ABSTRACT
Background: Allogeneic hematopoietic cell transplantation (HCT) with ex vivo T cell receptor (TCR) alphabeta+ T cell and CD19+ B cell depletion is an effective approach for children with primary immune deficiency disorders (PIDD) as it combines advantages of high CD34+ cell dose facilitating rapid engraftment with low risk of Graft Versus Host Disease (GVHD). The ideal pre-conditioning regimen that facilitates robust donor engraftment without increasing risk of transplant related mortality has not been well defined with this approach. Method(s): We report the outcomes of 4 pediatric subjects: Chronic Granulomatous Disease (CGD) (2), Wiskott Aldrich Syndrome (WAS) (1), and RAC2 deficient Severe Combined Immunodeficiency (1) who underwent haploidentical HCT with TCRalphabeta+ T cell/CD19+ depletion at Johns Hopkins All Children's Hospital/Moffitt Cancer Center from 2020-2022 (NCT04414046). Pre-conditioning regimen consisted of distal thymoglobulin (7.5 mg/kg), fludarabine (175 mg/m2), thiotepa (10 mg/kg) and pharmacokinetic guided busulfan targeting a cumulative area under curve (cAUC) (65-75 mgxhr/L). Rituximab (200 mg/m2) was administered on day +1. Result(s): The median age at HCT was 51 months (range 10-163 months). All patients received mobilized peripheral blood stem cells from HLA- haploidentical donors (paternal=1, maternal=1 sibling=2). Median busulfan cAUC for all patients was 69 mgxhr/L (range 65-76). Median CD34 and TCR alphabeta T cell dose was 9.13x106 cells/kg (range 7.0-18.9x106) and 0.7x105 cells/kg (range 0.09-1.0x105). Median times to neutrophil and platelet engraftment were 11 days (9-12) and 11 days (range 8-15), respectively. All 4 patients are alive with median follow-up of 19.5 months (range 7-24). One patient developed late VOD without organ dysfunction that resolved with defibrotide. At last follow up, peripheral T and myeloid chimerisms exceeded 90% in all 4 patients. Average time to CD4 recovery (> 200x106/L) was 142 days. Pre-existing inflammatory bowel disease in CGD (n=1) and WAS (n=1) patients resolved immediately following transplant. There was no graft failure, and none developed Grade III-IV acute or extensive chronic GVHD. Patient with WAS developed recurrent autoimmune cytopenias requiring corticosteroids, rituximab, sirolimus and daratumumab, and ultimately resolved. Viral reactivations included EBV (n= 1), adeno (n= 1), HHV6 (n= 2), BK (n=1), norovirus (n=1), and late HSV (n=1), all responded to antivirals without disease. All patients acquired SARS-Cov-2 after transplant and recovered without sequelae. Conclusion(s): TCR alphabeta+ and CD19+ depleted haploidentical transplantation using a reduced toxicity conditioning regimen with pharmacokinetic guided busulfan, fludarabine, thiotepa and thymoglobulin is well-tolerated in young children with PIDD that results in rapid, durable engraftment with low likelihood of GVHD and graft rejection.Copyright © 2023 American Society for Transplantation and Cellular Therapy
ABSTRACT
Background: T cells have been shown to play a role in the immune response to SARS-CoV-2. Identification of T cell epitopes and a better understanding of the T cell repertoire will provide important insights into how T cells impact antiviral immunity. Here, we identified T cell epitopes within the Spike (S), Nucleocapsid (N) and Membrane (M) proteins from SARS-CoV-2 convalescent individuals and performed TCR sequencing on epitope-specific T cells. Methods: Epitope mapping was performed by IFNγ ELISpot on PBMC from SARS-CoV-2 convalescent patients with mild/moderate disease (n = 19 for S;n=15 for N and M), and minimum epitopes were determined using truncated peptides and ICS. TCR sequence analysis was performed on a subset of individuals (n=9 donors;2-3 epitopes/donor), with longitudinal samples for 7 donors (2-3 time points/donor;33 to 236 days post-symptom onset). T cells were stimulated with individual peptides for 6 hours and sorted based on the expression of activation markers (CD4+: CD69, CD40L;CD8+: CD69, CD107a, surface TNF). scRNAseq was performed on sorted cells for TCR repertoire and transcriptome analysis. Results: We identified several peptides recognized by multiple individuals, including S42 (amino acids 165-179;7/19 donors), S302 (a.a. 1205-1219;6/19 donors), N27 (a.a. 106-120;6/14 donors) and M45 (a.a. 177-191;10/14 donors). S42 elicited both CD4+ (n=5) and CD8+ (n=1) T cell responses, with one individual having both a CD4+ and CD8+ response. The minimum epitope for S42 was determined to be a 9mer (FEYVSQPFL) for both CD4+ and CD8+ cells. TCR sequencing of S42-specific T cells identified a dominant gene pairing for TCRα across multiple donors (TRAV35;TRAJ42) and for both CD4+ and CD8+ T cells (Figure 1). In general, epitope-specific CD4+ responses (S42, M45) were more clonally diverse than CD8+ responses (S42, S302, N27). For both CD4+ and CD8+ T cells, conserved TCR gene usage and gene pairings could be identified within multiple donors responding to the same epitope. Conclusion: These data suggest that in SARS-CoV-2 convalescent people, epitope-specific CD4+ and CD8+ T cells can differ in their clonal diversity and that related TCRs can be identified across multiple donors. S42-specific T cell studies are ongoing to determine their transcriptional profile and pMHC presentation. Ongoing longitudinal analysis will provide a better understanding of different epitope-specific TCR repertoires and T cell transcriptional profiles, and how they evolve after infection.
ABSTRACT
Introduction: Since December 2019, COVID-19 has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.