IL-10 Is Critical for Regulation of Cytotoxic CD4+NKG7+ T Cells in Lung Allograft Rejection but Is Not Required for Allograft Acceptance.

Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell-mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-γ) responses along with elevated CD4+NKG7+ and CD4+CD107a+ responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection.

Lung Epithelium Releases Growth Differentiation Factor 15 in Response to Pathogen-mediated Injury.

GDF15 (growth differentiation factor 15) is a stress cytokine with several proposed roles, including support of stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the cellular sources and downstream effects of GDF15 during pathogen-mediated lung injury are unclear. We quantified GDF15 in lower respiratory tract biospecimens and plasma from patients with acute respiratory failure. Publicly available data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reanalyzed. We used mouse models of hemorrhagic acute lung injury mediated by Pseudomonas aeruginosa exoproducts in wild-type mice and mice genetically deficient for Gdf15 or its putative receptor, Gfral. In critically ill humans, plasma levels of GDF15 correlated with lower respiratory tract levels and were higher in nonsurvivors. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium, and lower respiratory tract GDF15 levels were higher in coronavirus disease (COVID-19) nonsurvivors. In mice, intratracheal P. aeruginosa type II secretion system exoproducts were sufficient to induce airspace and plasma release of GDF15, which was attenuated with epithelial-specific deletion of Gdf15. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and an altered lung transcriptional profile during injury induced by P. aeruginosa type II secretion system exoproducts, which was not recapitulated in mice deficient for Gfral. Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but increased circulating erythrocyte counts. Lung epithelium releases GDF15 during pathogen injury, which is associated with plasma levels in humans and mice and can increase erythrocyte counts in mice, suggesting a novel lung-blood communication pathway.

Exploring preferred binding domains of IgG1 mAbs to multimodal adsorbents using a combined biophysics and simulation approach.

In this work, we employ a recently developed biophysical technique that uses diethylpyrocarbonate (DEPC) covalent labeling and mass spectrometry for the identification of mAb binding patches to two multimodal cation exchange resins at different pH. This approach compares the labeling results obtained in the bound and unbound states to identify residues that are sterically shielded and thus located in the mAb binding domains. The results at pH 6 for one mAb (mAb B) indicated that while the complementarity determining region (CDR) had minimal interactions with both resins, the FC domain was actively involved in binding. In contrast, DEPC/MS data with another mAb (mAb C) indicated that both the CDR and FC domains were actively involved in binding. These results corroborated chromatographic retention data with these two mAbs and their fragments and helped to explain the significantly stronger retention of both the intact mAb C and its Fab fragment. In contrast, labeling results with mAb C at pH 7, indicated that only the CDR played a significant role in resin binding, again corroborating chromatographic data. The binding domains identified from the DEPC/MS experiments were also examined using protein surface hydrophobicity maps obtained using a recently developed sparse sampling molecular dynamics (MD) approach in concert with electrostatic potential maps. These results demonstrate that the DEPC covalent labeling/mass spectrometry technique can provide important information about the domain contributions of multidomain proteins such as monoclonal antibodies when interacting with multimodal resins over a range of pH conditions.

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.

Impact of enzymatic digestion on single cell suspension yield from peripheral human lung tissue.

An increasing number of translational investigations of lung biology rely on analyzing single cell suspensions obtained from human lungs. To obtain these single cell suspensions, human lungs from biopsies or research-consented organ donors must be subjected to mechanical and enzymatic digestion prior to analysis with either flow cytometry or single cell RNA sequencing. A variety of enzymes have been used to perform tissue digestion, each with potential limitations. To better understand the limitations of each enzymatic digestion protocol and to establish a framework for comparing studies across protocols, we performed five commonly published protocols in parallel from identical samples obtained from 6 human lungs. Following mechanical (gentleMACS™) and enzymatic digestion, we quantified cell count and viability using a Nexcelom Cellometer and determined cell phenotype using multiparameter spectral flow cytometry (Cytek™ Aurora). We found that all protocols were superior in cellular yield and viability when compared to mechanical digestion alone. Protocols high in dispase cleaved immune markers CD4, CD8, CD69, and CD103 and contributed to an increased monocyte to macrophage yield. Similarly, dispase led to a differential epithelial cell yield, with increased TSPN8+ and ITGA6+ epithelial cells and reduced CD66e+ cells. When compared to collagenase D, collagenase P protocols yielded increased AT1 and AT2 cells and decreased endothelial cells. These results provide a framework for selecting an enzymatic digestion protocol best suited to the scientific question and allow for comparison of studies using different protocols.

