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Primary graft dysfunction (PGD) is one of the leading causes of early mortality following lung transplantation (LTx). Here we investigated the correlation between PGD early after lung transplantation and the subsequent development of transplant atherosclerosis (TA) in a humanized mouse model. The pericardio-phrenic artery obtained from human donor lungs was implanted into the aorta of NOD/Rag-/-/IL-2rγc-/- mice reconstituted with either naive or primed allogeneic peripheral blood mononuclear cells (PBMC), that were obtained from the respective lung recipient either before or 21 days after transplantation. Parallel to the previously described experimental groups, there was another group. Here, T regs were enriched and additionally injected with the PBMC. Exactly as mentioned before, on day 0 and day 21. PGD was defined according to the 2017 ISHLT Guidelines. Histological assessment after 28 days showed significantly more intimal hyperplasia of arteries in mice reconstituted with PBMC from patients with PGD (24 hours after transplantation; score 2 or 3) compared to patients without PGD (score 0 or 1). The attached figure shows the measured occlusion in the individual groups. Enriching of PBMC with CD4+CD25high regulatory T cells (Treg) subpopulation, suppressed the development of TA in both PGD groups. TA was similarly suppressed by enriching of naive or alloantigen-primed Treg. Mice transplanted with arteries from donors undergoing PGD score 2 or 3 developed higher serum concentrations IFN-γ (PGD: 1129,9 conc. Pg/ml vs noPGD: 577,24 conc. Pg/ml), a pro-inflammatory TH1 signature cytokine. We conclude that both donor tissue inflammation and recipient T cell responses contribute to PGD, eventually triggering rejection. This inflammatory response can be suppressed by early supplementation with Treg cells, which suggests a potential target for future early interventions in lung transplantation. [ABSTRACT FROM AUTHOR]

After lung transplantation (LuTx), a transient chimerism of donor cells can be observed in recipient blood due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor T and NK cells and to investigate their potential origin from tissue-resident memory (TRM) T and NK cells. Lymphocyte dynamics in recipient blood were determined in n=97 LuTx patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry. Donor T and NK cells were analyzed by HLA class I allele-specific mAb in n=44 LuTx recipients. T and NK cell markers were used to discriminate circulating from TMR subsets in blood, organ storage solutions (perfusates, n=97), explanted lung parenchyma (n=28) and donor trachea (n=17). Single cell mRNA sequencing of explant lung parenchyma was conducted (n=16). In peripheral blood of all recipients, donor-derived T and NK cells were detected at T0, T24 and 3 wks after LuTx, showed higher CD69 expression compared to recipient cells (p=0.01 to 0.04) and were mostly CCR7− memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In recipient parenchyma and donor trachea, most CD69+ T and NK cells showed coexpression of TRM markers (CD103, CD49a, PD-1) with significant enrichment in trachea (p<0.05). These other TRM markers were not found in circulating donor T and NK cells or in perfusates, arguing for different memory T and NK cell subsets. Sc-mRNA sequencing confirmed distinct TRM and TRM-like T cell subsets in lung parenchyma according to separate expression profiles. The presence of TRM T and NK cells did not have an impact on primary graft dysfunction (PGD) 24h after LuTx. Yet, patients with high frequencies of donor T cells showed a trend towards CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). Our results demonstrate that donor T and NK cells found in recipient blood immediately after LuTx are separate subsets from circulating as well as pulmonary TRM T and NK cells, since they express CD69 but lack expression of other classical TRM markers. Donor T cells may be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features. [ABSTRACT FROM AUTHOR]

