Nancy Steward

Early posttransplant inflammation promotes the development of alloimmunity and chronic human lung allograft rejection

Background. Chronic human lung allograft rejection, represented by bronchiolitis obliterans syndrome (BOS), is the single most important factor that limits the long-term survival following lung transplantation (LT). However, the pathogenesis of BOS remains unclear. We hypothesized that the early posttransplant inflammation would promote the development of donor anti–human leukocyte antigen (HLA) alloimmunity and predispose to BOS. Methods. Serum levels of interleukin (IL)-1&bgr;, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, IP-10, MIG, MCP-1, MIP-1&agr;, MIP-1&bgr;, RANTES, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&agr;, IFN-&ggr;, granulocyte-macrophage colony-stimulating factor, IL-1R&agr;, and IL-2R were serially analyzed in 31 BOS+ and matched 31 BOS− patients using quantitative multiplex bead immunoassays. Donor-specific HLA class II cellular immunity was analyzed using enzyme-linked immunospot (ELISPOT) by testing recipient peripheral blood mononuclear cells against mismatched donor HLA-DR peptides. Anti-HLA class II antibodies were monitored using flow panel reactive antibodies. Results. There was early posttransplant elevation in basal serum levels of proinflammatory chemokines IP-10 and MCP-1 and Th1-cytokines IL-1&bgr;, IL-2, IL-12, and IL-15 in BOS+ patients, compared to BOS− and normal subjects. In addition, a threefold decline in IL-10 levels was found during BOS development. BOS+ patients revealed increased development of HLA class II alloantibodies and Th1-predominant donor-specific cellular immunity with high frequency of IFN-&ggr; and low IL-5 producing T-cells. Conclusion. Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.

Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts

BACKGROUND Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality after lung transplantation (LTx). We sought to better understand the relationship between alloimmune responses and autoimmunity and, subsequently, how autoimmunity leads to chronic rejection. METHODS We analyzed the development of donor-specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T- and ColV-specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELISPOT. RESULTS In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor-specific anti-HLA Abs, Abs to self-antigens and BOS (p < 0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor-specific Abs (DSA) and Abs to self-antigens. A total of 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS(+) patients had higher frequency of T cells secreting IL-17 (p < 0.01) and IFN-γ (p < 0.05) with decreased IL-10 (p < 0.05) when compared with BOS(-) patients. CONCLUSIONS Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be play a key role in preventing the development of chronic rejection.

Immunological Link Between Primary Graft Dysfunction and Chronic Lung Allograft Rejection

BACKGROUND Primary graft dysfunction (PGD) in the immediate post-lung transplant period strongly increases the risk of chronic rejection (broncholitis obliterans syndrome). Here, we hypothesized that PGD-induced inflammation augments alloimmunity, thereby predisposing to broncholitis obliterans syndrome. METHODS Primary graft dysfunction and broncholitis obliterans syndrome were diagnosed according to the established International Society for Heart and Lung Transplantation criteria. Anti-human leukocyte antigen (HLA) alloantibodies were analyzed using Flow-PRA. Donor HLA class II-specific T cells were analyzed using interferon (IFN)-gamma ELISPOT. Serum levels of 25 cytokines and chemokines were measured using LUMINEX. RESULTS Of the 127 subjects, 29 (22.8%) had no PGD (grade 0), 42 (33.2%) had PGD-1, 36 (28.3%) had PGD-2, and 20 (15.7%) had PGD-3. Patients with PGD grades 1 to 3 (PGD(1-3)) had elevated proinflammatory mediators MCP-1, IP-10, interleukin (IL)-1 beta, IL-2, IFN-gamma, and IL-12 in the sera during the early posttransplant period compared with patients with PGD grade 0 (PGD(0)). On serial analysis, PGD(1-3) patients revealed increased development of de novo anti-HLA-II (5 years: 52.2% versus PGD(0) 13.5%, p = 0.008). However, no difference was found in anti-HLA-I alloantibody development (PGD(1-3) patients 48% versus PGD(0) 39.6%, p = 0.6). Furthermore, PGD(1-3) patients had increased frequency of donor HLA class II-specific CD4(+) T cells [(91.4 +/- 19.37) x 10(-6) versus (23.6 +/- 15.93) x 10(-6), p = 0.003]. CONCLUSIONS Primary graft dysfunction induces proinflammatory cytokines that can upregulate donor HLA-II antigens on the allograft. Increased donor HLA-II expression along with PGD-induced allograft inflammation promotes the development of donor specific alloimmunity. This provides an important mechanistic link between early posttransplant lung allograft injury and reported association with broncholitis obliterans syndrome.

