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Biomedical subjects

W J Burlingham

Publications and source records attributed to W J Burlingham.

At least 19 recordsLinked to original sources
PubMed2006

Mouse strain and injection site are crucial for detecting linked suppression in transplant recipients by trans-vivo DTH assay.

Chemokine-driven accumulation of lymphocytes, mononuclear and polymorphonuclear proinflammatory cells in antigenic tissue sites is a key feature of several types of T-cell-dependent autoimmunity and transplant rejection pathology. It is now clear that the immune system expends considerable energy to control this process, exemplified by the sequential layers of regulatory cell input, both innate and adaptive, designed to prevent a classical Type IV or 'delayed-type' hypersensitivity (DTH) reaction from occurring in the visual field of the eye. Yet, despite an abundance of in vitro assays currently available to the human T-cell immunologist, none of them adequately models the human DTH response and its various control features. The theme of this article is that it is relatively easy to model the effector side of the human DTH response with xenogeneic adoptive transfer models. However, we show that in order to detect inhibition of a recall DTH in response to colocalized donor antigen (linked suppression)--a characteristic feature of peripheral tolerance to an organ transplant--both the challenge site and the immunocompetence of the mouse adoptive host are critical factors limiting the sensitivity of the trans-vivo DTH test.

Animals↗
PubMed2001

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.

Abatacept↗
PubMed2001

Human liver allograft acceptance and the "tolerance assay": in vitro anti-donor T cell assays show hyporeactivity to donor cells, but unlike DTH, fail to detect linked suppression.

Human allograft acceptance is associated with immune regulation, characterized by donor-antigen-linked suppression of delayed-type hypersensitivity (DTH). We wished to determine if "classical" in vitro assays of alloreactivity could also detect linked suppression and thus be useful in the clinical diagnosis of active immune regulation. We analyzed peripheral blood mononuclear cells from a group of eight liver transplant recipients, one of whom had stopped all immunosuppression 4.5 years ago yet continues to have good graft function (graft acceptor). The regulator phenotype was defined as the ability to suppress a DTH response to a recall antigen in the presence of donor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro mixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specifically hyporesponsive to donor compared with third-party B-LCL stimulators. However, in contrast to the linked suppression of DTH seen when a given B-LCL expressed donor-type HLA-B antigens, there was no evidence of linked suppression in vitro, either in CTL, proliferative, or interferon-gamma cytokine release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor beta or interleukin-10, which could restore a strong DTH response to donor B-LCL. We conclude that DTH analysis can readily detect donor antigen-linked suppression in liver transplant recipients. CTL and MLC tests failed to do so. These findings may be relevant to the development of a tolerance assay suitable for use in clinical trials.

Antigens↗
PubMed2000

Tumor necrosis factor-alpha and tumor growth factor-beta1 genotype: partial association with intragraft gene expression in two cases of long-term peripheral tolerance to a kidney transplant.

Genomic DNA was obtained from peripheral blood samples of patients JB and DS each of whom received a kidney transplant at 16 years of age from a serologically HLA-DR matched and HLA-class I -mismatched donor. Both patients discontinued immunosuppression after 1-2 years and retained good renal function for an additional 5 years or more. DNA was analyzed for genetic polymorphisms in the tumor necrosis factor-alpha (TNFalpha) and tumor growth factor-beta1 (TGFbeta1) loci. Biopsy samples obtained during stable function (DS, JB) and during rejection (JB) were analyzed by RT/PCR for cytokine gene expression. Both patients had a high responder genotype for TGFbeta1. DS had a low responder TNFalpha genotype, while JB and his donor were both genotypically TNFalpha intermediate responders. DS had a high TGFbeta1: TNFalpha mRNA ratio in two biopsies obtained during tolerance, while JB, who eventually lost his graft, had more TNFalpha than TGFbeta1 mRNA. The results suggest a possible role for cytokine immunogenetics in the stability of peripheral tolerance.

Adolescent↗
PubMed2000

Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient.

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

Amino Acid Sequence↗
PubMed2000

Soluble donor HLA class I and beta 2m-free heavy chain in serum of lung transplant recipients: steady-state levels and increases in patients with recurrent CMV infection, acute rejection episodes, and poor outcome.

We determined the concentration of donor sHLA/beta(2)m and total beta(2)m-free heavy chain (HC) in the serum of lung transplant recipients with ELISA assays. While we were unable to detect specific donor beta(2)m-free HCs due to a lack of available antibodies, we could determine if events that led to an increase in the release of beta(2)m-free HC also led to an increase in the release of donor sHLA/beta(2)m, particularly the 36 kDa, proteolytically cleaved form. We found that lung transplants constituitively release donor sHLA/beta(2)m at ng/ml levels. The levels (both of donor sHLA/beta(2)m and total beta(2)m-free HC) were significantly increased in CMV-sero-negative recipients (but not in CMV-sero-positive recipients) at the onset of post-transplant CMV disease. Acute rejection episodes were also associated with an increased release of donor sHLA/beta(2)m, but not of beta(2)m-free HC. However, in patients with particularly poor outcome (i.e., graft loss within 1 year) there was a significant release of beta(2)m-free HC. Analysis of one such patient showed a predominance of 36 kDa forms of donor-sHLA/beta(2)m. Our data are consistent with the hypothesis that the metalloproteinase that cleaves beta(2)m-free HC is active during uncontrolled CMV infection and acute rejection. However, recall responses to CMV and controlled immune responses to donor may result in little or no activation of sHLA class I release.

