ABSTRACT
Objetivos: As neoplasias Linfoproliferativas (NLP) T sao um grupo heterogeneo de doencas oncohematologicas cujo diagnostico e desafiador devido ao amplo espectro de achados clinicos, morfologicos e histologicos que frequentemente se sobrepoem aos encontrados em condicoes reacionais. A determinacao de clonalidade e necessaria e pode ser realizada por deteccao dos rearranjos de genes do TCR por PCR ou NGS. No entanto, tais tecnicas apresentam alto custo e tempo prolongado para resultado. A imunofenotipagem por citometria de fluxo (CMF) e utilizada rotineiramente para diagnostico das NLP. O TCR e composto por cadeias alfa e beta com dois dominios constante da cadeia beta, TRBC 1 e TRBC2 que sao mutuamente exclusivos. O anticorpo JOVI-1 apresenta alta especificidade contra o TRBC1. Celulas normais ou reacionais demonstram um misto de positividade e negatividade para TRBC1, ou seja, um perfil policlonal, enquanto celulas T monoclonais demonstram expressao restrita de algum TRBC. A inclusao do anticorpo JOVI-1 nos paineis de CMF e, portanto, atrativa para determinacao da clonalidade das NLP-T. O objetivo desse trabalho e avaliar e reportar a inclusao do JOVI-1 nos paineis de CMF em casos suspeitos de NLP-T. Materiais e Metodos: Avaliacao de perfil de expressao de TCR Beta 1 atraves da inclusao com anticorpo JOVI-1 aos paineis utilizados em pesquisa de NLP-T. O JOVI-1 foi tambem utilizado na avaliacao de Leucemias Agudas T (LLA) de forma a ser um grupo controle para o ensaio, uma vez que linfoblastos imaturos nao apresentam expressao de TCR. Resultados: O JOVI-1 foi realizada em 18 casos suspeitos de NLP. 6 amostras de medula ossea e 12 de sangue periferico. A idade mediana dos suspeitos foi de 50,5 anos (1-71 anos) e a proporcao de individuos do genero masculino para o feminino foi de 2,4:1,0. 10 casos (55%) demonstraram perfil policlonal sendo que em 3 desses casos confirmaram-se infeccoes virais (1 por Covid-19 e 2 por Citomegalovirus) e 7 tiveram outros diagnosticos que nao NLP. Ja em 45% dos casos observou-se perfil monoclonal: 4 diagnosticos de Leucemia de Grandes Linfocitos Granulares T, 2 diagnosticos de Linfoma/Leucemia de Celulas T do Adulto, 1 diagnostico de Sindrome de Sezary e 1 caso de Linfoma T gama-delta. Foram avaliados 6 casos de LLA T, no qual apenas um foi positivo para o marcador e tratava-se de LLA T madura. Discussao e Conclusao: Nesse trabalho demonstramos que a adicao do JOVI-1 ao painel habitual de diagnostico torna a CMF uma boa ferramenta diagnostica para NLP-T cronicas obtendo estudo de clonalidade de forma relativamente simples e rapida, similar ao que acontece nas NLP de linhagem B ha decadas. Copyright © 2022
ABSTRACT
Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic that has taken millions of lives around the globe. Treatment of patients with moderate and severe COVID-19 disease has included dexamethasone, tocilizumab, Remdesivir, convalescent plasma, and targeted antibodies, however, currently, there are no FDA approved targeted cellular therapies in the treatment of mild or moderate SARS-CoV-2 disease. Virus-specific cytotoxic T cell lymphocytes (vCTLs) have shown therapeutic efficacy in immunocompromised patients with viral infections. We developed a multicenter and multidisciplinary Viral Cytotoxic T-Cell Consortium (VIRCTLC) to investigate the use of vCTLs manufactured by direct enrichment using the Cytokine Capture System (CCS) on the CliniMACS® Prodigy device. SARS-CoV-2 specific PepTivator Peptides consist of overlapping peptides that span the entire sequence of the protein (Protein N and M), or the length of its immunodominant domain (Protein S). The peptides can bind to either MHC class I or MHC class II molecules and are therefore able to target both CD4 and CD8 T cells. Objective: To screen, manufacture, and characterize SARS-CoV-2 vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System on the CliniMACS® Prodigy device. Methods: Donor screening was done utilizing PBMNCs from 15 convalescent COVID-19 donors after informed consent. PBMNCs were stimulated with a mix of PepTivator peptides (Miltenyi Biotech®) contained in the S, M and N proteins. IFN-γ levels were examined in CD3, CD4, and CD8 T cells by flow cytometry analysis. After informed consent, PBMNCs from three convalescent COVID-19 donors who screened positively to the PepTivator® peptide pools of SARS-CoV-2 Proteins M, N and S were collected by apheresis using the SPECTRA Optia® apheresis instrument. PBMNCs were incubated with the PepTivator® peptide pools for 4 hours. After incubation, the SARS-CoV-2 vCTLs were enriched using the CliniMACS