Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate

Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumorassociated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells.

Low T Cell Responsiveness in the Early Phase Of COVID-19 Associates with Progression to Severe Pneumonia in Kidney Transplant Recipients

Purpose: Kidney transplant (KT) recipients are more prone to developing lifethreatening forms of COVID-19 than the general population. Little is known about the immunological mechanisms underlying disease severity in these patients receiving T-cell targeting immunosuppressive drugs. We investigated the relationship between T cell responsiveness at the beginning of the infection and the risk to subsequently progress to respiratory failure. Method(s): We performed a multicentric prospective study in 45 KT recipients with a positive RT-PCR COVID-19 test and only mild symptoms at inclusion. Blood samples were collected at baseline directly in a cell culture system containing T cell stimuli. We assessed T cell responsiveness by computing the ratio between the levels of Th1, Th2, Th17 and Treg cytokines produced after polyclonal stimulation and the number of blood lymphocytes. We then used an unsupervised classification approach to stratify patients into low and high T cell responders and a penalized logistic regression to identify variables independently associated with progression to severe COVID-19. Result(s): Forty-five kidney transplant patients were included. Unsupervised clusterization identified 31 low and 14 high T cell responders. Patients who progressed to severe pneumonia were all low T cell responders (p=0.01). In multivariate analysis, we found that low T cell responsiveness at baseline was the main risk factor for subsequent progression to severe pneumonia. Conclusion(s): Low T cell reactivity in the early phase of COVID-19 is strongly associated with progression to severe pneumonia. This study provides new insights into the mechanisms underlying COVID-19 severity in organ transplant recipients and data of interest to clinicians managing immunosuppressive drugs in these patients.

Myeloid-Dependent Immunosuppressive Features Drive and Predict IPF Progression

Rationale: Despite the availability of pharmacologic therapies, idiopathic pulmonary fibrosis (IPF) is still a clinical challenge with several unmet needs. Robust evidence supports monocytes as cellular biomarkers of progression in IPF. Yet, their precise role and whether specific subtypes might predict progression and drive disease is unknown. We reported, for the first time, that myeloidderived suppressor cells (MDSC), immature precursors of monocytes, are increased in numbers, functionally active in IPF. Monocytic MDSC is the predominant subtype in IPF, and yet, functional characterization and immune modulation properties have not been explored. Methods and Results: characterization of circulating myeloid populations in IPF by multicolor FACS confirmed the abundance of MDSC (Lin-, HLA-DRlo, CD33+, CD14+, S100A+, CD28L1+ and ICOSL+) in IPF (n=78) and fILD (n=83), also abundant in whole blood scRNA seq of severe Covid-19 patients that progressed into fibrosis, and not in mild Covid-19. Then, we prospectively followed 83 fILD patients (45% IPF, 55% non-IPF -EAA, CTD-ILD, NSIP-) over 1 year and immunophenotyped them every 3 months. Cross-sectional analysis showed that patients with a higher number circulating MDSC, had a higher GAP index (7-8) (p<0,001). Longitudinal follow-up showed that patients with constant higher circulating MDSC had lower transplant-free survival (p=0.0058). Primary isolated MDSC when co-cultured with autologous T cells induced CD8+ T cell exhaustion (PD1hi, Lag3hi, Tim3hi, TNFalpha lo, INFglo), and downregulation of co-stimulatory T cell signaling (CD28, ICOS, ITK, and LCK), preliminary data support the induction of de-novo FoxP3 Treg formation, creating a suppressive and immunosenescent microenvironment in IPF. FACS analysis of explanted lungs demonstrated the increase of tissue-resident MDSC in fibrosis (HP, NSIP, IPF) compared with donor lungs, as well as in bleomycin-induced fibrosis compared to PBS. Conclusion: Taking together, a high number of circulating MDSC reflects worse lung function and higher GAP index in cross-sectional analysis, and associates with lower transplant-free survival longitudinally. The role that immature and mature monocytes play during promotion of a suppressive microenvironment in IPF is an unexplored area that may lead to a paradigm shift in our understanding of the sequelae of exhaustion and immunosenescence, contributing to the identification of novel targets useful for therapeutic myeloid selection in IPF.

A Case of Acute Interstitial Pneumonia (AIP)

Introduction: Acute interstitial pneumonia (AIP) also known as Hamman-Rich Syndrome is an uncommon, acute, and rapidly progressive idiopathic pulmonary disease that often leads to acute respiratory distress syndrome (ARDS). We present a case of a 52-year-old male who developed this condition. Case: A 52-year-old male with no past medical history presented to the emergency department with a 3-day history of progressively worsening dyspnea, dry cough, and chills. Prior to symptom onset, he was in his usual state of health but did report having polyarthralgia mainly involving large joints with no other associated symptoms. He denied a history of sick contacts including COVID exposure, sexually transmitted infections, incarceration, intravenous drug abuse, or travel to tuberculosis endemic countries. He denied tobacco use and any other form of illicit drug use. On physical examination, he was afebrile, tachycardic, and hypoxic on room air. He appeared to be in no respiratory distress and chest was clear to auscultation. There were no joint abnormalities, skin rashes, or lymphadenopathy. Lab workup revealed elevated D-Dimer (2140 ng/mL), CRP (50 mg/L), lactate dehydrogenase (296 IU/L), ferritin (578 ng/mL). His SARSCoV2 PCR was negative. Chest X-ray and CT chest both revealed right pleural effusion and diffuse reticular and ground-glass opacities. He underwent thoracentesis and fluid analysis revealed lymphocytic exudate with negative cultures. Antibiotics and steroids were initiated. He underwent a complete rheumatologic workup including myositis panel, due to concern for possible autoimmune etiologies and it was negative. His respiratory status worsened, and he eventually required intubation. At this point given unclear etiology, he underwent bronchoscopy with transbronchial cryobiopsy. Cryobiopsy revealed evidence of organizing phase of diffuse alveolar damage (Figure 1) and in the setting of negative cultures, COVID-19 and autoimmune panel, there was a growing concern for acute interstitial pneumonia. The patient was started on pulse dose of steroids and transferred to a transplant center for lung transplantation evaluation. Discussion: Acute interstitial pneumonia is a rare idiopathic clinicopathological condition that is characterized clinically by rapid onset of respiratory failure in patients with no past medical history of pre-existing lung disease. Histopathological findings are identical to those of diffuse alveolar damage. Closely resembling ARDS, it is frequently confused with other clinical entities characterized by rapidly progressive interstitial pneumonia. Considering this a high index of suspicion is required to diagnose these patients and institute appropriate management as mortality is as high as 70%. (Figure Presented).