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Acute cellular rejection (ACR) is associated with the ultimate development of chronic lung allograft dysfunction. Transbronchial biopsy, the gold standard for the diagnosis of ACR, is associated with potential morbidity. We set out to define the correlation between the recipient-derived immune cell content in bronchoalveolar lavage (BAL) and biopsy proven early ACR (<4 months). We analyzed BAL samples from 8 lung transplant recipients with early ACR and 5 without ACR. Immunophenotyping was performed using spectral flow cytometry; cell origin (donor v recipient) was determined by staining for human leukocyte antigen. T test was used to identify differences in immune cell populations between those with and without ACR. Live CD45+ cells were defined as CD4+ and CD8+ T cells, B cells (CD19+, CD20+), CD56+ lymphocytes, and monocytes (CD14+CD206+/CD64+). BAL was obtained at 1.5 months (range 0.5-3.5) from transplant at time of ACR and at 1.6 months (range 0.5 - 2.8) for non-ACR. The presence of ACR was associated with a statistically significant decrease in the proportion of donor monocytes (Figure 1: Mean 85%, SD 15% v 34%, SD 16%; p <0.001) and donor-derived regulatory T cells (Tregs), defined as CD4+CD25+FOXP3+ (Mean 3%, SD 3% v Mean 9%, SD 3%; p = 0.004). When comparing absolute number of both donor and recipient immune cells found in the BAL, patients with ACR had a statistically significant increase in the number of recipient-derived CD4+ T cells (Mean 7%, SD 5% v 1%, SD 0.05%; p value = 0.02), and a trend towards increased recipient-derived CD8+ T cells (Mean 18%, SD 27% v <1%, SD 1%; p value = 0.17). Biopsy proven, early ACR is associated with a relative decline in donor monocytes and donor Tregs, and increased proportion of recipient-derived CD4+ T cells. These results support further investigation into the role of BAL immune cell phenotyping as a potential less-invasive biomarker for ACR. [ABSTRACT FROM AUTHOR]

Purpose Alveolar macrophages play vital roles in attenuating lung injury through immune modulatory effects and improving gas exchange by scavenging cellular debris within the alveoli. A recent analysis of lung biopsy specimens suggests that donor-derived macrophages persist within the allograft after lung transplantation. The phenotypic differences between donor and graft-infiltrating recipient macrophages remains unreported. Methods Single cell suspensions obtained from bronchoalveolar lavage (BAL) of 15 lung transplant recipients at various times following transplantation were analyzed by flow cytometry. Macrophages were defined as CD45+CD3-CD19-CD64+CD14+CD11chisinglets. Cell origin (donor versus recipient) was identified by staining for human leukocyte antigen discrepancies. Paired t test was used to compare cell surface marker expression between donor and recipient macrophages. Results The proportion of donor macrophages within the BAL was variable across patients but did not vary over the time following transplantation (p = 0.5). The median percentage of donor macrophages was 10.5% (range 1-64). Donor macrophages had increased expression of CD206 (Figure: Rep. flow cytometry plot from BAL one month following transplantation and cumulative data from 10 participants, p = 0.004), with trends in increased expression of HLADR (p = 0.07), and no difference in CD163 expression (p = 0.17). Conclusion The scavenger function of alveolar macrophages is vital in maintaining gas exchange in the setting of severe influenza infection. Following lung transplantation, persisting donor-derived alveolar macrophages have significantly increased expression of the scavenger receptor CD206 when compared to graft-infiltrating recipient-derived macrophages. Further investigation is warranted to identify whether this has implications for graft function in the perioperative setting. [ABSTRACT FROM AUTHOR]

Most memory T cells reside in mucosal and lymphoid organs as tissue resident memory T cells (TRM). During acute cellular rejection after lung transplantation, recipient-derived T cells recruited to the lung persist as long-lived TRM. Studies in cutaneous skin cancer suggest that systemic lymphodepletive therapies, like alemtuzumab, spare skin TRM. We aimed to determine the impact of alemtuzumab on lung TRM survival by comparing donor TRM persistence between lung transplant recipients receiving alemtuzumab versus basiliximab induction immunosuppression. A convenience cohort was identified of adult lung transplant recipients who had cryopreserved bronchoalveolar lavage (BAL) cells obtained between 0.5 to 2.8 months post-transplantation. Multiparameter spectral flow cytometry was used to phenotype T cells; donor versus recipient origin was determined by staining for unique human leukocyte antigen expression. To estimate total lung CD3+ T cell content, we stained simultaneously obtained transbronchial biopsies for CD3 using immunohistochemistry. Unpaired Mann Whitney U test was used to identify statistically significant differences in T cell phenotype and content in lungs after induction with alemtuzumab or basiliximab. We included BAL from 25 lung transplant recipients (15 alemtuzumab, 10 basiliximab); transbronchial biopsies were available for 12. Median age was 56 for alemtuzumab and 39 for basiliximab; interstitial lung disease was the primary indication in the alemtuzumab cohort versus cystic fibrosis in the basiliximab cohort. There was a M:F ratio of 3:2 for both cohorts. We found no difference in the proportion of persisting donor CD8+ or CD4+ T cells in the BAL based on induction immunosuppression (Figure, p = 0.99 for CD8, p = 0.28 for CD4). We also found no difference in the persistence of donor regulatory T cells (CD127loCD25hiFOXP3+). The percentage of CD3+ cells found on IHC ranged from 3 - 7% in all biopsies. There was a trend towards increased total T cell content of the lungs of those study participants who underwent induction with basiliximab (p = 0.1). Systemic lymphodepletive therapy with alemtuzumab spares TRM after lung transplantation, but a trend towards increased T cell content in the lungs of those who received basiliximab. This data suggests that systemic therapies may not adequately target potentially harmful TRM. [ABSTRACT FROM AUTHOR]

Circulating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury (rejection, infection, ischemia/reperfusion injury). In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction at 72 hours and acute cellular rejection in the first month after lung transplant. Thirty-five subjects undergoing double lung transplants at our institution between Jun.2020-Oct.2021 were included in this study. Primary Graft Dysfunction (PGD) was graded according to ISHLT criteria. Our protocol includes a surveillance bronchoscopy with transbronchial biopsy at 3 weeks post-transplant. Acute cellular rejection was diagnosed and graded by a pulmonary pathologist after evaluation of histopathology slides. Blood samples were collected at various time points before and after lung transplant. Dd-cfDNA in samples were determined using AlloSure dd-cfDNA test kits (CareDx, Inc.). We observed a rapid increase of dd-cfDNA in blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks (Figure A). The peak was observed within 72 hours after transplantation. The peak values of dd-cfDNA varied among subjects and had no correlation with PGD grade 3 occurrences at 72 hours. (Figure B, p=0.85). We observed an association between levels of dd-cfDNA from blood collected at the time of transbronchial biopsy and the histological diagnosis of ACR at 3 weeks. (Figure C, mean 2.7% versus 1.5%, p=0.015) This preliminary data shows that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD at 72hours. Plasma levels of dd-cfDNA may be a less invasive tool to estimate graft rejection after lung transplantation however larger studies are still necessary to better identify thresholds. [ABSTRACT FROM AUTHOR]