Patrick W. Adams

Clinical Significance of MHC‐Reactive Alloantibodies that Develop after Kidney or Kidney–pancreas Transplantation

The purpose of this study was to determine the relationships between acute rejection, anti‐major histocompatibility complex (MHC) class I and/or class II‐reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney‐pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post‐transplant (mean 2.6 years). The presence of anti‐MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC‐reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti‐MHC class II, but not anti‐MHC class I, alloantibodies detected post‐transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II‐reactive, but not MHC class I‐reactive, alloantibodies post‐transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post‐transplant detectable MHC class II‐reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post‐transplant alloantibody production, and avoidance of acute rejection episodes, may improve long‐term graft survival by reducing the incidence of chronic allograft rejection.

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

Purpose: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4ds role as a prognostic adjunct in lung allografts. Material and Methods: Twenty‐three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. Results: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p < .0001) compared to other the immunoreactants C1q, C5b‐9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon‐BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. Conclusions: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.

De novo Thrombotic Microangiopathy in Renal Allograft Biopsies—Role of Antibody‐Mediated Rejection

The most common cause of thrombotic microangiopathy (TMA) in renal allografts is thought to be calcineurin inhibitor toxicity. Antibody‐mediated rejection (AMR) can also cause TMA, but its true impact on de novo TMA is unknown. In a retrospective review of renal allograft biopsies from January 2003 to December 2008 at our institution, we determined the prevalence of TMA in patients with C4d positive (n = 243) and C4d negative (n = 715) biopsies. Over 90% of patients received cyclosporine in both groups. De novo TMA was seen in 59 (6.1%) patients; most of them (55%) with C4d positive biopsy. Among patients with C4d positive biopsies, 13.6% had TMA, as compared to only 3.6% patients with C4d negative biopsies (p < 0.0001). Incidence of graft loss between C4d positive and C4d negative TMA groups was not significantly different, but 70% of patients with C4d positive TMA who received plasmapheresis had slightly lower graft loss rate. In biopsies with AMR‐associated TMA, glomerulitis and peritubular capillaritis were significantly more prominent. AMR is the most common cause of TMA in renal allografts in our patient population. It is important to recognize AMR‐related TMA because plasmapheresis treatment may be beneficial.

T lymphocyte activation by cytomegalovirus-infected, allogeneic cultured human endothelial cells

Cytomegalovirus, a source of serious complications among immunosuppressed individuals, infects endothelial cells in vivo, and has been epidemiologically associated with allograft rejection and transplantation-associated accelerated arteriosclerosis (TxAA). As the interface between the host immune system and allograft tissues, the endothelium may be of primary importance in mediating pathogenic immune responses to CMV. We have therefore investigated T lymphocyte responses to CMV-infected allogeneic human umbilical vein endothelial cells (HUVE) in vitro. Proliferation assays demonstrated dramatically enhanced responses by CMV-seropositive donor-derived PBMC or purified T cells cocultured with CMV-infected HUVE, as compared with those elicited by noninfected stimulator cells. As determined by limiting dilution analysis of IL-2-producing cells, the frequency of T cells responding to infected HUVE was generally found to exceed by approximately one order of magnitude those responding to uninfected cells. Similar analyses of isolated T cell subsets revealed that these responses (proliferation and IL-2 production) were nearly entirely accountable to the CD4+ fraction. Responses of CD8+ populations, however, varied among donors. The marked activation of CD4+ cells is particularly intriguing since we have shown that CMV-infected HUVE do not express HLA

Evidence That Humoral Allograft Rejection in Lung Transplant Patients Is Not Histocompatibility Antigen‐Related

We have recently recognized humoral rejection (HR) in lung allograft recipients and its association with acute and chronic graft dysfunction. We have shown that C4d, a stable marker of classic complement activation, is deposited in lung allografts, correlating with clinical rejection and parenchymal injury. The antigenic target may be endothelium in the setting of recurrent acute rejection while varying components of the bronchial wall may be important in chronic graft dysfunction. We sought to establish whether there is a role for antibodies with histocompatibility antigen specificity in the lung humoral allograft phenomenon. Flow cytometric and ELISA assays to assess donor‐specific antigens were conducted on sera from 25 lung transplant recipients who had experienced one or more episodes of clinical rejection; in addition, the serum samples were tested for evidence of antiendothelial cell antibody activity. Morphologically, each case had biopsies showing septal capillary injury with significant deposits of immunoreactants with microvascular localization and positive indirect immunofluorescent antiendothelial cell antibody assay. Panel‐reactive antibody testing showed absence of MHC Class I/II alloantibodies; ELISA based crossmatch detecting donor‐specific MHC Class I/II specific antibodies was negative. HR can occur in the absence of antibodies with HLA specificity; antigenic targets may be of endothelial cell origin.

In vitro induction of endothelial HLA class II antigen expression by cytomegalovirus-activated CD4+ T cells.

CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system. Although endothelial HLA class II expression is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells (EC) and, indeed, renders them refractory to HLA DR induction by IFN-γ. Our earlier studies have demonstrated, however, that CMV-infected EC are capable of eliciting vigorous activation responses by allogeneic, CMV-seropositive donor-derived CD4+ T cells. We now show that T cells thus activated can induce HLA DR expression on noninfected EC. Human umbilical vein endothelial cells (HUVEC) were inoculated at low titer with CMV strain VHL/E, cocultured with allogeneic, CMV-seropositive or CMV-seronegative do

Frequency of human alloantigen-reactive T lymphocytes. II. Method for limiting dilution analysis of alloantigen-reactive helper T cells in human peripheral blood.

