Purpose Primary Graft Dysfunction (PGD) is a clinical syndrome that results from severe pulmonary ischemia reperfusion injury (IRI). We previously showed a role for Natural Killer (NK) cells recruited to airways in human PGD and mouse IRI. However, little is known of how NK cells traffic to airways during lung injury. Therefore, we hypothesized that NK cells home to airways via chemokine receptor signaling during pulmonary IRI. Methods We used the orthotopic lung transplant with prolonged ischemia (OLT-PCI) and hilar clamp (HC) mouse models of IRI (Figure A). Native lungs and sham surgeries served as controls, respectively. RNA sequencing was performed on OLT-PCI (n = 8) and control lungs (n = 8) with differential gene expression and pathway analyses. NK cells from lung, lymph node, blood and spleen among HC (n = 5) and sham (n = 5) mice were phenotyped by flow cytometry. Mice were treated with CCR5 blockade (n = 6, Maraviroc 10 mg/kg) or vehicle control (n = 6, 2% DMSO in castor oil) preceding IRI surgery. NK cells were assessed in bronchoalveolar (BAL) and lung digest. Statistical differences were determined by Kruskal-Wallis test with Mann-Whitney post-hoc testing and Bonferroni correction. Results Chemokine signaling pathways were increased in lung tissue during IRI experiments (Figure B, p < 0.0001). CCR5 and CCR1 receptor ligands (Figure C, CCL3 p = 0.007, CCL5 p = 0.02) were the most highly expressed chemokine transcripts. CCR1 (p = 0.007) and CCR5 (p = 0.01) receptors were increased on NK cells within the lung during IRI (Figure D). CCR5+ and CCR1+ NK cells co-expressed CD49a, a marker of tissue residence. In Maraviroc-treated mice, NK cell recruitment to the airways was blunted, as measured by BAL frequency (p = 0.02) and total cell counts (Figure E, p = 0.03) when compared to vehicle control. BAL tissue-resident NK cells (CD49a+, p = 0.005) and CCR5+ NK cells (p = 0.008) were decreased by CCR5 blockade. Conclusion NK cell airway trafficking during IRI is mediated by CCR5, suggesting a novel pathway for clinical intervention.

Purpose Telomere dysfunction may persist in the immune system following lung transplantation for idiopathic pulmonary fibrosis (IPF) and impact responses to infections or donor antigens. Mechanistically, critically short telomeres can activate p53 and block cell proliferation. We hypothesized that p53 activation might be associated with the impaired proliferation to alloantigens for IPF lung transplant recipients. Methods We analyzed 5 IPF lung transplant recipients with telomere dysfunction and 6 age-matched non-IPF controls, 18 months after transplantation, and 4 healthy donors. We quantified CD8+ T cell proliferation to pooled, donor-derived, stimulated B cells (sBc) with or without treatment with KML001, a reagent that binds and erodes telomeres. Expression of p53 and proportions of Annexin V+ apoptotic cells were quantified by flow cytometry. Statistical comparisons were performed using 2-way ANOVA with Dunnett's post-test. Results IPF lung transplant recipients (Fig. A) demonstrated impaired CD8+ T cell proliferation relative to non-IPF patients (Fig. B) and healthy controls (Fig. C&D, P≤0.0002). This proliferation was further impaired by telomere erosion (Fig. C&E, P<0.0001). P53 expression was increased in cells responding to sBc stimulation (MFI change 183, 95% CI 159-207). Among T cells that did not proliferate to allo-stimulation (Fig. F), p53 expression was higher in IPF subjects versus non-IPF (P=0.049) or healthy referents (P=0.002). Apoptotic cells were not different between groups. Conclusion P53 upregulation is associated with impaired CD8+ T cell proliferation to alloantigens in lung transplant recipients with IPF and may mediate proliferative arrest in CD8+T cells for IPF lung transplant recipients. These findings may explain sensitivity of some recipients to p53 activators, such as mycophenolic acid, that may synergize with telomere dysfunction to drive cell cycle arrest and leukopenia.

