Background Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality post lung transplantation (LTx). We sought to determine 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 ELSIPOT. 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. 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 IFNγ (p<0.05) with decreased IL-10 (p<0.05) compared to BOS- patients. Conclusion 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 important in preventing the development of chronic rejection.

Background Primary graft dysfunction (PGD) is a known risk factor for bronchiolitis obliterans syndrome (BOS) following 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. PGD and BOS were diagnosed based on ISHLT guidelines. Antibodies to self-antigens K alpha-1 tubulin, collagen type V and collagen I, were quantitated using standardized ELISA while cytokines were analyzed using Luminex. HLA-antibodies were measured using Flow-PRA. Results Lung transplant recipients with pre-transplant antibodies to self-antigens had increased risk of PGD (Odds 3.09, 95% CI 1.2 – 8.1, p=0.02) compared to those without. Conversely, in patients with PGD, 34.7% were positive for pre-transplant antibodies while in the PGD negative group only 14.6% had antibodies (p=0.03). Antibody positive patients demonstrated high levels of pro-inflammatory cytokines IL-1 (2.1 fold increase), IL-2 (3.0), IL-12 (2.5), IL-15 (3.0) and chemokines IP-10 (3.9) and MCP-1 (3.1, p<0.01 for all). On 5-yr follow-up, patients without antibodies showed greater freedom from development of HLAantibodies compared to those with antibodies (Class I:67% versus 38%, p=0.001; Class II: 71% Vs 41%, p<0.001 ). Patients with pre-transplant antibodies were found to have an independent relative risk of 2.3 (95% CI 1.7 – 4.5, p=0.009) for developing BOS. Conclusions Presence of antibodies to self-antigens pre-transplant increases the risk of PGD immediately post-transplant period and BOS on long-term follow-up. PGD is associated with an inflammatory cascade that augments the alloimmune (anti-HLA) response that predisposes to BOS.

Background-Primary graft dysfunction (PGD) in the immediate post-lung transplant period strongly increases the risk of chronic rejection (BOS). Here we hypothesized that PGD-induced inflammation augments alloimmunity, thereby predisposing to BOS.

Chronic human lung allograft rejection is manifested by bronchiolitis obliterans syndrome (BOS).

Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations. The ability to enrich for chrondroprogenitors and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications.

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection. The method involves the initial development of a panel of probes in which each probe will specifically target a unique sequence on human leucocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR (ddPCR) used as a quantitative assay to accurately track the trace amount of DcfDNA in an ample excess of recipient DNA background. The average false positive rate noted was about 1 per 800,000 molecules. Serially 2-fold diluted cfDNA, representing donor fractions of cfDNA, were spiked into a constant level of cfDNA representing the recipient cfDNA. The fraction of spiked cfDNA was measured and quantitative linearity was observed across seven serially diluted cfDNA samples. We were able to measure the minor portion of cfDNA as low as 0.2% of total cfDNA. We subsequently applied the method to a pilot set of 18 LTx recipients grouped into biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or stable groups. Serial plasma samples were used to identify the percentage of DcfDNA over total cfDNA. The level of DcfDNA was significantly elevated in patients diagnosed with acute rejection (10.30 ± 2.80, n=18), compared to that from stable (1.71 ± 0.50, n=24) or from BOS patients (2.52 ± 0.62, n=20). In conclusion, we present results validating the application of digital PCR to quantify DcfDNA assay in primary clinical specimens, which demonstrate that DcfDNA can be used as an early non-invasive biomarker for acute lung allograft rejection.