Brief research report: in-depth immunophenotyping reveals stability of CD19 CAR T-cells over time.

Variability or stability might have an impact on treatment success and toxicity of CD19 CAR T-cells. We conducted a prospective observational study of 12 patients treated with Tisagenlecleucel for CD19+ B-cell malignancies. Using a 31-color spectral flow cytometry panel, we analyzed differentiation stages and exhaustion markers of CAR T-cell subsets prior to CAR T-cell infusion and longitudinally during 6 months of follow-up. The majority of activation markers on CAR T-cells showed stable expression patterns over time and were not associated with response to therapy or toxicity. Unsupervised cluster analysis revealed an immune signature of CAR T-cell products associated with the development of immune cell-associated neurotoxicity syndrome. Warranting validation in an independent patient cohort, in-depth phenotyping of CAR T-cell products as well as longitudinal monitoring post cell transfer might become a valuable tool to increase efficacy and safety of CAR T-cell therapy.

Spectral flow cytometry cluster analysis of therapeutic donor lymphocyte infusions identifies T cell subsets associated with outcome in patients with AML relapse.

Identification of immune phenotypes linked to durable graft-versus-leukemia (GVL) response following donor lymphocyte infusions (DLI) is of high clinical relevance. In this prospective observational study of 13 AML relapse patients receiving therapeutic DLI, we longitudinally investigated changes in differentiation stages and exhaustion markers of T cell subsets using cluster analysis of 30-color spectral flow cytometry during 24 months follow-up. DLI cell products and patient samples after DLI were analyzed and correlated to the clinical outcome. Analysis of DLI cell products revealed heterogeneity in the proportions of naïve and antigen experienced T cells. Cell products containing lower levels of effector memory (eff/m) cells and higher amounts of naïve CD4+ and CD8+ T cells were associated with long-term remission. Furthermore, investigation of patient blood samples early after DLI showed that patients relapsing during the study period, had higher levels of CD4+ eff/m T cells and expressed a mosaic of surface molecules implying an exhausted functional state. Of note, this observation preceded the clinical diagnosis of relapse by five months. On the other hand, patients with continuous remission retained lower levels of exhausted CD4+ eff/m T cells more than four months post DLI. Moreover, lower frequencies of exhausted CD8+ eff/m T cells as well as higher amounts of CD4+temra CD45RO+ T cells were present in this group. These results imply the formation of functional long-term memory pool of T cells. Finally, unbiased sample analysis showed that DLI cell products with low levels of eff/m cells both in CD4+ and CD8+ T cell subpopulations associate with a lower relapse incidence. Additionally, competing risk analysis of patient samples taken early after DLI revealed that patients with high amounts of exhausted CD4+ eff/m T cells in their blood exhibited significantly higher rates of relapse. In conclusion, differentially activated T cell clusters, both in the DLI product and in patients post infusion, were associated with AML relapse after DLI. Our study suggests that differences in DLI cell product composition might influence GVL. In-depth monitoring of T cell dynamics post DLI might increase safety and efficacy of this immunotherapy, while further studies are needed to assess the functionality of T cells found in the DLI.

Longitudinal Tracking of Immune Responses in COVID-19 Convalescents Reveals Absence of Neutralization Activity Against Omicron and Staggered Impairment to Other SARS-CoV-2 Variants of Concern.

Evaluating long-term protection against SARS-CoV-2 variants of concern in convalescing individuals is of high clinical relevance. In this prospective study of a cohort of 46 SARS-CoV-2 patients infected with the Wuhan strain of SARS-CoV-2 we longitudinally analyzed changes in humoral and cellular immunity upon early and late convalescence. Antibody neutralization capacity was measured by surrogate virus neutralization test and cellular responses were investigated with 31-colour spectral flow cytometry. Spike-specific, isotype-switched B cells developed already during the disease phase, showed a memory phenotype and did not decrease in numbers even during late convalescence. Otherwise, no long-lasting perturbations of the immune compartment following COVID-19 clearance were observed. During convalescence anti-Spike (S1) IgG antibodies strongly decreased in all patients. We detected neutralizing antibodies against the Wuhan strain as well as the Alpha and Delta but not against the Beta, Gamma or Omicron variants for up to 7 months post COVID-19. Furthermore, correlation analysis revealed a strong association between sera anti-S1 IgG titers and their neutralization capacity against the Wuhan strain as well as Alpha and Delta. Overall, our data suggest that even 7 month after the clearance of COVID-19 many patients possess a protective layer of immunity, indicated by the persistence of Spike-specific memory B cells and by the presence of neutralizing antibodies against the Alpha and Delta variants. However, lack of neutralizing antibodies against the Beta, Gamma and Omicron variants even during the peak response is of major concern as this indicates viral evasion of the humoral immune response.