Cytokine Capture System as we have previously described (Flower/Cairo, et al, ASTCT, 2020). Samples were taken from the enriched vCTLs and tested in gram stains, sterility cultures, cell counts, viability and IFN-γ cytokine staining (CD3/CD4/CD8/IFN-γ marker panel) by flow cytometry. Amplification and sequencing of TCRβ CDR3 regions of pre-stimulated PBMNC, stimulated PBMNCs samples taken from the QC bag (QC samples) and the enriched SARS-CoV-2 vCTLs were performed on the ImmunoSEQ platform using ImmunoSEQ® TCRB Assay kit (Adaptive Biotechnologies, Seattle, WA, USA). Characterization of immune subsets was done by mass cytometry analysis with 41 Immunophenotypic markers. Transcriptome of the immune landscape of QC samples, and enriched vCTLs was compared with the pre samples using the human nCounter PanCancer Immune Profiling Panel on the nCounter system. Results: We demonstrate that 93.3% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that consisted of 79% + 21% (mean + SEM) IFNγ + T cells (Fig.1). TCRβ sequencing showed that convalescent COVID-19 donors have a highly diverse TCR repertoire and we identified TCRβ CDR3 clones that are known to be associated with SARS-CoV-2 T cell responses. Immunophenotyping analysis demonstrated more CD4 T cells than CD8 T cells in the SARS CoV-2 vCTLs, an increase in memory CD8 and CD4 cells, especially CD8 T EM, CD4 T cm and CD4 T EMRA cells (Fig.2) and an increase DC cells in the SARS CoV-2 vCTL products as compared to pre-stimulated PBMNCs. Expression of the exhaustion markers was not enhanced in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Transcriptome analysis showed increased gene expression in T-cell function, interleukin, pathogen defense, and TNF superfamily pathway genes in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Conclusion: Our study demonstrates that highly functional SARS-CoV-2 vCTLs can be rapidly generat d by direct cytokine enrichment from convalescent donor peripheral blood mononuclear cells. These data serve as pre-clinical validation for an ongoing clinical trial utilizing related HLA-matched and haplo-identical SARS CoV-2 vCTLs for the treatment of patients with mild and moderate SARS-CoV-2 disease (IND #27260, NCT# 04896606). [Formula presented] Disclosures: Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding;Courier Therapeutics: Current holder of individual stocks in a privately-held company. Johnson: Miltenyi Biotec: Research Funding. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen: Speakers Bureau;Sanofi: Speakers Bureau;Servier: Speakers Bureau;Sobi: Speakers Bureau;Omeros: Membership on an entity's Board of Directors or advisory committees;Nektar: Membership on an entity's Board of Directors or advisory committees.
ABSTRACT
Introduction: At the end of April 2020, European clinicians warned the Public Health Agencies about an abnormal increase of Kawasakilike diseases and myocarditis requiring critical care support in the context of the ongoing COVID-19 epidemic in children. American clinicians also reported a large outbreak of severe inflammation in children following COVID-19 infection, a condition that is now named Pediatric Inflammatory Multisystemic Syndrome (PIMS) or Multisystem Inflammatory Syndrome in children (MIS-C). Objectives: As MIS-C combines clinical features of Kawasaki disease (KD) and Toxic Shock Syndrome (TSS), we aimed to compare the immunological profile of pediatric patients with these different conditions. Methods: We analysed blood cytokine expression, and the T cell repertoire and phenotype in 36 MIS-C cases, which were compared to 16 KD, 58 TSS, and 42 COVID-19 cases. Results: We observed an increase of serum inflammatory cytokines (IL-6, IL-10, IL-18, TNF-a, IFNg, CD25s, MCP1, IL-1RA) in MIS-C, TSS and KD, contrasting with low expression of HLA-DR in monocytes. We detected a specific expansion of activated T cells expressing the Vβ21.3 T cell receptor β chain variable region in both CD4 and CD8 subsets in 75% of MIS-C patients and not in any patient with TSS, KD, or acute COVID-19;this correlated with the cytokine storm detected. The T cell repertoire returned to baseline within weeks after MIS-C resolution. Vβ21.3+ T cells from MIS-C patients expressed high levels of HLA-DR, CD38 and CX3CR1 but had weak responses to SARS-CoV- 2 peptides in vitro. Consistently, the T cell expansion was not associated with specific classical HLA alleles. Conclusion: Thus, our data suggested that MIS-C is characterized by a polyclonal Vβ21.3 T cell expansion not directed against SARS-CoV-2 antigenic peptides, which is not seen in KD, TSS and acute COVID-19.