Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing.

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 (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65-76.

Tuning the Response of Synthetic Mechanogenetic Gene Circuits using Mutations in TRPV4.

Conventional gene therapy approaches for drug delivery generally rely on constitutive expression of the transgene and thus lack precise control over the timing and magnitude of delivery. Synthetic gene circuits with promoters that are responsive to user-defined stimuli can provide a molecular switch that can be utilized by cells to control drug production. Our laboratory has previously developed a mechanogenetic gene circuit that can deliver biologic drugs, such as interleukin-1 receptor antagonist (IL-1Ra), on-demand via the activation of TRPV4, a mechanosensory ion channel that has been shown to be activated transiently in response to physical stimuli such as physiologic mechanical loading or hypo-osmotic stimuli. The goal of this study was to use mutations in TRPV4 to further tune the response of this mechanogenetic gene circuit. Human iPSC-derived chondrocytes harboring targeted gain-of-function mutations of TRPV4 were chondrogenically differentiated. Both mutants - V620I and T89I - showed greater total IL-1Ra production compared to wild type following TRPV4 agonist treatment as well as mechanical or osmotic loading, but with altered temporal dynamics. Gene circuit output was dependent on the degree of TRPV4 activation secondary to GSK101 concentration or strain magnitude during loading. V620I constructs secreted more IL-1Ra compared to T89I across all experimental conditions, indicating that two mutations that cause similar functional changes to TRPV4 can result in distinct circuit activation profiles that differ from wild-type cells. In summary, we successfully demonstrate proof-of-concept that point mutations in TRPV4 that alter channel function can be used to tune the therapeutic output of mechanogenetic gene circuits.

A Damaging COL6A3 Variant Alters the MIR31HG-Regulated Response of Chondrocytes in Neocartilage Organoids to Hyperphysiologic Mechanical Loading.

The pericellular matrix (PCM), with its hallmark proteins collagen type VI (COLVI) and fibronectin (FN), surrounds chondrocytes and is critical in transducing the biomechanical cues. To identify genetic variants that change protein function, exome sequencing is performed in a patient with symptomatic OA at multiple joint sites. A predicted damaging variant in COL6A3 is identified and introduced by CRISPR-Cas9 genome engineering in two established human induced pluripotent stem cell-derived in-vitro neocartilage organoid models. The downstream effects of the COL6A3 variant on the chondrocyte phenotypic state are studied by a multi-omics (mRNA and lncRNA) approach in interaction with hyper-physiological mechanical loading conditions. The damaging variant in COL6A3 results in significantly lower binding between the PCM proteins COLVI and FN and provokes an osteoarthritic chondrocyte state. By subsequently exposing the neocartilage organoids to hyperphysiological mechanical stress, it is demonstrated that the COL6A3 variant in chondrocytes abolishes the characteristic inflammatory signaling response after mechanical loading with PTGS2, PECAM1, and ADAMTS5, as central genes. Finally, by integrating epigenetic regulation, the lncRNA MIR31HG is identified as key regulator of the characteristic inflammatory signaling response to mechanical loading.

Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells.

The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis.

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.

Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.

BACKGROUND: Articular cartilage shows little or no capacity for intrinsic repair, generating a critical need of regenerative therapies for joint injuries and diseases such as osteoarthritis. Human-induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering and in vitro human disease modeling; however, off-target differentiation remains a challenge during hiPSC chondrogenesis. Therefore, the objective of this study was to identify cell surface markers that define the true chondroprogenitor population and use these markers to purify iPSCs as a means of improving the homogeneity and efficiency of hiPSC chondrogenic differentiation. METHODS: We used a CRISPR-Cas9-edited COL2A1-GFP knock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population. Single-cell RNA sequencing was then used to analyze the distinct clusters within the population. An unpaired t test with Welch's correction or an unpaired Kolmogorov-Smirnov test was performed with significance reported at a 95% confidence interval. RESULTS: Chondroprogenitors expressing CD146, CD166, and PDGFRß, but not CD45, made up an average of 16.8% of the total population. Under chondrogenic culture conditions, these triple-positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single-cell RNA sequencing with fewer clusters (9 clusters in unsorted vs. 6 in sorted populations) closer together. Additionally, there was more robust and homogenous matrix production (unsorted: 1.5 ng/ng vs. sorted: 19.9 ng/ng sGAG/DNA; p < 0.001) with significantly higher chondrogenic gene expression (i.e., SOX9, COL2A1, ACAN; p < 0.05). CONCLUSIONS: Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146+/CD166+/PDGFRß+/CD45- and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.