Purpose The potential for heterogenous pathobiology underlying physical frailty might explain the inconsistent associations observed between frailty and poor outcomes in lung transplantation. We hypothesize that molecular phenotyping could refine frailty measurement and ultimately tailor interventions. Methods In a three-center cohort of lung transplant candidates, we measured frailty by the Short Physical Performance Battery (SPPB); demographic and clinical variables; body composition by bioelectrical impedance, and 17 serum biomarkers reflecting commonly cited causes of frailty including inflammation, sarcopenia, adiposity, mitochondrial and neurohormonal dysfunction. Among those pre-frail or frail (SPPB <12), we first applied latent class modelling to the baseline data and then followed participants forward in time. The best-fitting model was determined by the Bayesian Information Criteria and the Vuong-Lo-Mendell-Rubin test. We tested predictive validity of these classes with disability by the Lung Transplant Valued Life Activities scale and by candidate delisting for becoming too ill or death. Results In 442 participants, a 3 latent class model best fit (p = 0.046; Figure 1A). Type A was characterized by higher serum biomarkers of inflammation and sarcopenia (lower muscle mass [ASMI] and grip strength); Type B by sarcopenia, high adiposity (VFA), and lack of inflammation, and Type C was relatively fit (higher ASMI and grip strength) and without inflammation (Figure 1B). Amongst the Type A endotype, 13% of participants died or were delisted compared to 6% in both Type B and C (p = 0.15). Participants comprising Type A reported worse disability (LT-VLA 1.7) than Types B and C (1.4 and 1.3, respectively) (p < 0.001) and had shorter time to delisting or death (1.4 vs 8.0 and 4.0 months, respectively) (p < 0.001). Conclusion We identified distinct biological phenotypes of frailty. A frailty phenotype characterized by systemic inflammation and sarcopenia appears to be associated with worse clinical outcomes.

BACKGROUND: Telomere dysfunction is associated with idiopathic pulmonary fibrosis (IPF) and worse outcomes following lung transplantation. Telomere dysfunction may impair immunity by upregulating p53 and arresting proliferation, but its influence on allograft-specific immune responses is unknown. We hypothesized that subjects undergoing lung transplantation for IPF would have impaired T cell pro-liferation to donor antigens. METHODS: We analyzed peripheral blood mononuclear cells (PBMC) from 14 IPF lung transplant recipients and 12 age-matched non-IPF subjects, before and 2 years after transplantation, as well as PBMC from 9 non-transplant controls. We quantified T cell proliferation and cytokine secretion to donor antigens. Associations between PBMC telomere length, measured by quantitative PCR, and T cell proliferation to alloantigens were evaluated with generalized estimating equation models. RESULTS: IPF subjects demonstrated impaired CD8+ T cell proliferation to donor antigens pre-trans -plant (p < 0.05). IL-2, IL-7, and IL-15 cytokine stimulation restored T cell proliferation, while p53 upregulation blocked proliferation. IPF subjects had shorter PBMC telomere lengths than non-IPF sub-jects (p < 0.001), and short PBMC telomere length was associated with impaired CD8+ T cell prolifer-ation to alloantigens (p = 0.002). CONCLUSIONS: IPF as an indication for lung transplant is associated with short PBMC telomere length and impaired T cell responses to donor antigens. However, the rescue of proliferation following cyto-kine exposure suggests that alloimmune anergy could be overcome. Telomere length may inform immunosuppression strategies for IPF recipients. J Heart Lung Transplant 2022;41:641-653 (c) Published by Elsevier Inc.