CD4+25+ Regulatory T Cells Limit Th1-Autoimmunity by Inducing IL-10 Producing T Cells Following Human Lung Transplantation

Chronic human lung allograft rejection is manifested by bronchiolitis obliterans syndrome (BOS). BOS has a multifactorial etiology. Previous studies have indicated that both cellular and humoral alloimmunity play a significant role in the pathogenesis of BOS. Recently, autoimmunity has also been demonstrated to contribute to lung allograft rejection in animal models. However, the significance of autoimmunity in BOS remains unknown. In this report, we investigated the role of naturally occurring CD4+CD25+ regulatory T cells (T‐regs) in modulating cellular autoimmunity to collagen type V (col‐V), a ‘sequestered’ yet immunogenic self‐protein present in the lung tissue, following lung transplantation (LT). We demonstrated that col‐V reactive CD4+ T cells could be detected in the peripheral blood of lung transplant recipients. There was a predominance of IL‐10 producing T cells (TIL‐10) reactive to col‐V with significantly lower levels of IFN‐γ and IL‐2 producing T cells (Th1 cells). The col‐V specific TIL‐10 cells suppressed the proliferation and expansion of col‐V specific Th1 cells by IL‐10‐dependent and contact‐independent pathways. The TIL‐10 cells were distinct but their development was dependent on the presence of T‐regs. Furthermore, during chronic lung allograft rejection there was a significant decline of TIL‐10 cells with concomitant expansion of col‐V‐specific IFN‐γproducing Th1 cells.

Antibodies to Self-Antigens Predispose to Primary Lung Allograft Dysfunction and Chronic Rejection

BACKGROUND Primary graft dysfunction (PGD) is a known risk factor for bronchiolitis obliterans syndrome (BOS) after lung transplantation. Here, we report that preformed antibodies to self-antigens increase PGD risk and promote BOS. METHODS Adult lung transplant recipients (n = 142) were included in the study. Primary graft dysfunction and BOS were diagnosed based on International Society for Heart and Lung Transplantation guidelines. Antibodies to self-antigens k-alpha-1 tubulin, collagen type V, and collagen I were quantitated using standardized enzyme-linked immunosorbent assays, and cytokines were analyzed using Luminex immunoassays (Biosource International, Camirillo, CA). Human leukocyte antigen (HLA) antibodies were measured using Flow-PRA (One Lambda, Canoga Park, CA). RESULTS Lung transplant recipients with pretransplant antibodies to self-antigens had increased risk of PGD (odds ratio 3.09, 95% confidence interval: 1.2 to 8.1, p = 0.02) compared with recipients without. Conversely, in patients with PGD, 34.7% were positive for pretransplant antibodies whereas in the PGD negative group, only 14.6% had antibodies (p = 0.03). Antibody positive patients demonstrated high levels of proinflammatory cytokines interleukin (IL)-1β (2.1-fold increase), IL-2 (3.0), IL-12 (2.5), IL-15 (3.0), and chemokines interferon-inducible protein-10 (3.9) and monocyte chemotactic protein-1 (3.1; p < 0.01 for all). On 5-year follow-up, patients without antibodies showed greater freedom from development of HLA antibodies compared with patients who had antibodies (class I: 67% versus 38%, p = 0.001; class II: 71% versus 41%, p < 0.001). Patients with pretransplant antibodies were found to have an independent relative risk of 2.3 (95% confidence interval: 1.7 to 4.5, p = 0.009) for developing BOS. CONCLUSIONS Presence of antibodies to self-antigens pretransplant increases the risk of PGD immediately after transplant period and BOS on long-term follow-up. Primary graft dysfunction is associated with an inflammatory cascade that augments the alloimmune (anti-HLA) response that predisposes to BOS.

