Blood Of Lung Transplant Recipients Research Articles

Defining the Role of B Cells in Graft Rejection Following Lung Transplantation

Humoral immunity is maintained by long lived memory B and plasma cells, both of which arise from the activation and differentiation of naïve B cells, and require T cell help. In the transplantation setting, humoral immune responses that target the donor tissue are undesirable and can lead to antibody mediated rejection (AMR). AMR has remained challenging to prevent and overcome, with current immunosuppressive therapies predominantly focused on T cells. However, the presence of antibodies to donor tissue human leukocyte antigen (HLA), aka donor-specific antibody (DSA), are associated with a poorer prognosis in transplantation. DSA can be either pre-existing (generated through previous transplants, blood transfusions or multiple pregnancies) or generated de novo after transplantation. Thus, understanding and characterizing B cell responses in transplantation may reveal novel immune monitoring and/or treatment. This study is a retrospective longitudinal and comparative analysis of B and T cells in the peripheral blood of lung transplant recipients at the Alfred Hospital (Melbourne, Australia), and aims to measure HLA Class II antibody responses. In addition to healthy control donors, a total of 22 lung transplant recipients (DSA- n=11; DSA+ (de novo) n=11) have had cryopreserved peripheral blood mononuclear cells immunophenotyped at several time points, including pre-transplant. Comparative analysis of lymphocytes between the two lung transplant cohorts revealed that patients who developed DSA had a higher frequency of B cells at 3 months post-transplantation in compared than those who did not. Further to this, at 6 weeks and 3 months pre-transplant, CD4+ T cells were more abundant than CD8+ T cells in patients with DSA, and these frequencies deviated from both their DSA- counterparts and healthy blood donors. In addition to B and T cell ex vivo characterization, we have developed a unique in vitro memory B cell culture system, design for low cell seed numbers that expand and differentiate memory B cells into antibody secreting cells. From these cultures, we are able to collect and characterize antibody secreted in the supernatant. To date, we have completed this work assessing tetanus toxoid antibody responses and are now assessing HLA II antibody responses. This study provides impetus to study the role of humoral immunity in lung transplantation.

Circulating Follicular Helper T Cells are Decreased in Chronic Lung Allograft Dysfunction

Humoral immunity is a fundamental element of chronic lung allograft dysfunction (CLAD) as shown by the presence of B cells and lymphoid follicles in the lung allograft. The formation of lymphoid follicles is under control of follicular helper T (TFH) cells, which also help B cells to differentiate into high-affinity alloantibody-producing plasma cells and memory B cells. The presence and role of circulating TFH cells in CLAD has not yet been investigated and is therefore the objective of our cross-sectional study. Whole blood samples were collected from healthy controls (HC) (n=7) and lung transplant recipients (n=31: stable n=12, CLAD n=19). After peripheral blood mononuclear cell isolation, quantification of CD3+ T cells, CD4+ T helper (TH) cells and CXCR5+PD1+ TFH cells was performed using flow cytometry (LSR Fortessa, BD Bioscience). One-way ANOVA with multiple comparisons was used for statistical analysis. No differences were found in CD3+ T lymphocytes between groups, but percentages of CD4+ TH cells within the T cell population were decreased in CLAD versus HC (p=0.005) and the same trend was seen in stable patients versus HC (p=0.07). The number of TFH cells within the TH cells was lower in HC (p=0.044) and in CLAD patients (p<0.0001) versus stable transplant patients. Circulating TFH cells are measurable in blood of lung transplant recipients by flow cytometry and are significantly decreased in patients with CLAD compared to stable lung transplant patients. TFH may play a role in immune activation after lung transplantation, however, further investigation is needed.