Purpose In previous studies, we identified donor lymphocytes in peripheral blood of recipients directly after transplantation and persisting at least three weeks that were characterized by the tissue retention marker CD69. In order to determine their origin, we hypothesized that donor T and NK cells have a peculiar tissue-resident memory phenotype that is shared between cells derived from lung perfusates, trachea parenchyma and lymph nodes. Methods Donor lymphocytes in recipient blood were determined in 27 lung transplant patients at T0, T24, 3 weeks by staining of donor HLA class I molecules in combination with lineage- and tissue-specific markers using flow cytometry. The phenotype of T and NK cells in perfusates (n=30), donor trachea (n=5), and lymph node (n=10), recipient explanted parenchyma (n=15) was compared to circulating cells using the same markers. Results In peripheral blood of all lung transplant recipients, donor derived T and NK cells were detected at T0, T24 and 3 weeks and had higher CD69 expression compared to recipient cells (p=0.001 to 0.03) and were mostly CD25−. This phenotype was similar to T and NK cells in corresponding perfusates, with significantly increased CD69 expression compared to circulating PBMCs (all p<0.005) without CD25. Perfusate, trachea and donor NK cells, were CD56dim CD16+, in contrast to lung-draining lymph nodes that only possess CD56bright CD16− NK cells. T cells from trachea and lymph node showed also high CD69 expression and a significant enrichment of effector memory (CCR7− CD45RO+) T cells (all p<0.03). In donor trachea and recipient parenchyma, CD69+ T cells showed coexpression of other tissue residency markers such as CD103, CD49a and PD-1, while these were not found in perfusates, highlighting the differences between these compartments. Conclusion Our results suggest that donor T and NK cells found in the periphery of lung transplant recipients are derived from lung parenchyma and represent tissue-resident memory cells. This transient chimerism in recipient blood might be clinically relevant for tolerance induction after transplantation due to unique features of TRM T and NK cells. [ABSTRACT FROM AUTHOR]

Purpose Regulatory T cells (Treg) may counteract chronic lung allograft dysfunction (CLAD). In the present study, we analyzed Treg in peripheral blood at 3 weeks and at 3, 6, 12 and 24 months after lung transplantation and correlated the percentages of 3 Treg subpopulations at 3 weeks with the later incidence of CLAD and graft survival. Methods Among lung-transplanted patients between January 2009 and October 2018, only adult patients with Treg measurements after transplantation were included into the study. Tregs were detected in peripheral blood and defined as CD4+CD25highT cells and further analyzed for CD127lowand FoxP3+ using FACS analysis. CD4+CD25highIL2+ T cells were also evaluated. Associations of Treg with CLAD and graft survival, defined as patient survival and freedom from re-transplantation, were evaluated using Cox analysis. Results Among the 1,089 lung-transplanted adult patients at our institution, 830 (76%) patients had Treg measurements after transplantation. Frequencies (%) of CD127low and IL2+ but not of FoxP3+ cells were significantly higher in patients without CLAD than in patients with CLAD at each time point after transplantation (Figure 1). Similar results were found for graft survival. Treg frequencies were higher in patients treated with tacrolimus vs. cyclosporine. Follow-up was 42±29 months. Freedom (%) from CLAD was 66 at 5 years. At the Cox analysis, increasing frequencies of CD127low (HR=0.988, p<0.001) and of IL2+ Tregs (HR=0.989, p=0.016) but not of FoxP3+ (HR=0.991, p=0.11) were protective against CLAD. Graft survival (%) amounted to 68 at 5 years. Only increasing frequencies of CD127low Tregs (HR=0.992, p=0.032) were protective against mortality or retransplantation. Conclusion A higher frequency of CD127low Tregs early after lung transplantation is protective against CLAD and associated with better survival. The role of CD4+CD25highIL2+ T cells and the effect of tacrolimus on Treg deserve further investigation. [ABSTRACT FROM AUTHOR]

After lung transplantation (LuTx), a transient chimerism of donor cells exists in the blood of recipients due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor CD8+/CD4+ T and NK cells and to investigate whether they might represent tissue-resident memory (TRM) cells. Lymphocyte dynamics in recipient blood were determined in 97 LuTx patients directly (T0), 24h (T24) and 3 (wks) weeks after LuTx using flow cytometry with lineage-, tissue-, and donor HLA class I allele-specific mAb. The same makers were used to determine the phenotype of lymphocytes present in perfusate (n=102), recipient explanted lung parenchyma (n=28) and donor trachea (n=17). In peripheral blood of all recipients, donor-derived CD4+/CD8+ T and CD56dim NK cells were detected at T0, T24 and 3 wks after LuTx and had higher CD69 expression compared to recipient cells (p=0.01 to 0.04), were mostly CCR7− memory cells and CD25−. This phenotype was similar to T and NK cells in corresponding perfusates. In recipient parenchyma and donor trachea, most CD69+ T and NK cells showed coexpression of other tissue residency markers such as CD103, CD49a and PD-1 with significant enrichment in trachea (p<0.05). In contrast, these markers were not found in circulating donor lymphocytes and perfusates, indicating that they represent distinct memory T and NK subsets. Donor T and NK cells showed higher IFN-g production with and w/o PMA/Iono stimulation compared to recipient cells (p<0.05). The presence of these particular TRM cells did not have an impact on the development of PGD 24h after transplantation. However, patients with high frequencies of donor T cells showed a trend towards a CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). Our results demonstrate that donor T and NK cells found in the periphery of LuTx recipients are a distinct subset from circulating lymphocytes and TRM cells present in lung tissue, since they express CD69 but lack expression of other classical TRM markers. They display a higher functional capacity despite the onset of immunosuppression. Donor T cells might be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features. [ABSTRACT FROM AUTHOR]