Cytolytic T lymphocytes from human renal allograft biopsies are tissue specific.

The cytolytic activity of T lymphocytes infiltrating renal allografts from recipients undergoing episodes of acute cellular rejection was studied. These T-cell populations, composed of both CD4+ and CD8+ cells, demonstrated significant cytolytic activity against both donor-derived KCLs and B-LCLs. In five of 21 biopsy-derived lines greater cytolytic activity was measured against donor KCLs compared to donor B-LCLs, suggesting the presence of kidney antigen-specific, MHC-restricted clones. Clones developed by stimulation with donor B-LCLs lysed both donor B-LCLs and KCLs while clones developed on donor KCLs as stimulator cells showed tissue specificity. Three of 26 clones recognized tissue-specific antigens in the context of donor MHC class I antigens lysing donor KCLs but not B-LCLs. These data demonstrate that a subpopulation of T cells recognizing kidney-specific antigens are present in biopsies of renal allograft recipients undergoing acute cellular rejection. This subpopulation of tissue-specific cytotoxic T lymphocytes may prove to contribute significantly to the pathology of allograft rejection.

Indirect Recognition of Porcine Swine Leukocyte Ag Class I Molecules Expressed on Islets by Human CD4+ T Lymphocytes

Xenotransplantation of porcine islets is considered a viable alternative treatment for type 1 diabetes mellitus. Therefore, we characterized human PBL responding to porcine islets both in vitro by coculture and in vivo using SCID mice reconstituted with human PBLs (HuPBL-SCID) and transplanted with porcine islets. T cell lines generated in vitro and graft-infiltrating T cells obtained from HuPBL-SCID mice were CD4+-proliferated specifically to porcine islets cultured with autologous APC. This proliferation was abrogated by an anti-human class II Ab. These T cell lines also proliferated to purified swine leukocyte Ag (SLA) class I molecules in the presence of self-APC, indicating that the primary xenoantigens recognized are peptides derived from SLA. This CD4+ T cell line lysed porcine islets but not splenocytes. CD4+ T cell clones with Th0, Th1, and Th2 cytokine profiles were isolated. The Th0 and Th1 clones lysed porcine islets, whereas the Th2 clone that secreted a large amount of IL-4 was not lytic. These results demonstrate that human T cells responding to porcine islets are primarily CD4+ and recognize porcine xenoantigens by the indirect Ag pathway presentation. These activated T cells produce cytokines that lyse islets. Furthermore, we demonstrate that the major porcine xenoantigens recognized are SLA class I molecules.

Differentiation and functional maturation of human CD14(+) adherent peripheral blood monocytes by xenogeneic endothelial cells: up-regulation of costimulation, cytokine generation, and toll-like receptors.