Early posttransplant inflammation promotes the development of alloimmunity and chronic human lung allograft rejection.

BACKGROUND: Chronic human lung allograft rejection, represented by bronchiolitis obliterans syndrome (BOS), is the single most important factor that limits the long-term survival following lung transplantation (LT). However, the pathogenesis of BOS remains unclear. We hypothesized that the early posttransplant inflammation would promote the development of donor anti-human leukocyte antigen (HLA) alloimmunity and predispose to BOS. METHODS: Serum levels of interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, IP-10, MIG, MCP-1, MIP-1alpha, MIP-1beta, RANTES, tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-gamma, granulocyte-macrophage colony-stimulating factor, IL-1Ralpha, and IL-2R were serially analyzed in 31 BOS+ and matched 31 BOS- patients using quantitative multiplex bead immunoassays. Donor-specific HLA class II cellular immunity was analyzed using enzyme-linked immunospot (ELISPOT) by testing recipient peripheral blood mononuclear cells against mismatched donor HLA-DR peptides. Anti-HLA class II antibodies were monitored using flow panel reactive antibodies. RESULTS: There was early posttransplant elevation in basal serum levels of proinflammatory chemokines IP-10 and MCP-1 and Th1-cytokines IL-1beta, IL-2, IL-12, and IL-15 in BOS+ patients, compared to BOS- and normal subjects. In addition, a threefold decline in IL-10 levels was found during BOS development. BOS+ patients revealed increased development of HLA class II alloantibodies and Th1-predominant donor-specific cellular immunity with high frequency of IFN-gamma and low IL-5 producing T-cells. CONCLUSION: Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.

Elevated soluble CD30 characterizes patients with hepatitis C virus-induced liver allograft cirrhosis.

Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) significantly accelerates progression to allograft cirrhosis. Current biochemical parameters to monitor progression of chronic HCV after OLT have yielded low specificity and sensitivity. Here we investigated the HCV-specific immunity and serum levels of soluble CD30 (sCD30), a novel marker of Th2 immunity, in patients with and without allograft cirrhosis. Patients with hepatic inflammation but no cirrhosis (HIN, n=20) revealed elevated serum interferon (IFN)-gamma and high frequency of IFN-gamma producing CD4 T(h1) cells compared to those with hepatic cirrhosis (HFC, n=20) that had high interleukin (IL)-5 and IL-5 producing CD4 T(h2) cells. Patients with HFC, but not HIN, were found to have significantly higher levels of sCD30. Therefore, we conclude that lack of optimal Th1-type CD4 T cells is associated with HCV-induced allograft cirrhosis. Further, sCD30 may represent a novel marker for surveillance of hepatic cirrhosis in transplant recipients with chronic HCV infection.

Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch.

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 of a human leukocyte 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.

Role of Circulating MicroRNAs in the Immunopathogenesis of Rejection After Pediatric Lung Transplantation.