Acute Disease↗
PubMed2000

Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis.

We studied late graft rejection in a patient who had received a kidney transplant 9-10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DR beta 1 locus (donor: DR beta 1*1104; recipient: DR beta 1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient's creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient's peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the development of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DR beta 1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DR beta 1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DR beta 1*1104 and DR beta 1*1101 but not with the recipient DR beta 1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this "indirect" alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DR beta 1*1104 subtype.

Adolescent↗
PubMed2000

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

Animals↗
PubMed1999

Donor dendritic cell persistence in organ allograft recipients in the absence of immunosuppression.

Donor-derived leukocytes are known to persist in the peripheral blood of organ allograft recipients after withdrawal of all immunosuppressive drug therapy and can exert a donor-specific veto effect. Antigen-presenting cells (APC), in particular dendritic cells (DC), have been proposed as a candidate for this veto leukocyte. Myeloid DC were derived from the peripheral blood of two ion-compliant organ transplant recipients: D. S., a heart transplant recipient, and J. M., a liver transplant recipient. Donor-specific signal was enriched in the cultured DC fraction relative to whole blood for both patients. The clinical outcome in each patient was different: D. S. lost his heart allograft due to biopsy-proven acute and chronic rejection 2.5 years after discontinuing anti-rejection medication; J. M. continues to maintain adequate liver function. The results have important implications for the planned withdrawal of immunosuppression in tolerance protocols as DC may play a role either in the maintenance of tolerance or immune activation.

Adult↗
PubMed1999

Peptide-conformed beta2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase.

Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection.

Cell Line, Transformed↗
PubMed1998

HLA (A*0201) mimicry by anti-idiotypic monoclonal antibodies.

Soluble MHC Ags and anti-Id (anti-anti-MHC) Abs have both been shown to inhibit MHC alloantigen-specific B cell responses in vivo. We hypothesized that some anti-idiotypic Abs function as divalent molecular mimics of soluble HLA alloantigen. To test this idea, we studied two well-defined anti-idiotypic mAbs, T10-505 and T10-938, elicited in syngeneic BALB/c mice by immunization with CRll-351, an HLA-A2,24,28-specific mAb. Each anti-Id induced "Ab-3" Abs in rabbits that cross-reacted with HLA-A2 but not with HLA-B Ags. Furthermore, each anti-Id could bind to and block Ag recognition by Ha5C2.A2, a human homologue of mAb CRll-351. Both anti-Id mAb displayed weak reactivity with the human mAb SN66E3, which recognized an overlapping but distinct determinant of HLA-A2 Ags; neither reacted with human mAb MBW1, which recognized a nonoverlapping HLA-A2 determinant. Amino acid sequence comparison of mAb CRII-351 heavy and light chain variable region complementarity-determining regions (CDRs) with those of mAb Ha5C2.A2 and SN66E3 revealed short regions of homology with both human mAb; a large insert in the light chain CDR1 of mAb SN66E3 distinguished it from both CRll-351 and Ha5C2.A2. The amino acid sequences of mAb T10-505 and T10-938, which differed markedly from each other, revealed no homology to the alpha2 domain sequence of HLA-A*0201 that contains the CRll-351 mAb-defined epitope. We conclude that structurally different anti-Id Abs can mimic a polymorphic conformational epitope of an HLA Ag. In the case of T10-505 and T10-938 mimicry was not based on exact replication of the epitope by the hypervariable loops of the anti-Id mAb.

Alleles↗
PubMed1998

The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors.

BACKGROUND: During pregnancy and nursing, a baby's developing immune system is intimately exposed to the mother's antigens. To determine whether this exposure is of clinical benefit to patients who later receive an allograft as an adult, we analyzed the outcome of primary renal transplantations from sibling donors. METHODS: We retrospectively studied graft survival and rejection episodes in 205 patients who had received renal transplants at nine centers between 1966 and 1996 from sibling donors bearing maternal or paternal HLA antigens not inherited by the recipient. The sibling donors were categorized by analysis of family HLA-typing data. RESULTS: In the multicenter analysis, graft survival was higher at 5 years and at 10 years after transplantation in recipients of kidneys from siblings expressing maternal HLA antigens not inherited by the recipient than in recipients of kidneys from siblings expressing paternal HLA antigens not inherited by the recipient (86 percent vs. 67 percent at 5 years and 77 percent vs. 49 percent at 10 years, P=0.006 for both). Paradoxically, there was a higher incidence of early rejection in the former group, suggesting that fetal and neonatal exposure to maternal antigens results in immunologic priming. Pretransplantation transfusions of donor blood reduced the incidence of acute rejection while preserving the beneficial effect of tolerance to noninherited maternal antigens on graft survival. Since 1986, new immunosuppressive drugs have lessened the short-term, but not the long-term, survival advantage of grafts expressing maternal HLA antigens not inherited by the recipient. CONCLUSIONS: In the transplantation of a kidney from a sibling donor who is mismatched with the recipient for one HLA haplotype, graft survival is higher when the donor has maternal HLA antigens not inherited by the recipient than when the donor has paternal HLA antigens not inherited by the recipient.

