a population of cells transfected with the plasmid containing the corresponding gene plus 100-fold as many plasmids containing an irrelevant gene. In addition, the transiently transfected COS cells can specifically restimulate CTL as detected by a standard microcytotoxicity assay using the target cell line. Overall, the results suggest that COS cells could be employed for the direct screening of an antigen or antigen gene library by immune CTL.

STUDY OF HLA CLASS I/II AND T LYMPHOCYTE SUBSETS IN KUWAITI VITILIGO PATIENTS

HLA polymorphisms of class I and class II MHC were investigated in 40 Kuwaiti vitiligo patients and in 40 controls using microcytotoxicity assay. HLA-B21, Cw6 and DR53 were increased significantly in patients compared to controls (P = 0.00001, 0.00001 and P = 0.0053 respectively) while HLA-A19, DR52, were significantly decreased (P = 0.00236, 0.05, respectively). Total T-cells, T4 and T8 were measured as CD2, CD4 and CD8 respectively by flow cytometry. Vitiligo patients showed significant increase in CD4 compared to controls (P = 0.03). Our findings suggest that HLA-B21 and Cw6 and DR53, are susceptible genes of vitiligo, while A19 and DR52 are protective genes in the Kuwaiti population.

Pre-transplant anti-epithelial cell antibodies and graft failure after single lung transplantation

Eighty-five patients undergoing single lung transplantation have been studied to determine the presence of anti-epithelial cell antibodies (AECA) prior to transplantation using the human lung carcinoma epithelial cell line A549 in a microcytotoxicity assay. In addition, 29 healthy volunteers were also assayed for the presence of AECA. Twenty-seven of the 85 recipients exhibited AECA prior to transplantation compared to none of the 29 control subjects ( p = 0.0001). Actuarial graft survival at 1 year was 78% for the AECA negative group compared to 56% for AECA positive recipients ( p = 0.01). No correlation was seen between the presence of AECA and graft rejection as determined by transbronchial biopsy. However, there was an association between AECA and post-transplant infection ( p = NS) where 16 (64%) of the AECA positive recipients had postoperative infection episodes compared to 25 (47%) of the negative recipients. Sodium dodecylsulphate polyacrylamide gel electrophoresis and Western blotting was also performed for 68 of the recipients and antibody reactivity was detected in 22 patients compared to 26 patients exhibiting AECA detectable by microcytotoxicity. The presence of AECA demonstrable by Western blotting did not correlate with graft survival, rejection or infection. In conclusion, AECA detectable prior to single lung transplantation are associated with a decrease in graft survival and with postoperative infections.

NATURAL-KILLER CELL-MEDIATED CYTOTOXICITY OF BLOOD-LYMPHOCYTES FROM PATIENTS WITH MALIGNANT MESOTHELIOMA TREATED BY INTRAPLEURAL INTERLEUKIN-2

The most impressive biological effect of recombinant Interleukin-2 (rIL-2) is the generation of nonspecific killer cells that have lytic activity for a variety of tumor cells. Numerous studies have shown that these non specific killer cells might be of NK cell lineage even though they are different from resident NK-cell. We have examined the kinetics of the NK cell-mediated cytotoxicity of blood lymphocytes in patients after intrapleural rIL-2 administered for the treatment of pleural cancer. Escalating doses of rIL-2 were administered by intrapleural route to treat 11 patients with malignant pleural effusions due to malignant pleural mesothelioma (4 stage I, 4 stage II, 2 stage III, 1 stage IV). Two patients received respectively 3 cycles and 2 cycles of treatment. Peripheral blood lymphocyte cytotoxicity was assessed by an in vitro, chromium release microcytotoxicity assay against K562 cell line. Preliminary results indicate: (i) an important and prolonged increase in the cytotoxic response of blood lymphocytes in all patients but one having a clinical response and (ii) a lack of cytotoxicity or a baseline cytotoxic response of blood lymphocytes in all patients but one with no clinical response. These results likely point out the significance of NK-activity in the IL-2-induced antitumoral response and the interest of this in vitro assay for screening patients for further cycles of treatment.