Bioinspired, Non-Enzymatic, Aqueous Synthesis of Size-Tunable CdS Quantum Dots for Sustainable Optoelectronic Applications.

The enzymatic production of hydrogen sulfide (H2S) from cysteine in various metabolic processes has been exploited as an intrinsically "green" and sustainable mode for the aqueous biomineralization of functional metal sulfide quantum dots (QDs). Yet, the reliance on proteinaceous enzymes tends to limit the efficacy of the synthesis to physiological temperature and pH, with implications for QD functionality, stability, and tunability (i.e., particle size and composition). Inspired by a secondary non-enzymatic biochemical cycle that is responsible for basal H2S production in mammalian systems, we establish how iron(III)- and vitamin B6 (pyridoxal phosphate, PLP)-catalyzed decomposition of cysteine can be harnessed for the aqueous synthesis of size-tunable QDs, demonstrated here for CdS, within an expanded temperature, pH, and compositional space. Rates of H2S production by this non-enzymatic biochemical process are sufficient for the nucleation and growth of CdS QDs within buffered solutions of cadmium acetate. Ultimately, the simplicity, demonstrated robustness, and tunability of the previously unexploited H2S-producing biochemical cycle help establish its promise as a versatile platform for the benign, sustainable synthesis of an even wider range of functional metal sulfide nanomaterials for optoelectronic applications.

Incidence of Acute Cellular Rejection After Granulocyte Colony-Stimulating Factor in Lung Transplant Recipients.

BackgroundNeutropenia is a common complication in lung transplant recipients (LTRs). Filgrastim may be used to treat neutropenia in LTRs, but its consequences on acute cellular rejection (ACR) remain controversial. Objective: The purpose was to examine the association between filgrastim and incidence of ACR 6 months after filgrastim administration in LTRs. Secondary outcomes included burden of ACR, infections, chronic lung allograft dysfunction (CLAD), and survival. Methods: This was a matched cohort study of patients transplanted between January 2010 and October 2019. LTRs who received filgrastim for neutropenia were compared to a cohort who did not. LTRs were matched on transplant indication, sex, age, and time post-transplant and multivariable logistic regression models were used to evaluate the likelihood of ACR. Results: 212 patients were included in the analysis (106 in each group). 50 patients (47.2%) in the filgrastim group experienced ACR compared to 37 patients (34.9%) in the no filgrastim group (P = .070). In multivariable analysis, filgrastim use was not associated with ACR at 6 months (OR 1.409, 95% CI 0.772-2.571). Time to first ACR was shorter (P = .049) and 6-month ACR score was higher in the filgrastim group (.49 vs .33, P = .047). LTRs in the filgrastim group had higher incidence of bacterial pneumonia and 1-year mortality. Conclusions: Although not associated with increased likelihood of ACR at 6 months, our study found that filgrastim is associated with increased ACR burden and decreased time to ACR. This study can help inform clinicians of ACR risk after filgrastim use in LTRs.

Rabbit Antithymocyte Globulin for Treatment of Corticosteroid Refractory Acute Cellular Rejection After Lung Transplantation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) remains a major cause of death after the first year posttransplant, with acute cellular rejection (ACR) being a major risk factor for CLAD. We evaluated the use of rabbit antithymocyte globulin (rATG) for corticosteroid refractory ACR in lung transplant recipients. METHODS: We retrospectively identified 112 adult lung transplant recipients who received rATG for refractory ACR after lung transplantation. The primary endpoint was the incidence of ACR on follow-up transbronchial biopsy. Secondary endpoints included freedom from ACR within 1 y post-rATG, CLAD progression at 1 y post-rATG, and all-cause mortality at 1 y post-rATG. RESULTS: A complete resolution of ACR was observed in 60.2% of patients, an improvement but not complete resolution in 22.1%, and no response on follow-up biopsy in 17.8%. Mean A grade 1 y post-rATG was 0.51 in complete responders, 1.01 in partial responders, and 2.19 in nonresponders ( P < 0.001). Complete responders had significantly less new or worsening CLAD at 1 y than partial responders (17% versus 40%; P = 0.02). All-cause mortality rate was 14.9% in complete responders, 40% in partial responders, and 30% in nonresponders ( P < 0.01). CONCLUSIONS: rATG appears to be an effective treatment of refractory ACR in lung transplant recipients. Failure to respond to rATG carries an increased risk of early CLAD and death.