Quantitative immunologic techniques for analysis of human alloreactivity are currently lacking in transplantation immunology. We report a rapid, sensitive, and quantitative limiting dilution analysis technique that provides a minimal estimate of the number of peripheral blood mononuclear cells (PBMC) capable of secreting interleukin-2 (operationally defined as helper T lymphocytes) when cultured in vitro with allogeneic PBMC bearing serlogically identified MHC disparities. Using this LDA technique, we have estimated that approximately 1/500 to 1/2000 (0.2% to 0.05%) of the PBMC from various individuals can secrete IL-2 after in vitro contact with completely major-histocompatibility-complex-disparate PBMC. Under normal conditions the HTL frequency in human peripheral blood appears quite stable, based on serial analysis of HTL frequency in a healthy human donor. This LDA technique is more rapid and informative than the MLR, and may be useful for pretransplant evaluation and posttransplant monitoring of donor reactivity in transplant recipients.

High incidence of donor-reactive delayed-type hypersensitivity reactivity in transplant patients.

Evidence of transplant recipient cellular sensitization towards donor antigens has rarely been directly measured. Rather, sensitization has been generally inferred by the presence of detectable allo‐reactive or donor‐reactive antibodies. In this study a newly developed delayed‐type hypersensitivity assay was used to directly determine the incidence of post‐transplant donor‐reactive T‐cell sensitization in a large cohort of kidney and simultaneous kidney‐pancreas recipients. These results were compared with the presence of detectable circulating alloantibodies and with patient clinical outcome. We found an unexpectedly high incidence (52%) of donor‐reactive delayed‐type hypersensitivity reactivity in our study patients. Donor‐reactive delayed‐type hypersensitivity reactivity occurred at a much higher frequency than detectable alloantibodies (20%). Further, we found no correlation between the presence of alloantibodies and donor‐reactive delayed‐type hypersensitivity reactivity. We also found no correlation between the development of donor‐reactive delayed‐type hypersensitivity reactivity and the degree of donor and recipient HLA matching. Finally, the presence of detectable donor‐reactive delayed‐type hypersensitivity reactivity did not correlate with a worse clinical outcome at the time of these analyses. We conclude that in transplant recipients, the presence of circulating alloantibodies is a poor indicator of previous T‐cell sensitization to donor antigens. We also conclude that our current immunosuppression strategies are relatively ineffective at blocking T‐cell allosensitization, but are very effective at blocking the biological consequences of that allosensitization.

Limiting dilution analysis of human, tetanus-reactive helper T lymphocytes: A rapid method for the enumeration of helper T lymphocytes with specificity for soluble antigens☆

The number of helper T lymphocytes (HTL) in human peripheral blood with specificity for the soluble protein, tetanus toxoid, was estimated by limiting dilution analysis (LDA). HTL were detected via antigen-induced interleukin-2 (IL-2) production, as measured by incorporation of tritiated thymidine by an IL-2-dependent indicator cell line, CTLL-20. Culture conditions optimizing assay sensitivity are described, and the ability to detect antigen-specific HTL using this LDA technique are demonstrated. Observed HTL frequencies in healthy human donors tested for tetanus-reactive helper T cells ranged from less than 1 HTL/268,749 peripheral blood mononuclear cells (PBMC) (undetectable) to 1 HTL/1486 PBMC. The LDA technique was also used to detect frequency shifts in human peripheral blood HTL following challenge with antigen. This assay offers distinct advantages over proliferative LDA techniques in that it is rapid (requiring only 2 days), and defines an antigen-reactive T cell subset with defined function (IL-2 secretion). Furthermore, the LDA technique can be adapted for the detection of other soluble protein antigens, such as PPD and Candida albicans. In general, this LDA technique provides a reliable, quantitative index of human HTL reactivity to any of a variety of soluble protein antigens, and has clinical as well as experimental applicability.

Cytolytic activity against allogeneic human endothelia: resistance of cytomegalovirus-infected cells and virally activated lysis of uninfected cells.

BACKGROUND Cytomegalovirus (CMV) has been implicated as an exacerbating agent in the development of transplant vascular sclerosis; however, specific etiologic mechanisms remain unresolved. Based upon our previous observations that CMV-infected endothelial cells (ECs) stimulate proliferation and cytokine production by allogeneic T cells, we now test the hypothesis that CMV-driven cytolytic activity may contribute to graft endothelial injury. METHODS Limiting dilutions of CMV-seropositive or -seronegative donor-derived T cells were stimulated with CMV-infected or uninfected allogeneic ECs in the presence of interleukin-2. T-cell proliferation was monitored by assay of [3H]thymidine incorporation and stimulated T cells were tested for lytic activity against CMV-infected or uninfected radiolabeled EC targets by 51Cr release assay. Natural killer (NK) cell activity was examined by incubating freshly isolated peripheral blood mononuclear cells with 51Cr-labeled targets, followed by assay of radiolabel release. RESULTS CMV-infected ECs were resistant to T cell- and NK-mediated cytolysis regardless of donor serostatus, nature of stimulation, or level of T-cell proliferation. In contrast, although uninfected ECs were unharmed by NK cells, these targets experienced significant lysis by T cells stimulated with either uninfected or CMV-infected ECs. CONCLUSIONS These results implicate CMV-infected graft endothelium as a persistent source of infectious virus, a chronic stimulus for potentially destructive host inflammatory activity, and a potential trigger for the generation of lytic injury to uninfected bystander endothelia, suggesting multiple mechanisms by which this virus might perturb equilibrium at the graft/host interface.