GLA/DRST real-world outcome analysis of CAR T-cell therapies for large B-cell lymphoma in Germany.

CD19-directed chimeric antigen receptor (CAR) T cells have evolved as a new standard-of-care (SOC) treatment in patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL). Here, we report the first German real-world data on SOC CAR T-cell therapies with the aim to explore risk factors associated with outcomes. Patients who received SOC axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for LBCL and were registered with the German Registry for Stem Cell Transplantation (DRST) were eligible. The main outcomes analyzed were toxicities, response, overall survival (OS), and progression-free survival (PFS). We report 356 patients who received axi-cel (n = 173) or tisa-cel (n = 183) between November 2018 and April 2021 at 21 German centers. Whereas the axi-cel and tisa-cel cohorts were comparable for age, sex, lactate dehydrogenase (LDH), international prognostic index (IPI), and pretreatment, the tisa-cel group comprised significantly more patients with poor performance status, ineligibility for ZUMA-1, and the need for bridging, respectively. With a median follow-up of 11 months, Kaplan-Meier estimates of OS, PFS, and nonrelapse mortality (NRM) 12 months after dosing were 52%, 30%, and 6%, respectively. While NRM was largely driven by infections subsequent to prolonged neutropenia and/or severe neurotoxicity and significantly higher with axi-cel, significant risk factors for PFS on the multivariate analysis included bridging failure, elevated LDH, age, and tisa-cel use. In conclusion, this study suggests that important outcome determinants of CD19-directed CAR T-cell treatment of LBCL in the real-world setting are bridging success, CAR-T product selection, LDH, and the absence of prolonged neutropenia and/or severe neurotoxicity. These findings may have implications for designing risk-adapted CAR T-cell therapy strategies.

Six-year experience with treatment of early donor-specific anti-HLA antibodies in pediatric lung transplantation using a human immunoglobulin-based protocol.

OBJECTIVES: Experience with the treatment of early donor-specific anti-HLA antibodies (eDSA) after lung transplantation in children is very limited. At our institution, we have treated patients with eDSA since 2013 with successive infusions of intravenous human immunoglobulins (IVIG), combined in some cases with a single dose of Rituximab and plasmapheresis (therapeutic plasma exchange [tPE]) or immunoabsorption. The aim of this study was to present the 6-year results of IVIG-based therapy in pediatric lung recipients. METHODS: Records of pediatric (<18 years old) patients transplanted at our institution between 01/2013 and 03/2019 were reviewed. Outcomes were compared between patients with eDSA treated with IVIG (IVIG group) and without eDSA (control group). Median (interquartile range [IQR]) follow-up amounted to 28 (12-52) months. RESULTS: During the study period, 66 lung-transplanted pediatric patients were included, of which 27 (41%) formed the IVIG group and 38 (57%) the control group. Among the IVIG patients, 14 (52%) patients showed concomitant graft dysfunction (possible clinical antibody-mediated rejection). The median time to eDSA detection was 24 (14-63) days after transplantation. eDSA were cleared in 25 (96%) of the 26 patients which completed treatment. At 3 years, graft survival (%) was 73 vs 85 (P = .65); freedom (%) from chronic lung allograft rejection (CLAD) was 89 vs 78 (P = .82); and from infection 47 vs 31 (P = .15), in IVIG vs control patients, respectively. CONCLUSIONS: After lung transplantation, an IVIG-based treatment for eDSA yielded high eDSA clearance. IVIG and control patients showed similar CLAD-free and graft survival.