Background. Dendritic cells (DCs) can be generated from peripheral blood mononuclear cells (PBMC) after stimulation with exogenous granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Further, extravasation of monocytes through human endothelial cells can also cause differentiation, maturation, and expression of DC-specific phenotype. However, it is unclear whether human DCs can be generated from monocytes under the influence of xenogeneic endothelial cells in the absence of exogenous cytokines. We therefore analyzed and compared the effect of human and porcine endothelial cells on the differentiation of human monocytes into DC. Methods. Adherent peripheral blood CD14+ monocytes were cultured in the presence of different cytokine combinations, human or porcine endothelial cells, and smooth muscle cells, and analyzed for DC-specific antigen expression, antigen-presenting capacity, cytokine and chemokine generation, and expression of Toll-like receptors by flow cytometry and reverse transcription polymerase chain reaction. Results. Human monocytes express a DC-specific surface phenotype and efficiently present allo- and xenoantigens to allogeneic T cells after co-culturing with allogeneic and xenogeneic endothelial cells, respectively. Differentiation of monocytes under different stimulating conditions is also accompanied by the up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), human leukocyte antigen (HLA)-DR, synthesis of cytokines tumor necrosis factor-&agr;, IL-12p70, IL-10, and differential expression of message for Toll-like receptors. Conclusions. Porcine aortic endothelial cells can provide immunostimulatory signals to human peripheral blood adherent monocytes similar to allogeneic endothelial cells through the participation of innate immune mechanisms.

Respiratory Virus-Induced Dysregulation of T-Regulatory Cells Leads to Chronic Rejection

BACKGROUND Lower respiratory viral infections predispose to bronchiolitis obliterans syndrome (BOS). In addition, there is emerging evidence to support the role of autoimmunity in the pathogenesis of BOS. Because CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Treg) control autoimmunity, we tested the hypothesis that respiratory virus-induced Treg dysfunction leads to BOS. METHODS Treg frequency was monitored using flow cytometry. Apoptosis, cytokines, and antibodies were analyzed using annexin V assay, LUMINEX, and enzyme-linked immunosorbent assay, respectively. Murine studies were performed using the orthotopic tracheal transplant model. RESULTS (A) Human studies: Treg troughs (decrease >50% of baseline) were found in 13 (43.3%) of 30 lung transplant recipients. Treg isolated during troughs revealed increased apoptosis (37.8%). Patients with Treg troughs had increased prevalence of antibodies to self-antigens collagen type I (23.1% vs 5.8% pretrough), collagen V (7.7% vs 0%), and k-alpha tubulin (30.7% vs 11.7%, p < 0.01) at 6 months post-trough. Increased number of Treg troughs correlated with more rapid onset of BOS. (B) Murine studies: Infection of tracheal transplant recipients with murine parainfleunza sendai virus led to increased Treg apoptosis (50.5%) in the draining lymph nodes. Vaccination against sendai virus prior to transplant abrogated apoptosis of Treg. In vitro, sendai virus-infected, but not naive, tracheal epithelial cells demonstrated upregulation of FasL (>3.5-fold) and induction of co-cultured Treg apoptosis (5.6-fold increase). CONCLUSIONS Respiratory viral infections cause Treg apoptosis which leads to the development of de novo autoimmunity that may play a role in the pathogenesis of BOS.

Elevated soluble CD30 correlates with development of bronchiolitis obliterans syndrome following lung transplantation

Background. The long-term function of lung transplants is limited by chronic rejection (bronchiolitis obliterans syndrome, BOS). Due to lack of specific markers, BOS is diagnosed clinically. Because there is strong evidence that alloimmunity plays a significant role in the pathogenesis of BOS, we investigated whether soluble CD30 (sCD30), a T-cell activation marker, would correlate with BOS. Methods. Sera collected serially from BOS+ (n=20) and matched BOS− (n=20) lung transplant (LT) patients were analyzed for sCD30 by enzyme-linked immunosorbent assay. Pretransplant sera and sera from normal donors were also analyzed. Results. PreLT levels were comparable to normal subjects. However, posttransplant there was a significant elevation in sCD30 levels during BOS development in all BOS+ patients, compared to BOS− (mean 139.8±10.7 vs. 14.8±2.7 U/ml, P<0.001). sCD30 levels declined in the BOS+ patients but were still elevated compared to BOS− (48.52±5.04 vs. 7.19±2.9, P<0.0001). Conclusions. We conclude that sCD30 may represent a novel marker to monitor the development of BOS.