Brian Duffy

Peripheral blood microchimerism in human liver and renal transplant recipients : Rejection despite donor-specific chimerism

BACKGROUND Development of donor-specific microchimerism (DSM) has been proposed as one of the possible mechanisms for induction and maintenance of allograft tolerance. The aim of this study was to determine: (1) the state of DSM in liver transplant (LTx) and renal transplant (RTx) recipients, (2) whether the persistent presence of an allograft is a requirement for maintenance of chimerism, and (3) whether donor-specific blood transfusions (DST) facilitate chimerism development in RTx recipients and whether this correlates with allograft function. METHODS Qualitative and quantitative analysis of DSM in peripheral blood of LTx and RTx recipients was assessed by polymerase chain reaction and competitive polymerase chain reaction using HLA-DR probes for mismatched antigens between the donor and recipient. RESULTS LTx recipients (11 of 12) who had or were having rejection were positive for DSM in circulation compared with 4 of 11 with normal allograft function (P<0.01). The number of donor cells did not correlate with allograft function. LTx recipients (4 of 4) who lost their first allograft and underwent retransplantation retained DSM for the first donors. RTx recipients who received DST (8 of 8) were positive for DSM compared with 6 of 12 of nontransfused recipients (P<0.045). CONCLUSIONS The results suggest that LTx and RTx recipients undergo rejection despite DSM. The development of DSM may not be a prerequisite for normal allograft function. Once DSM is established, the presence of the allograft is not required for maintenance of chimerism. DST facilitated the development of DSM in RTx recipients. Direct correlation was not observed between the development of DSM and allograft function in either DST or nontransfused RTx recipients.

The association of HLA class II with pars planitis.

PURPOSE To investigate the association of HLA class II alleles with pars planitis. METHODS Blood samples were obtained from 28 patients with pars planitis seen in the Department of Ophthalmology and Visual Sciences and the Barnes Retina Institute at Washington University, St. Louis, Missouri. RESULTS HLA-DR15, one of the allelic subtypes of HLA-DR2, was present in 18 (64.3%) of 28 patients vs. 10 (20%) of 50 controls (OR = 7.20, CI = 2.28--23.20, P =.0001). HLA-DR51 (HLA-DRB 5) was present in 16 (57.1%) of 28 patients vs. 6 (12%) of 50 controls (OR = 9.78, CI = 2.79--36.42, P =.0001). HLA-DR17 was present in eight (28.6%) of 28 patients vs. one (2%) of 50 controls (OR = 19.60, CI = 2.29--886.7, P =.0001). CONCLUSION Pars planitis is associated with an increased frequency of the HLA-DR2 suballele, -DR15, HLA-DR51, and HLA-DR17. These results suggest an immunogenic predisposition exists to pars planitis.

ATHLATES: accurate typing of human leukocyte antigen through exome sequencing

Human leukocyte antigen (HLA) typing at the allelic level can in theory be achieved using whole exome sequencing (exome-seq) data with no added cost but has been hindered by its computational challenge. We developed ATHLATES, a program that applies assembly, allele identification and allelic pair inference to short read sequences, and applied it to data from Illumina platforms. In 15 data sets with adequate coverage for HLA-A, -B, -C, -DRB1 and -DQB1 genes, ATHLATES correctly reported 74 out of 75 allelic pairs with an overall concordance rate of 99% compared with conventional typing. This novel approach should be broadly applicable to research and clinical laboratories.

Cytolytic effector mechanisms of human CD4+ cytotoxic T lymphocytes

To elucidate mechanisms by which human CD4+ cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4+ clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4+ CTLs, whereas the absence of extracellular Ca+2 or the presence of Ca+2 channel blockers partially inhibited cytotoxicity. CD4+ CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4+ clones synthesized perforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4+ clones: (1) a Ca+2-dependent mechanism associated with the cytotoxic granules and (2) a Ca+2-insensitive mechanism.