Impact of Human HLA-E and NKG2C Variants and of HCMV Ul40 Genotype Populations on HCMV Replication in Lung Transplant Recipients

Human Cytomegalovirus (HCMV) is an important viral pathogen in lung-transplant recipients (LTRs), which may cause tissue-invasive disease. The risk for HCMV-replication depends on HCMV-specific immune responses, which may limit viral spread after lung-transplantation. NKG2C+ Natural-Killer (NK-) cells limit HCMV replication, even under immunosuppressive therapy. NK-cell activation can be mediated by the NKG2C receptor, which binds to HLA-E molecules on the surface of HCMV-infected cells, and these are stabilized by an HCMV-encoded UL40 peptide. NKG2C, HLA-E and UL40 occur naturally as different variants. We investigated whether these variants differently influence the individual NK-cell response and have impact on the extent of HCMV-replication after lung transplantation. We included 137 lung transplant recipients (LTRs), who received the transplant between 2013 and 2016 at the Medical University of Vienna and who had either no or low HCMV replication (N=69) or developed high level viremia (>1000 copies HCMV DNA/ml plasma, N=68) post-transplantation. We assessed donor and recipient NKG2Cwt/del and HLA-E*0101/0103 variants as well as the UL40 peptide variants of the infecting HCMV strains. We further investigated UL40 kinetics by NGS and UL40 dependent NK-cell cytotoxicity and proliferation. In total, we detected 18 different UL40 peptides in low- and high viremic LTRs. Specific UL40 peptide variants were differently distributed in the blood of LTRs and the VMAPRTLIL variant occurred more frequently in highly viremic patients, while the VMTPRTLIL variant was detectable preferably in patients with low viremia (p=0.004). This difference was not associated with altered cytotoxicity, but with differences in the proliferation of NK-cells (p=0.002). In homozygous HLA-E recipients, UL40 strains seem to emerge over time towards low affinity. In addition, recipient encoded NKG2Cwt/del and donor encoded heterozygous HLA-E*0101/0103 variants were associated with development of high-level viremia in the follow-up (p=0.01 and p=0.007, respectively). The present data provide evidence that the NKG2C/UL40/HLA-E axis and variations thereof have a substantial impact on the patients' individual NK-cell response and on the development of high-level viremia post-transplantation.

Analysis of long term CD4+CD25highCD127- T-reg cells kinetics in peripheral blood of lung transplant recipients

BackgroundThe role of CD4+CD25highCD127− T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables.MethodsFrom jan 2009 to dec 2014, 137 lung Tx recipients were submitted to an immunological follow up (median: 105.9 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD4+CD25highCD127− T and FOXP3+ cells. We tested the association between T-reg and relevant variables by linear OR regression models for repeated measures, adjusting for time from Tx. Also, by ordered logistic models for panel data, the association between Chronic Lung Allograft Dysfuncton (CLAD) onset/progression and T-reg counts in the previous 3 months was tested.ResultsAmong all variables analyzed at multivariate analysis: Bronchiolitis Obliterans Syndrome (OR −6.51, p < 0.001), Restrictive Allograft Syndrome (OR −5.19, p = 0.04) and Extracorporeal photopheresis (OR −5.65, p < 0.001) were significantly associated to T-reg cell. T-reg cell counts progressively decreased according to the severity of CLAD. Furthermore, patients with higher mean T-reg counts in a trimester had a significantly lower risk (OR 0.97, p = 0.012) of presenting CLAD or progressing in the graft dysfunction in the following trimester.ConclusionsOur present data confirm animal observations on the possible role of T-reg in the evolution of CLAD.

(1164) - Preservation Solution Affects the Immunological Milieu in Lung Transplantation and Alters the Inflammatory Balance

The pro-/ anti-inflammatory balance in organ transplantation is shaped by the organ-specific immune compartment with its predominating receptor repertoire of NK and T cells. However, it remains unknown if the respective perfusion solution influences this microenvironment. Therefore, perfusion solution (PS) (Perfadex®, PER respectively Celsior®, CEL) but also peripheral blood of lung transplant recipients were analysed for their lymphocyte distributions and cytokine levels at different time points during lung transplantation.

The Presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection.

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.