Psychological Predictors of Medical Students' Involvement in Pro Bono.

THEORY: Medical pro bono, in which medical professionals provide no (or low) cost services, is one approach to addressing unmet healthcare needs. Prior efforts to understand who chooses to take part in pro bono and why they might do so have been primarily atheoretical in their approach. The current investigation focuses on students in medical school and draws on relevant theory and research in psychology to identify predictors of their intentions to engage in medical pro bono service during and after medical school.Hypotheses:Four major approaches to identifying predictors of medical pro bono are examined: the role of demographic variables as predictors of medical pro bono, conceptualizing medical pro bono as a form of volunteerism, viewing medical pro bono as an expression of personality, and medical pro bono as a reflection of role identities and expectations. Each of these approaches can be characterized as being about medical students' individual attributes or aspects of the situation they are in. METHODS: A total of 278 medical students from 15 different medical schools in the United States of America completed a web-based survey (8/4/2020-9/22/2020). The students completed measures of pro bono identity and expectations, intentions to engage in medical pro bono activities, prosocial personality, volunteer motivation, exposure to volunteering, general traits of personality, and demographic variables (in this order). We used linear regression analyses to separately predict three measures of intentions (general medical school intentions, intentions toward medical pro bono trips during medical school, and general post medical school intentions). RESULTS: The strongest predictors of intentions to engage in medical pro bono were one's identity and expectations related to pro bono. Medical students who had incorporated medical pro bono into aspects of their identity and/or considered medical pro bono to be an expectation indicated higher intentions to engage in medical pro bono work. Conversely, volunteer motivation/exposure, personality, and demographic variables were much weaker predictors of medical pro bono. CONCLUSIONS: The findings of the present study have implications for ways that medically oriented volunteering may be increased by individual-level interventions and/or changes in medical education. Individual-level interventions could leverage the importance of identity and expectations to craft persuasive messaging to appeal to identity and expectations as drivers of engagement in medical pro bono. Program level interventions could work toward the institutionalization of medical pro bono by the inclusion/promotion of medical pro bono into the program's co-curricular and/or extracurricular activities.

Single center study investigating the clinical association of donor-derived cell-free DNA with acute outcomes in lung transplantation.

Background: Circulating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury. In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction (PGD) and graft rejection after lung transplant. Methods: A total of 40 subjects undergoing de-novo lung transplants at our institution were recruited in this study. Blood samples were collected at various time points before and after lung transplant for 1 year. Dd-cfDNA in samples was determined using AlloSure assay (CareDx Inc.). The correlation of the value of %dd-cfDNA was investigated with the incidence of PGD, acute cellular rejection (ACR), and donor-specific antibody. Results: We observed a rapid increase of %dd-cfDNA in the blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks. The peak was observed within 72 h after transplantation. The peak values of %dd-cfDNA varied among subjects and did not correlate with severe PGD incidence. 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. Conclusion: Our data show that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD. 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.

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.

Sequential, low-temperature aqueous synthesis of Ag-In-S/Zn quantum dots via staged cation exchange under biomineralization conditions.

The development of high quality, non-toxic (i.e., heavy-metal-free), and functional quantum dots (QDs) via 'green' and scalable synthesis routes is critical for realizing truly sustainable QD-based solutions to diverse technological challenges. Herein, we demonstrate the low-temperature all-aqueous-phase synthesis of silver indium sulfide/zinc (AIS/Zn) QDs with a process initiated by the biomineralization of highly crystalline indium sulfide nanocrystals, and followed by the sequential staging of Ag+ cation exchange and Zn2+ addition directly within the biomineralization media without any intermediate product purification. Therein, we exploit solution phase cation concentration, the duration of incubation in the presence of In2S3 precursor nanocrystals, and the subsequent addition of Zn2+ as facile handles under biomineralization conditions for controlling QD composition, tuning optical properties, and improving the photoluminescence quantum yield of the AIS/Zn product. We demonstrate how engineering biomineralization for the synthesis of intrinsically hydrophilic and thus readily functionalizable AIS/Zn QDs with a quantum yield of 18% offers a 'green' and non-toxic materials platform for targeted bioimaging in sensitive cellular systems. Ultimately, the decoupling of synthetic steps helps unravel the complexities of ion exchange-based synthesis within the biomineralization platform, enabling its adaptation for the sustainable synthesis of 'green', compositionally diverse QDs.