PurposePrimary graft dysfunction (PGD) occurs in 1/3 of all lung transplants. It is defined by epithelial dysfunction and innate immune cell infiltration. Natural killer (NK) cells are innate lymphocytes that are activated by NKG2D receptor binding of specific stress molecules. We hypothesized that NK cells mediate PGD by recognizing stress molecules induced on lung epithelial cells and causing injury via direct cytotoxicity.MethodsIn an established experimental PGD model, left hilar clamp (HC) was compared to sham (S) surgery in C57BL/6 mice. Stress molecules (RAE-1, MULT1) and NK cell receptors were measured by flow cytometry median fluorescent intensity (MFI) on dissociated lung cells. Mice were given blocking anti-NKG2D or isotype control antibodies preceding HC. Human stress molecules (MICA, MICB, ULBP1, 3, and 2/5/6) were measured by flow cytometry on bronchial epithelial cells incubated for 4 hours in hypoxia (1% O2) versus normoxia conditions. The Mann-Whitney U test was applied for pairwise comparisons.ResultsNK cells were increased as a percent of lymphocytes (p = 0.008) and by count (p = 0.04) in HC (n = 5) versus S (n = 5) lungs and infiltrated airways (Figure 1A). The NKG2D receptor was increased on NK cells following HC (p = 0.005). The stress molecules MULT1 (p = 0.0002) and RAE-1 (Figure 1B, p = 0.002) were increased on epithelial cells in HC compared to S lungs. NKG2D blockade (n = 9) resulted in less pulmonary edema following HC compared to isotype control antibody (Figure 1C, n = 9, p = 0.04). Hypoxic human bronchial epithelial cells (n = 6) had increased MICB (p = 0.008), ULBP-1 (p = 0.05), and ULBP-2,5,6 (Figure 1 D, p = 0.004) compared to normoxic cells (n = 6).ConclusionPGD induces NK cell stress ligands leading to NK cell recruitment that contributes to PGD. NKG2D blockade, under investigation in clinical trials of other diseases, resulted in decreased lung injury. The induction of human NKG2D stress molecules during hypoxia suggests that this NK cell receptor-ligand interaction may mediate PGD. Primary graft dysfunction (PGD) occurs in 1/3 of all lung transplants. It is defined by epithelial dysfunction and innate immune cell infiltration. Natural killer (NK) cells are innate lymphocytes that are activated by NKG2D receptor binding of specific stress molecules. We hypothesized that NK cells mediate PGD by recognizing stress molecules induced on lung epithelial cells and causing injury via direct cytotoxicity. In an established experimental PGD model, left hilar clamp (HC) was compared to sham (S) surgery in C57BL/6 mice. Stress molecules (RAE-1, MULT1) and NK cell receptors were measured by flow cytometry median fluorescent intensity (MFI) on dissociated lung cells. Mice were given blocking anti-NKG2D or isotype control antibodies preceding HC. Human stress molecules (MICA, MICB, ULBP1, 3, and 2/5/6) were measured by flow cytometry on bronchial epithelial cells incubated for 4 hours in hypoxia (1% O2) versus normoxia conditions. The Mann-Whitney U test was applied for pairwise comparisons. NK cells were increased as a percent of lymphocytes (p = 0.008) and by count (p = 0.04) in HC (n = 5) versus S (n = 5) lungs and infiltrated airways (Figure 1A). The NKG2D receptor was increased on NK cells following HC (p = 0.005). The stress molecules MULT1 (p = 0.0002) and RAE-1 (Figure 1B, p = 0.002) were increased on epithelial cells in HC compared to S lungs. NKG2D blockade (n = 9) resulted in less pulmonary edema following HC compared to isotype control antibody (Figure 1C, n = 9, p = 0.04). Hypoxic human bronchial epithelial cells (n = 6) had increased MICB (p = 0.008), ULBP-1 (p = 0.05), and ULBP-2,5,6 (Figure 1 D, p = 0.004) compared to normoxic cells (n = 6). PGD induces NK cell stress ligands leading to NK cell recruitment that contributes to PGD. NKG2D blockade, under investigation in clinical trials of other diseases, resulted in decreased lung injury. The induction of human NKG2D stress molecules during hypoxia suggests that this NK cell receptor-ligand interaction may mediate PGD.

Purpose Extracorporeal photopheresis (ECP) has been advocated as a therapy for chronic lung allograft dysfunction (CLAD), despite an absence of randomized controlled trials to support its use. ECP is postulated to alter the recipient lymphocyte population favoring regulatory over effector T cells, as well as altering cytokine profiles. We hypothesized that ECP would result in selective deletion of donor specific T cells, as identified by T-cell receptor (TCR) sequences. Methods For five subjects undergoing ECP, we performed TCR sequencing on peripheral blood collected peri-transplant, (0-14 weeks) pre-ECP, and (6-20 weeks) post-ECP. Donor-reactive recipient TCR sequences within the peri-transplant sample were identified by sequencing the cells which expanded during a mixed lymphocyte reaction against donor stimulated B cells. We compared the overlap with donor reactive sequences, Jaccard similarity (intersection over union) to the peri-transplant sample, Shannon alpha-diversity, and evenness between pre-and post-ECP samples using Pearson's correlation and paired Student's t-tests. Results The donor-reactive TCR proportions were correlated (r=0.91, p=0.03) but unchanged (p=0.51, Figure 1A) following ECP. TCR similarity to the peri-transplant sample (Figure 1B, p=0.12), alpha-diversity (p=0.37) and evenness (p=0.59) were also unchanged. Conclusion TCR sequencing demonstrated stability of donor-reactive T cell clones, which were readily identifiable in before and after ECP peripheral blood samples from this cohort. While these findings do not support the hypothesis that ECP results in deletion of donor-reactive T cell clones in patients with CLAD, more subtle effects might be apparent from larger cohorts.