BACKGROUND: Acute rejection (AR) and development of chronic rejection, bronchiolitis obliterans syndrome (BOS) remain major limiting factors for lung transplantation (LTx). This retrospective study is to identify differentially expressed circulating microRNAs (miRNAs) that associate with development of AR and BOS in pediatric lung transplant recipients (LTxR). METHODS: We determined the circulating levels of 7 selected candidate miRNAs in 14 LTxR with AR, 7 with BOS, and compared them against 13 stable pediatric LTxR at 1, 6, and 12 months after LTx. In addition, 6 AR, 7 BOS, and 8 stable pediatric LTxR, 16 AR, 17 BOS, and 16 stable adult LTxR were included for validation. RESULTS: MiR-10a, -195, -133b were significantly lower in AR and miR-144, -142-5p, -155 were higher in AR compared to stable (P < 0.05). In addition, circulating levels of miR-134, -10a, -195, -133b were significantly lower and miR-144, -142-5p, -155 were higher (P < 0.05) with development of BOS. The receiver-operating characteristic demonstrated that miR-142-5p, miR-155, and miR-195 strongly discriminated patients with AR from stable LTxR (P < 0.001 for all comparisons): miR-142-5p (area under the curve [AUC], 0.854), miR-155 (AUC, 0.876), and miR-195 (AUC, 0.872). Further, miR-10a, miR-142-5p, miR-144, and miR-155 strongly discriminated BOS from stable LTxR (P < 0.001 for all comparisons). CONCLUSIONS: We demonstrated that differential expression of circulating miRNAs occurs in LTxR with AR and BOS, suggesting that they can provide not only important clues to pathogenesis but also may serve as potential noninvasive biomarkers for AR and BOS after pediatric LTx.

Elevated soluble CD30 correlates with development of bronchiolitis obliterans syndrome following lung transplantation.

BACKGROUND: The long-term function of lung transplants is limited by chronic rejection (bronchiolitis obliterans syndrome, BOS). Due to lack of specific markers, BOS is diagnosed clinically. Because there is strong evidence that alloimmunity plays a significant role in the pathogenesis of BOS, we investigated whether soluble CD30 (sCD30), a T-cell activation marker, would correlate with BOS. METHODS: Sera collected serially from BOS+ (n = 20) and matched BOS- (n = 20) lung transplant (LT) patients were analyzed for sCD30 by enzyme-linked immunosorbent assay. Pretransplant sera and sera from normal donors were also analyzed. RESULTS: PreLT levels were comparable to normal subjects. However, posttransplant there was a significant elevation in sCD30 levels during BOS development in all BOS+ patients, compared to BOS- (mean 139.8+/-10.7 vs. 14.8+/-2.7 U/ml, P < 0.001). sCD30 levels declined in the BOS+ patients but were still elevated compared to BOS- (48.52+/-5.04 vs. 7.19+/-2.9, P < 0.0001). CONCLUSIONS: We conclude that sCD30 may represent a novel marker to monitor the development of BOS.

Differentiation and functional maturation of human CD14(+) adherent peripheral blood monocytes by xenogeneic endothelial cells: up-regulation of costimulation, cytokine generation, and toll-like receptors.

BACKGROUND: Dendritic cells (DCs) can be generated from peripheral blood mononuclear cells (PBMC) after stimulation with exogenous granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Further, extravasation of monocytes through human endothelial cells can also cause differentiation, maturation, and expression of DC-specific phenotype. However, it is unclear whether human DCs can be generated from monocytes under the influence of xenogeneic endothelial cells in the absence of exogenous cytokines. We therefore analyzed and compared the effect of human and porcine endothelial cells on the differentiation of human monocytes into DC. METHODS: Adherent peripheral blood CD14(+) monocytes were cultured in the presence of different cytokine combinations, human or porcine endothelial cells, and smooth muscle cells, and analyzed for DC-specific antigen expression, antigen-presenting capacity, cytokine and chemokine generation, and expression of Toll-like receptors by flow cytometry and reverse transcription polymerase chain reaction. RESULTS: Human monocytes express a DC-specific surface phenotype and efficiently present allo- and xenoantigens to allogeneic T cells after co-culturing with allogeneic and xenogeneic endothelial cells, respectively. Differentiation of monocytes under different stimulating conditions is also accompanied by the up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), human leukocyte antigen (HLA)-DR, synthesis of cytokines tumor necrosis factor-alpha, IL-12p70, IL-10, and differential expression of message for Toll-like receptors. CONCLUSIONS: Porcine aortic endothelial cells can provide immunostimulatory signals to human peripheral blood adherent monocytes similar to allogeneic endothelial cells through the participation of innate immune mechanisms.