After lung transplantation (LuTx), a transient chimerism of donor cells exists in the blood of recipients due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor CD8+ and CD4+ T and NK cells and to investigate whether they might represent tissue-resident memory (TRM) cells. Lymphocyte dynamics in recipient blood were determined in 97 lung transplant patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry with lineage-, tissue-, and donor HLA class I allele-specific mAb. The same makers were used to determine the phenotype of lymphocytes present in organ storage solution (perfusate, n=102), recipient explanted lung parenchyma (n=28) and donor trachea (n=17). In peripheral blood of all recipients, donor-derived CD4+ and CD8+ T and CD56dim NK cells were detected at T0, T24 and 3 wks after LuTx and had higher CD69 expression compared to recipient cells (p<0.05) and were mostly CD25− CCR7− memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In lung parenchyma and trachea, most CD69+ T and NK cells showed coexpression of other tissue residency markers such as CD103, CD49a and PD-1 with significant enrichment in trachea (p<0.05). These markers were not found in circulating donor lymphocytes and perfusates, indicating that they represent distinct memory T and NK subsets. Donor T and NK cells showed higher IFN-γ production with PMA/ionomycin stimulation compared to recipient cells (p<0.05). The presence of these particular TRM cells did not have an impact on the development of PGD 24h after LuTx. However, patients with high frequencies of donor T cells showed a trend towards a CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). Our results demonstrate that donor T and NK cells found in the blood of LuTx recipients are a distinct subset from circulating lymphocytes and TRM cells present in lung tissue, since they express CD69 but lack expression of other classical TRM markers. They display a higher functional capacity despite the onset of immunosuppression. Donor T cells might be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features. [ABSTRACT FROM AUTHOR]

Purpose The appearance of passenger donor lymphocytes in recipient blood after double lung transplantation has been described decades ago. However, neither their early kinetics, nor distribution of lymphocyte subsets and clinical relevance have been addressed in detail. Therefore, we investigated frequencies and phenotypes of donor T and NK cells within the first 24 hours and at 3 weeks after lung transplantation and correlated them to important clinical outcomes. Methods Blood and perfusion solutions of 59 lung recipients (30 male, 29 female, median age 51) were analysed pre Tx, at T0, T24 and at three weeks (3wk) for NK and T cell subsets. In a subset of 20 patients with standard cold donor lung preservation and 9 preserved with portable ex vivo lung preservation (OCS Lung), donor T and NK cells were identified in blood by staining of donor HLA alleles (HLA-A1,A2,A11,A24) combined with lineage markers. Frequencies of donor lymphocytes were correlated to cold ischemic time (CIT), primary graft dysfunction (PGD) and chronic lung allograft dysfunction (CLAD). Results In all lung recipients, the NK cell frequency was significantly increased at T0 and T24 (p=0.04), CD4 T cells decreased and CD8 T cells remained stable. No significant differences were seen between recipients of standard vs OCS preserved lungs. Donor NK cells comprised 18.8% at T0, 17.1% at T24 and 7.8% at 3 wk (p<0.001) of circulating NK cells. Frequencies were for donor CD8+ T cells 8.3%, 6.6% and 2.6%, and for CD4+ donor T cells 6.4%, 4.6% and 1.3% of the respective subset. At T0, significantly less donor NK cells were detected in recipients of OCS lungs (p<0.008), while T cells were reduced non-significantly. No correlation between donor NK or T cell frequencies was observed for CIT or PGD. In the limited number of patients at risk, a trend towards higher early donor T cell frequencies in recipients not developing CLAD at two years after Tx was observed (p=0.04). Conclusion Donor T and NK cells are detectable in blood of all lung recipients during the first 3 weeks after Tx and did not correlate with CIT or PGD. Preservation with portable EVLP resulted in decreased NK cell frequencies which might be explained by their ex vivo mobilization. Furthermore, transient donor chimerism might have a protective effect against CLAD development. [ABSTRACT FROM AUTHOR]