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

BACKGROUND 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). METHODS 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. RESULTS 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. CONCLUSIONS 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.

Airway epithelial cell damage mediated by antigen-specific T cells: implications in lung allograft rejection.

The aim of this study is to assess the mechanisms associated with airway epithelial cell (AEC) injury, which may have implications in lung allograft rejection. Three AEC lines, KDI-650, Beas-2B and A549 were analyzed. Effect of cytokines on the expression of Fas, HLA class I, and HLA class II were assessed by flow cytometry. AEC-specific T cells were generated in vitro and assessed for lysis by (51)Cr release assay. HLA class I and Fas were expressed on all AEC lines. Beas-2B and A549 expressed low levels of class II compared with KDI-650, which lack this expression. Expression of HLA class II was augmented on KDI-650 and Beas-2B by IFN-gamma treatment. AEC-specific T cells generated in vitro were predominantly CD8(+) and lysed relevant AEC targets. Anti-HLA class I monoclonal antibodies inhibited the lysis of AEC by specific T cells while anti-Fas and anti-HLA class II monoclonal antibodies did not have any effect on the T cell induced lysis of AECs. AECs cultured with supernatant derived from T-cell cultures induced the expression of Fas, HLA class I, as well as HLA class II. These results suggest AEC damage is mediated by AEC-specific T cells primarily by the conventional HLA class I/peptide complex and TCR interaction. Further, the factors released by these T cells also induce the expression of Fas, as well as HLA class I and class II, which may have implications on the outcome of the immune response against AECs.

Lack of T-cell tolerance of noninherited maternal HLA antigens in normal humans

Recent clinical reports of nonresponsiveness to noninherited maternal human leukocyte antigens have led to speculation that humans may acquire tolerance of noninherited maternal antigens through exposure to maternal cells neonatally or in utero. To test this hypothesis, we measured the responsiveness of normal subjects to their noninherited maternal and paternal antigens using cell-mediated lympholysis assays and mixed leukocyte reactions. All individuals exhibited cell-mediated lympholysis and mixed leukocyte reaction responses to the maternal cells that were comparable to those to the paternal cells. Limiting dilution analyses revealed significant cytotoxic T-lymphocyte precursor frequencies to both sets of parental antigens. To exclude the possibility that tolerance of individual noninherited maternal antigens was masked by the response to other antigens expressed on the same target cell, we raised cytotoxic T lymphocytes to the maternal cells and then tested for reactivity to a panel of targets that expressed single noninherited maternal HLA antigens. In all cases, each noninherited maternal antigen expressed on the maternal cells elicited a significant cell-mediated lympholysis response. An analysis of clinical data showed that pretransplant mixed lymphocyte reactions to maternal cells are not significantly lower than those to paternal cells. These data suggest that the reported B-cell tolerance of noninherited maternal antigens is not mediated by clonal deletion of T cells induced by exposure to the maternal cells neonatally or in utero.

Chimerism in peripheral blood of sensitized patients waiting for renal transplantation: clinical implications.

BACKGROUND Potential renal transplant recipients with preformed antibodies to HLA resulting from previous transplants, pregnancy, and/or transfusions are unlikely to receive an allograft. The factors contributing to the long-term maintenance of antibody titers in these individuals are still unknown. In the present study, we sought to determine whether chimerism in the blood correlates with maintenance of HLA sensitization in highly sensitized patients. METHODS Qualitative analysis of chimerism in blood of sensitized patients was assessed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) to HLA-DR. PCR single-strand conformation polymorphism (PCR-SSCP) was used to confirm the extra HLA-DR antigens detected by PCR-SSOP. RESULTS Fourteen of 36 patients (38.9%) were positive for more than two HLA-DR indicative of chimerism. The presence of extra HLA-DR was confirmed by PCR-SSCP. When patients were analyzed on the basis of their panel-reactive antibody (PRA) status, 10 of 15 (66.7%) were positive for chimerism in the sensitized group, compared with only two of eight (25%) in the unsensitized group. Of the five males in the sensitized group who had received a blood transfusion but not a transplant, three were positive for chimerism. An association was observed between chimerism and maintenance of sensitization. None of the eight normal subjects studied demonstrated chimerism. CONCLUSIONS The results obtained with sensitized patients suggest an association between blood chimerism and maintenance of HLA sensitization. We speculate that chimerism may lead to long-term maintenance of anti-HLA antibody titers. This finding implies that abolition of chimerism could result in the eventual elimination of antigenic stimuli for antibody production against HLA antigens.

