Impact of age and telomere length on circulating T cells and rejection risk after lung transplantation for idiopathic pulmonary fibrosis.

BACKGROUND: Most idiopathic pulmonary fibrosis (IPF) lung transplant recipients (IPF-LTRs) have short telomere (ST) length. Inherited mutations in telomere-related genes are associated with the development of T cell immunodeficiency. Despite this, IPF-LTRs with telomere-related rare variants are not protected from acute cellular rejection (ACR). We set out to determine the impact of both age and telomere length on the circulating T cell compartment and ACR burden of IPF-LTRs. METHODS: We identified 106 IPF-LTRs who had telomere length testing using flowFISH (57 with short telomeres and 49 with long telomeres) as well as a subset from both cohorts who had cryopreserved PBMC at least 1 time point, 6 months posttransplantation. Circulating T cells from before transplantation and at 6 and 12 months posttransplantation were analyzed using multiparameter flow cytometry to study phenotype and functional capacity, and bulk T cell receptor sequencing was performed to study repertoire diversity. Linear regression was used to study the relationship of age and telomere length on early (within 1 year) and late (between 1 and 2 years) ACR. RESULTS: IPF-LTRs with ST were found to have premature "aging" of their circulating T cell compartment, with age-agnostic elevations in posttransplant terminal differentiation of CD8+ T cells, increased granzyme B positivity of both CD8+ and CD4+ T cells, upregulation of the exhaustion marker, CD57, and chemotactic protein CCR5, and enhanced T cell receptor clonal expansion. Additionally, we found a significant decline in early ACR burden with increasing age, but only in the ST cohort. CONCLUSIONS: IPF-LTRs with ST have premature "aging" of their circulating T cell compartment posttransplantation and a clear age-related decline in ACR burden.

Lung transplant recipients with telomere-mediated pulmonary fibrosis have increased risk for hematologic complications.

Idiopathic pulmonary fibrosis lung transplant recipients (IPF-LTRs) are enriched for short telomere length (TL) and telomere gene rare variants. A subset of patients with nontransplant short-TL are at increased risk for bone marrow (BM) dysfunction. We hypothesized that IPF-LTRs with short-TL and/or rare variants would be at increased risk for posttransplant hematologic complications. Data were extracted from a retrospective cohort of 72 IPF-LTRs and 72 age-matched non-IPF-LTR controls. Genetic assessment was done using whole genome sequencing or targeted sequence panel. TL was measured using flow cytometry and fluorescence in-situ hybridization (FlowFISH) and TelSeq software. The majority of the IPF-LTR cohort had short-TL, and 26% of IPF-LTRs had rare variants. Compared to non-IPF controls, short-TL IPF-LTRs were more likely to have immunosuppression agents discontinued due to cytopenias (P = .0375), and BM dysfunction requiring BM biopsy was more prevalent (29% vs 4%, P = .0003). IPF-LTRs with short-TL and rare variants had increased requirements for transfusion and growth factor support. Multivariable logistic regression demonstrated that short-TL, rare variants, and lower pretransplant platelet counts were associated with BM dysfunction. Pretransplant TL measurement and genetic testing for rare telomere gene variants identified IPF-LTRs at increased risk for hematologic complications. Our findings support stratification for telomere-mediated pulmonary fibrosis in lung transplant candidates.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

CD4+ T-Cell Dysfunction in Severe COVID-19 Disease Is Tumor Necrosis Factor-α/Tumor Necrosis Factor Receptor 1-Dependent.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

Lung transplantation for idiopathic pulmonary fibrosis enriches for individuals with telomere-mediated disease.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most common indication for lung transplantation in North America and variants in telomere-maintenance genes are the most common identifiable cause of IPF. We reasoned that younger IPF patients are more likely to undergo lung transplantation and we hypothesized that lung transplant recipients would be enriched for individuals with telomere-mediated disease due to the earlier onset and more severe disease in these patients. METHODS: Individuals with IPF who underwent lung transplantation or were evaluated in an interstitial lung disease specialty clinic who did not undergo lung transplantation were examined. Genetic evaluation was completed via whole genome sequencing (WGS) of 426 individuals and targeted sequencing for 5 individuals. Rare variants in genes previously associated with IPF were classified using the American College of Medical Genetics guidelines. Telomere length from WGS data was measured using TelSeq software. Patient characteristics were collected via medical record review. RESULTS: Of 431 individuals, 149 underwent lung transplantation for IPF. The median age of diagnosis of transplanted vs non-transplanted individuals was significantly younger (60 years vs 70 years, respectively, p<0.0001). IPF lung transplant recipients (IPF-LTRs) were twice as likely to have telomere-related rare variants compared to non-transplanted individuals (24% vs 12%, respectively, p=0.0013). IPF-LTRs had shorter telomeres than non-transplanted IPF patients (p=0.0028) and >85% had telomeres below the age-adjusted mean. Post-transplant survival and CLAD were similar amongst IPF-LTRs with rare variants in telomere-maintenance genes compared to those without, as well as in those with short telomeres versus longer telomeres. CONCLUSIONS: There is an enrichment for telomere-maintenance gene variants and short telomeres among IPF-LTRs. However, transplant outcomes of survival and CLAD do not differ by gene variants or telomere length within IPF-LTRs. Our findings support individual with telomere-mediated disease should not be excluded from lung transplantation and focusing research efforts on therapies directed toward individuals with short-telomere mediated disease.

Idiopathic pulmonary fibrosis lung transplant recipients are at increased risk for EBV-associated posttransplant lymphoproliferative disorder and worse survival.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (EBV-PTLD) is a serious complication in lung transplant recipients (LTRs) associated with significant mortality. We performed a single-center retrospective study to evaluate the risks for PTLD in LTRs over a 7-year period. Of 611 evaluable LTRs, we identified 28 cases of PTLD, with an incidence of 4.6%. Kaplan-Meier analysis showed a decreased freedom from PTLD in idiopathic pulmonary fibrosis (IPF)-LTRs (P < .02). Using a multivariable Cox proportional hazards model, we found IPF (hazard ratio [HR] 3.51, 95% confidence interval [CI] 1.33-8.21, P = .01) and alemtuzumab induction therapy (HR 2.73, 95% CI 1.10-6.74, P = .03) as risk factors for PTLD, compared to EBV mismatch (HR: 34.43, 95% CI 15.57-76.09, P < .0001). Early PTLD (first year) was associated with alemtuzumab use (P = .04), whereas IPF was a predictor for late PTLD (after first year) (P = .002), after controlling for age and sex. Kaplan-Meier analysis revealed a shorter time to death from PTLD in IPF LTRs compared to other patients (P = .04). The use of alemtuzumab in EBV mismatch was found to particularly increase PTLD risk. Together, our findings identify IPF LTRs as a susceptible population for PTLD. Further studies are required to understand the mechanisms driving PTLD in IPF LTRs and develop strategies to mitigate risk.