In vitro T cell proliferation from kidney allograft biopsies with unremarkable pathology: new strategies for an old problem.

Acute rejection of renal allografts is mediated by infiltrating alloreactive T cells. The goals of this study were to correlate T cell proliferation with rejection and to determine whether T cell proliferation in the absence of rejection would predict future rejection episodes. Toward this, kidney biopsies (n=100) were cultured in the presence of interleukin-2. Cultures were examined at 4, 24, and 48 hr for T cell proliferation. A strong correlation was observed between T cell proliferation at any time point and rejection. There was not a significant correlation between T cell proliferation in biopsies with no rejection and the occurrence of a rejection episode within 2 months. However, T cell proliferation after 4 hr was a better predictor of the occurrence of rejection within 2 months compared with observations after 24 and 48 hr. Therefore, a subgroup of patients with unremarkable biopsies but T cell proliferation may be at risk for rejection and warrant closer observation and possible tailoring of immunosuppression.

Correlation between interleukin-15 and granzyme B expression and acute lung allograft rejection.

The levels of interleukin (IL)-15 and granzyme B mRNA expression have been correlated with acute rejection episodes of kidney and heart allografts. Thus, the purpose of this study was to determine whether a correlation exists between the expression of IL-15 and granzyme B and acute lung allograft rejection. Toward this, the levels of IL-15 and granzyme B mRNA expression were determined in bronchoalveolar lavage-derived alveolar macrophages and total cells, respectively, from lung transplant patients with stable lung allograft function and patients undergoing acute rejection episodes. The expression levels of IL-15 mRNA was significantly higher in the patients undergoing acute rejection as compared to patients with stable lung function (P=0.02). The expression levels of granzyme B mRNA was also significantly higher in the patients undergoing acute rejection as compared to patients with stable lung function (P=0.005). The Receiver-Operating-Characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 94% and specificity of 67% with the use of a cutoff value of 3.1 fg of granzyme B mRNA per microgram of total RNA (or 71% sensitivity and 75% specificity of a cutoff value of 9.1 fg/microg). These data indicate that IL-15 secreted by activated alveolar macrophages and granzyme B secreted by activated CD8+ cytotoxic T lymphocytes play important roles in the process of acute lung allograft rejection.

Elimination of socioeconomic and racial disparities related to lung cancer: closing the gap at a high volume community cancer center.

BACKGROUND: Healthcare disparities have afflicted the healthcare industry for decades and there have been many campaigns in recent years to identify and eliminate disparities. The purpose of this study was to identify disparities in the lung cancer population of a single community cancer center and to report the results in accordance with industry goals. METHODS: This was a retrospective cohort study of data on non-small cell lung cancer patients recorded in the Christiana Care Tumor Registry (CCTR) in Delaware. Gender, age, race, socioeconomic status and insurance status were used as potential variables in identifying disparities. RESULTS: We found no significant disparities between sexes, race or patients who were classified as having socioeconomic status 1-3. There was a lower survival rate associated with having the poorest socioeconomic status and in patients who used Medicare. Uninsured patients had the best survival outcomes and patients with Medicare had the poorest survival outcomes. CONCLUSION: Although we have closed the gap on sex and racial disparities, there remains a difference in survival outcomes across socioeconomic classes and insurance types.

Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality after lung transplantation (LTx). We sought to better understand 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 ELISPOT. 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. A total of 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 IFN-γ (p < 0.05) with decreased IL-10 (p < 0.05) when compared with BOS(-) patients. CONCLUSIONS: 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 play a key role in preventing the development of chronic rejection.