Fathers↗
PubMed1998

Stable lung allograft outcome correlates with the presence of intragraft donor-derived leukocytes.

BACKGROUND: The role of bone marrow-derived "passenger" leukocytes in the outcome of solid organ transplantation remains controversial. This study tested the relationship between high levels of donor-derived leukocytes within the transplanted organ and clinical outcome after lung transplantation. METHODS: Sequential bronchoalveolar lavage samples were obtained from human lung allograft recipients. Leukocytes of donor origin in the bronchoalveolar lavage fluid were detected using two-color immunofluorescence, and the results were correlated with multiple clinical parameters. RESULTS: Mean donor leukocyte levels for the first 200 days after transplantation were higher in patients with a good transplantation outcome compared with those patients who lost their grafts due to acute rejection (AR) or developed bronchiolitis obliterans syndrome. The presence of low numbers of donor-derived leukocytes for the first 200 days after transplantation was found to be a significant risk factor for graft loss due to either acute or chronic rejection (P=0.032). Nearly all patients (85%) experienced AR episodes. However, the time to onset of severe AR episodes was significantly longer (P=0.049), and the incidence of these episodes reduced, in patients who maintained high numbers of donor-derived leukocytes for the first 200 days after transplantation. CONCLUSIONS: The presence of high numbers of donor-derived leukocytes, particularly macrophages, in the transplanted lung in the first 200 days after transplantation was associated with stable graft function. Donor-derived leukocytes were reduced or absent in patients with a poor transplantation outcome. These findings rule out a negative influence of persisting donor leukocytes and are consistent with the emerging two-way models of transplant tolerance.

Bronchoalveolar Lavage Fluid↗
PubMed1998

The metalloproteinase-mediated pathway is essential for generation of soluble HLA class I proteins by activated cells in vitro: proposed mechanism for soluble HLA release in transplant rejection.

We and others have found donor-derived soluble beta2m-associated HLA class I proteins (sHLA/beta2m) in the serum of allograft recipients with acute and chronic rejection. Whether appearance of sHLA/beta2m and upregulated expression of donor cell-bound HLA/beta2m during allograft rejection are related events is unknown. Activation-induced upregulation of in vitro HLA/beta2m expression correlates with the surface expression of another form of HLA class I, namely beta2m-free HLA heavy chains (beta2m-free HC). We have shown that beta2m-free HC, but not beta2m-associated HC, are then cleaved by a specific membrane-bound metalloproteinase and released into supernatants as soluble 36 kDa proteins. We show now that activated peripheral blood lymphocytes produce predominantly the 36 kDa form of sHLA proteins which is present in supernatants as both beta2m-free HC and sHLA/beta2m. Importantly, the metalloprotease inhibitor BB-94 blocked not only the release of soluble beta2m-free HC, but also the appearance of sHLA/beta2m in cell supernatants. Low levels of 36 kDa beta2m-free HC were also present in human plasma of healthy donors. These data suggest an important role for the HLA class I-specific metalloproteinase in vivo in healthy individuals and during allograft rejection in the generation of soluble beta2m-free and beta2m-associated HLA proteins.

Cell-Free System↗
PubMed1997

Soluble HLA class I in epithelial lining fluid of lung transplants: associations with graft outcome.

We hypothesized that the small amounts of donor HLA-A and HLA-B proteins detected in the serum during organ allograft rejection are indicative of higher local releases within the graft itself. We determined the concentrations of total HLA class I (HLA-I) and, in selected cases, specific donor and host HLA-A and HLA-B proteins, in the epithelial lining fluid (ELF) sampled by bronchoalveolar lavage (BAL) of lung transplant recipients (n = 37) and of normal controls (n = 25). We found that 1) HLA-I proteins were enriched in the lung ELF relative to other proteins; 2) the concentration of HLA-I in the ELF of well-functioning transplants was similar to that in normal lungs; 3) HLA-I proteins and total proteins were elevated in the ELF of patients who developed chronic rejection or refractory acute rejection; 4) the concentration of HLA-I was correlated with the percentage of neutrophils but not with the percentage of lymphocytes in the ELF of transplanted lungs; and 5) only the percentage of lymphocytes was elevated in the ELF of transplant patients with active CMV infections. Total HLA-I from the ELF was found to contain a mixture of both donor- and recipient-type HLA-A and HLA-B proteins and the donor-type HLA-A2 was found to be highly enriched in the ELF relative to serum.

Acute Disease↗