Increased Numbers of Circulating CD8 Effector Memory T Cells before Transplantation Enhance the Risk of Acute Rejection in Lung Transplant Recipients

The effector and regulatory T cell subpopulations involved in the development of acute rejection episodes in lung transplantation remain to be elucidated. Twenty-seven lung transplant candidates were prospectively monitored before transplantation and within the first year post-transplantation. Regulatory, Th17, memory and naïve T cells were measured in peripheral blood of lung transplant recipients by flow cytometry. No association of acute rejection with number of peripheral regulatory T cells and Th17 cells was found. However, effector memory subsets in acute rejection patients were increased during the first two months post-transplant. Interestingly, patients waiting for lung transplant with levels of CD8+ effector memory T cells over 185 cells/mm3 had a significant increased risk of rejection [OR: 5.62 (95% CI: 1.08-29.37), p=0.04]. In multivariate analysis adjusted for age and gender the odds ratio for rejection was: OR: 5.89 (95% CI: 1.08-32.24), p=0.04. These data suggest a correlation between acute rejection and effector memory T cells in lung transplant recipients. The measurement of peripheral blood CD8+ effector memory T cells prior to lung transplant may define patients at high risk of acute lung rejection.

Prospective Study of Biomarkers of Immune Response in Lung Transplant Recipients

IntroductionStudies on biomarkers of tolerance in organ transplantation have been widely performed during the last decade. AimTo assess biomarkers in relation to evolution of the immune response among lung transplant recipients. MethodsThis multicenter study included 27 lung transplant recipients followed before as well as at 7, 14, 30, 60, 90, and 180 days posttransplantation. Biomarkers of the immune response based on flow cytometry technology were validated in each center. They included intracellular cytokine expression, regulatory T-cell level, as well as lymphocyte surface antigen and CD28 expressions. ResultsThe 13 patients who developed acute rejection episodes showed increased numbers of regulatory T cells at 12 months posttransplant. Sixteen patients experiencing infections displayed decreased expression of CD69 on CD8 T cells within the first year of follow-up. ConclusionHigh Treg levels in the peripheral blood of lung transplant recipients were associated with an increased risk of rejection but not infection. Inversely, we observed low levels of activated CD8 T cells in infected patients.

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 (T(IL-10)) reactive to col-V with significantly lower levels of IFN-gamma and IL-2 producing T cells (Th1 cells). The col-V specific T(IL-10) cells suppressed the proliferation and expansion of col-V specific Th1 cells by IL-10-dependent and contact-independent pathways. The T(IL-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 T(IL-10) cells with concomitant expansion of col-V-specific IFN-gammaproducing Th1 cells.

Kinetics of US28 Gene Expression during Active Human Cytomegalovirus Infection in Lung‐Transplant Recipients

Targeting viral proteins early during infection may limit exacerbation of human cytomegalovirus infection. The viral chemokine-receptor homologue US28 interferes with leukocyte trafficking and, possibly, viral replication. Because US28 molecules are abundant on the surface of infected cells, this homologue is a potential target for antiviral therapy. To assess the relationship between US28 and disease activity, we measured, by quantitative reverse-transcription polymerase chain reaction, the levels of US28 and immediate-early (IE) 1 gene transcripts in the blood of lung-transplant recipients. We found that, during primary and secondary infection, the IE1 and US28 genes have early transcription kinetics and are expressed at similar levels. This may render US28 an attractive target for antiviral therapy.

325: Impact of Campath-1H conditioning on DC subsets in peripheral blood of lung transplant recipients

Dendritic cells (DC) play a key role in immunity and in tolerance induction. Recent publications have shown that immunosuppressive drugs may target and modulate DC function both in vitro and in vivo. In this study, we have prospectively analyzed in peripheral blood of lung transplant patients, the impact of Campath-1H preconditioning combined with FK-506 and low dose Prednisone maintenance therapy on DC subset frequencies and kinetics.

Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients: correlation with transplant outcome.

The subset of CD4+CD25+ regulatory T cells, recently identified in humans, may play a central role in the regulation of immune tolerance to graft survival. This study assesses the frequency and functional profile of CD4+CD25+CD69- cells in the peripheral blood of lung transplant recipients (>3 years from transplantation), 10 of whom were in a stable clinical condition and 11 of whom demonstrated chronic rejection (bronchiolitis obliterans syndrome). We also studied a group of seven healthy subjects. The frequency of CD4+ T cells expressing CD25 (CD4+CD25+) and the highest levels (CD25) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P < or = 0.01). Purified CD4+CD25+ cells exhibited a regulatory functional profile in vitro: they were hyporesponsive, suppressed the proliferation of CD4+CD25- cells, and produced interleukin-10. These results provide in vivo evidence that peripheral CD4+CD25+ T cells may represent an important regulatory subset in lung transplantation.