Antibodies to self-antigens predispose to primary lung allograft dysfunction and chronic rejection.

BACKGROUND: Primary graft dysfunction (PGD) is a known risk factor for bronchiolitis obliterans syndrome (BOS) after 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. Primary graft dysfunction and BOS were diagnosed based on International Society for Heart and Lung Transplantation guidelines. Antibodies to self-antigens k-alpha-1 tubulin, collagen type V, and collagen I were quantitated using standardized enzyme-linked immunosorbent assays, and cytokines were analyzed using Luminex immunoassays (Biosource International, Camirillo, CA). Human leukocyte antigen (HLA) antibodies were measured using Flow-PRA (One Lambda, Canoga Park, CA). RESULTS: Lung transplant recipients with pretransplant antibodies to self-antigens had increased risk of PGD (odds ratio 3.09, 95% confidence interval: 1.2 to 8.1, p = 0.02) compared with recipients without. Conversely, in patients with PGD, 34.7% were positive for pretransplant antibodies whereas in the PGD negative group, only 14.6% had antibodies (p = 0.03). Antibody positive patients demonstrated high levels of proinflammatory cytokines interleukin (IL)-1ß (2.1-fold increase), IL-2 (3.0), IL-12 (2.5), IL-15 (3.0), and chemokines interferon-inducible protein-10 (3.9) and monocyte chemotactic protein-1 (3.1; p < 0.01 for all). On 5-year follow-up, patients without antibodies showed greater freedom from development of HLA antibodies compared with patients who had antibodies (class I: 67% versus 38%, p = 0.001; class II: 71% versus 41%, p < 0.001). Patients with pretransplant antibodies were found to have an independent relative risk of 2.3 (95% confidence interval: 1.7 to 4.5, p = 0.009) for developing BOS. CONCLUSIONS: Presence of antibodies to self-antigens pretransplant increases the risk of PGD immediately after transplant period and BOS on long-term follow-up. Primary graft dysfunction is associated with an inflammatory cascade that augments the alloimmune (anti-HLA) response that predisposes to BOS.

Respiratory virus-induced dysregulation of T-regulatory cells leads to chronic rejection.

BACKGROUND: Lower respiratory viral infections predispose to bronchiolitis obliterans syndrome (BOS). In addition, there is emerging evidence to support the role of autoimmunity in the pathogenesis of BOS. Because CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Treg) control autoimmunity, we tested the hypothesis that respiratory virus-induced Treg dysfunction leads to BOS. METHODS: Treg frequency was monitored using flow cytometry. Apoptosis, cytokines, and antibodies were analyzed using annexin V assay, LUMINEX, and enzyme-linked immunosorbent assay, respectively. Murine studies were performed using the orthotopic tracheal transplant model. RESULTS: (A) Human studies: Treg troughs (decrease >50% of baseline) were found in 13 (43.3%) of 30 lung transplant recipients. Treg isolated during troughs revealed increased apoptosis (37.8%). Patients with Treg troughs had increased prevalence of antibodies to self-antigens collagen type I (23.1% vs 5.8% pretrough), collagen V (7.7% vs 0%), and k-alpha tubulin (30.7% vs 11.7%, p < 0.01) at 6 months post-trough. Increased number of Treg troughs correlated with more rapid onset of BOS. (B) Murine studies: Infection of tracheal transplant recipients with murine parainfleunza sendai virus led to increased Treg apoptosis (50.5%) in the draining lymph nodes. Vaccination against sendai virus prior to transplant abrogated apoptosis of Treg. In vitro, sendai virus-infected, but not naive, tracheal epithelial cells demonstrated upregulation of FasL (>3.5-fold) and induction of co-cultured Treg apoptosis (5.6-fold increase). CONCLUSIONS: Respiratory viral infections cause Treg apoptosis which leads to the development of de novo autoimmunity that may play a role in the pathogenesis of BOS.