Immune and epithelial determinants of age-related risk and alveolar injury in fatal COVID-19.

Respiratory failure in COVID-19 is characterized by widespread disruption of the lung's alveolar gas exchange interface. To elucidate determinants of alveolar lung damage, we performed epithelial and immune cell profiling in lungs from 24 COVID-19 autopsies and 43 uninfected organ donors ages 18-92 years. We found marked loss of type 2 alveolar epithelial (T2AE) cells and increased perialveolar lymphocyte cytotoxicity in all fatal COVID-19 cases, even at early stages before typical patterns of acute lung injury are histologically apparent. In lungs from uninfected organ donors, there was also progressive loss of T2AE cells with increasing age, which may increase susceptibility to COVID-19-mediated lung damage in older individuals. In the fatal COVID-19 cases, macrophage infiltration differed according to the histopathological pattern of lung injury. In cases with acute lung injury, we found accumulation of CD4+ macrophages that expressed distinctly high levels of T cell activation and costimulation genes and strongly correlated with increased extent of alveolar epithelial cell depletion and CD8+ T cell cytotoxicity. Together, our results show that T2AE cell deficiency may underlie age-related COVID-19 risk and initiate alveolar dysfunction shortly after infection, and we define immune cell mediators that may contribute to alveolar injury in distinct pathological stages of fatal COVID-19.

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.

Modulation of tissue resident memory T cells by glucocorticoids after acute cellular rejection in lung transplantation.

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.

Rate of recipient-derived alveolar macrophage development and major histocompatibility complex cross-decoration after lung transplantation in humans.

Alveolar macrophages (AM) play critical roles in lung tissue homeostasis, host defense, and modulating lung injury. The rate of AM turnover (donor AM replacement by circulating monocytes) after transplantation has been incompletely characterized. Furthermore, the anatomic pattern of recipient-derived lung macrophages repopulation has not been reported, nor has their ability to accumulate and present donor major histocompatibility complex (a process we refer to as MHC cross-decoration). We longitudinally characterized the myeloid content of bronchoalveolar lavage (BAL) and biopsy specimens of lung transplant recipients and found a biphasic rate in AM turnover in the allograft, with a rapid turnover perioperatively, accelerated by both the type of induction immunosuppression and the presence of primary graft dysfunction. We found that recipient myeloid cells with cell surface AM phenotype repopulated the lung in a disorganized pattern, comprised mainly of large clusters of cells. Finally, we show that recipient AM take up and present donor peptide-MHC complexes yet are not able to independently induce an in vitro alloreactive response by circulating recipient T cells.

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography.

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography-mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.

Geographic disparities in lung transplantation in the United States before and after the November 2017 allocation change.

BACKGROUND: The primary lung allocation unit was expanded from the donation service area to a 250-mile radius in 2017. Prior to the change, geographic disparities in donor lung availability impacted waitlist outcomes. We sought to determine if the new allocation system improved these disparities. METHODS: We conducted a retrospective cohort study comparing the 2-year period before and after the change. Donor lung availability was defined as the ratio of donor lungs to waitlist candidates in the primary allocation unit. Transplant centers were divided into quartiles by donor lung availability. Multivariable competing risk models were used to determine the association between lung availability and waitlist outcomes. Multivariable Cox proportional hazards models compared post-transplant survival. RESULTS: Prior to the allocation change, the unadjusted transplant rate at centers in the lowest and highest quartiles was 132 and 607 transplants per 100 waitlist years. Candidates in the lowest quartile of donor lung availability had a 61% adjusted lower transplantation rate compared to candidates in highest quartile (sub-hazard ratio [sHR]: 0.39, 95% confidence interval [CI]: 0.34-0.44). After the allocation change, the disparity decreased resulting in an unadjusted transplant rate of 141 and 309 among centers in the lowest and highest quartiles. Candidates in the lowest quartile had a 38% adjusted lower transplantation rate compared to those in the highest (sHR: 0.62, 95% CI: 0.57-0.68). There was no significant difference in 1-year post-transplant survival. CONCLUSIONS: Although the expansion of the primary allocation unit improved disparities in waitlist outcomes without any change in post-transplant survival, there still remain significant differences due to geography.