Association of the HLA-DR15/HLA-DQ6 haplotype with development of choroidal neovascular lesions in presumed ocular histoplasmosis syndrome ☆

Associations of human leukocyte antigen DR2 (HLA-DR2) and HLA-B7 with presumed ocular histoplasmosis syndrome (POHS) in the United States has been previously described. However, these associations were determined by means of low-resolution, complement-dependent cytotoxicity assays for HLA-A, HLA-B, and HLA-DR molecules. To determine whether POHS is associated with other HLA alleles within the HLA-A, HLA-B, HLA-DR, and HLA-DQ loci, we performed a case control study of 34 patients diagnosed with macular choroidal neovascular membrane secondary to POHS and 45 healthy control individuals. Peripheral blood-derived DNA from the study patients was typed for HLA genes by means of sequence-specific primers that gave low-medium allele resolution. Significant associations were observed between HLA-B7 (X2 = 14.30, pc = 0.004, relative risk = 8.23), HLA-DR15 (X2 = 29.08, pc = 0.000001, relative risk = 27.50), and HLA-DQ6 (X2 = 23.09, pc = 0.00001, relative risk = 27.43) and POHS. Because there are strong linkage disequilibria between HLA-DR15 (a subtype of HLA-DR2) and HLA-B7 as well as HLA-DQ6, the significantly higher association of HLA-DR15 and HLA-DQ6 with POHS as compared to HLA-B7 suggests that the former alleles mediate susceptibility to the disease. In conclusion, there is a significant association between the HLA-DR15/HLA-DQ6 haplotype and development of choroidal neovascular lesions in POHS.

Activation of human dendritic cells by porcine aortic endothelial cells: transactivation of naïve T cells through costimulation and cytokine generation.

Background. Dendritic cells (DC) are the most potent antigen-presenting cells in the immune system. To define the role of human DC in human anti-porcine immune responses, we defined the interaction of human DC with porcine aortic endothelial cells (PAEC). Methods. To determine the immune responses, both monocyte-derived and peripheral blood DC were cultured with porcine and human endothelial cells. We analyzed the role of CD11a, CD11b, and CD54 in a cell-to-cell adhesion assay using antibodies against these molecules. The expression pattern of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and intracellular cytokines (interleukin-12p70 and tumor necrosis factor [TNF]-&agr;) in DC after interaction with endothelial cells was determined by immunofluorescence. Results. Human DC significantly adhered to PAEC (38–40%), and this adhesion was augmented (>50%) upon treatment with either recombinant swine interferon-&ggr; or recombinant human TNF-&agr;. Addition of human DC to PAEC was blocked by pretreatment of DC with antibodies specific to human leukocyte function-associated antigen-1 or CD54. Adhesion of DC to PAEC also resulted in the activation of DC, which was manifested by up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and HLA-DR. PAEC-activated human DC provided proliferative signals to the naïve autologous CD4+ T cells and synthesized interleukin-12p70 and TNF-&agr;. However, activated DCs failed to lyse PAEC in such interaction. Conclusion. Human DC effectively adhered to PAEC and were activated by xenoantigen, resulting in highly efficient antigen presentation and proliferation of CD4+ T cells. Further, this interaction of human DC to PAEC is regulated by the participation of costimulatory and adherence molecules and cytokines.