Rapid GMP-Compliant Expansion of SARS-CoV-2-Specific T Cells From Convalescent Donors for Use as an Allogeneic Cell Therapy for COVID-19

Abstract

COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 1010 to 1011 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients.

Keywords: CD4; CD8; COVID-19; T cell; adoptive T cell immunotherapy; memory T cell.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Cooper, Fraser, Smith, Burgoyne, Imlach, Jarvis, Turner, Zahra, Turner and Campbell.

Figures

Figure 1

Analysis of COVID-19 convalescent donor buffy coat-derived PBMCs. (A) Buffy coat-derived PBMCs from COVID-19 convalescent donors (CCD, n = 15, orange circles) and healthy uninfected donors (HD, n = 17, clear circles) were assessed for leukocyte lineage by flow cytometry (see Figure S1A for gating strategy). No correlation was seen between age of COVID-19 convalescent donors with (B) NK cells or (C) B cells, but significant correlation between (D) SARS-CoV-2 serum antibody content and the percentage of B cells between donors (p = 0.04, r = 0.534, Pearson correlation coefficient). (E) Analysis of the T cell compartment (see Figure S1B for gating strategy) shows comparable mean levels of T cell subtypes, as well as (F) CD4+ and (G) CD8+ memory populations between HD and CCD. Data is represented as mean ± SEM. Significance determined by unpaired t-test with Holm-Sidak correction for multiple comparisons. DN double negative (CD4−/CD8−), DP double positive (CD4+/CD8+), TNaive (CD62L+/CD45RA+/CD45RO−), TCM central memory (CD62L+/CD45RA−/CD45RO+), TEM effector memory (CD62L−/CD45RA−/CD45RO+), TEMRA terminal effector memory CD45RA revertant (CD62L−/CD45RA+/CD45RO−). **p ≤ 0.01 and ***p ≤ 0.001.

Figure 2

PBMC responses to SARS-CoV-2 peptides. PBMCs derived from buffy coats were incubated with SARS-CoV-2 peptides (Spike + Nucleocapsid + Membrane) for 5 h and corrected against a no antigen control well for positive expression of cytokines and activation markers. (A) Representation of flow cytometric analysis from a healthy uninfected donor (HD) and COVID-19 convalescent donor (CCD), note all flow analyses were gated on lymphocytes/single cells/live cells and subsequently quantified for percentage CD3+/IFN-γ+ cells, CD3+/TNF-α+ cells and CD4+/CD154+ cells (S2 for gating strategy). The percentage of SARS-CoV-2 VSTs in the CCD PBMC population (i.e. CD3+/IFN-γ+ cells reactive to pooled S+N+M peptides corrected to no antigen control) significantly correlated with (B) antibody titer at donation (p = 0.0381, r = 0.5391) and (C) days from resolution of symptoms to donation (p = 0.0021, r = 0.6275). Calculation was performed using Pearson correlation coefficient. (D) Mean percentages of CD3+/IFN-γ+ cells, CD3+/TNF-α+ cells and CD4+/CD154+ cells for individual and pooled peptides corrected to no antigen control were compared between HD (n = 12, clear circles) and CCD (n = 15, orange circles). Data is represented as mean ± SEM. Statistical significance was determined using unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.

Figure 3

Phenotypic analysis of isolated and expanded SARS-CoV-2 VSTs. The percentages of leukocytes, T cell subpopulations and CD4/CD8 differentiation status were quantified for (A) IFNγ+ target cells directly after SARS-CoV-2 peptide-mediated cytokine capture system (CCS) isolation and (B) following expansion in culture for 14 days. All data is represented as mean ± SEM.

Figure 4

Cultured SARS-CoV-2 VST peptide specificity. Isolated and expanded SARS-CoV-2 VST (n = 5) at day 14 culture were co-cultured with peptide-loaded mature autologous DC. (A) Flow cytometric analysis on either CD3/CD4+ or CD3/CD8+ cells for IFN-γ, TNF-α, CD154, CD107a, and CD137 is shown for negative controls (VSTs Only, and VSTs + Unloaded DCs), and VST with pooled SARS-CoV-2 peptide loaded DCs (VST + SNM-loaded DCs). (B) The mean percentage of CD4+ and CD8+ positive for each marker of DC-stimulated VST was compared using paired t-test Holm-Sidak correction for multiple comparisons, *p ≤ 0.05. (C, D) Individual donor VST were assessed for T cell response (% CD3+/IFN-γ+ and % CD3+/TNF-α+ respectively) against DCs loaded with individual SARS-CoV-2 peptide pools: spike, nucleocapsid and membrane. (E) Collated responses to the individual peptides in the total CD3+ population indicated no significant difference. (F, G) A significantly higher CD8+/IFN-γ+ cells response was seen with nucleocapsid stimulation than with the other peptide pools (significance determined using RM one-way ANOVA with Geisser-Greenhouse correction **p ≤ 0.01). (H) CD4+/IFN-γ+ cells responded similarly to all the three peptide pools. All data represented as mean ± SEM.

Figure 5

SARS-CoV-2 VST culture optimization. (A) Isolated SARS-CoV-2 VST from donors C19BC8-14 had a two- to three-log expansion over 21 day culture using an optimized culture expansion protocol. Variation in the start numbers of VST reflect donor variation in initial buffy coat PBMC numbers. Fold expansion between (B) day 0 and day 8 and (C) day 0 and day 14 was assessed in cultures after supplementation with IL-2, IL-7, and human platelet lysate (PL). Donors C19B9 (square), C19BC11 (triangle), C19BC12 (circle), and C19BC13 (diamond) were divided to compare medium supplementation condition. (D) Representative culture C19BC9 by day 21 without re-stimulation indicated some transition of CD4 TCM to CD4 TEM (Day 21 top panel). CD4 CM phenotype was retained when cultures re-stimulated at day 14 with autologous irradiated feeders (Day 21 bottom panel). (E) VST cultures were split at day 14 to directly compare standard continuation in culture (control) and re-stimulation with autologous irradiated feeders. Data is represented as mean T cell count ± SEM (n = 5). (F) VST from a single donor buffy coat were compared for optimal cell yields at day 14 (grey), and day 21 with feeder re-stimulation at day 14 (Day 21 FR, blue). (G–J) Final product phenotype and T cell memory status was compared at both harvest time-points: Day 14 (grey triangles) and Day 21 FR (blue triangles). Data is represented as mean ± SEM. No significant differences were observed using paired t-tests with Holm-Sidak correction for multiple comparisons.