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. 2022 Jun 9:25:439-447.
doi: 10.1016/j.omtm.2022.04.013. Epub 2022 Apr 29.

Transcriptomic analysis reveals optimal cytokine combinations for SARS-CoV-2-specific T cell therapy products

Jessica Durkee-Shock  1   2 , Christopher A Lazarski  1 , Mariah A Jensen-Wachspress  1 , Anqing Zhang  3 , Aran Son  2 , Vaishnavi V Kankate  1 , Naomi E Field  1 , Kathleen Webber  1 , Haili Lang  1 , Susan R Conway  1 , Patrick J Hanley  1   3   4 , Catherine M Bollard  1   3   4 , Michael D Keller  1   3   5 , Daniella M Schwartz  2
Affiliations

Transcriptomic analysis reveals optimal cytokine combinations for SARS-CoV-2-specific T cell therapy products

Jessica Durkee-Shock et al. Mol Ther Methods Clin Dev. .

Abstract

Adoptive T cell immunotherapy has been used to restore immunity against multiple viral targets in immunocompromised patients after bone-marrow transplantation and has been proposed as a strategy for preventing coronavirus 2019 (COVID-19) in this population. Ideally, expanded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-virus-specific T cells (CSTs) should demonstrate marked cell expansion, T cell specificity, and CD8+ T cell skewing prior to adoptive transfer. However, current methodologies using IL-4 + IL-7 result in suboptimal specificity, especially in CD8+ cells. Using a microexpansion platform, we screened various cytokine cocktails (IL-4 + IL-7, IL-15, IL-15 + IL-4, IL-15 + IL-6, and IL-15 + IL-7) for the most favorable culture conditions. IL-15 + IL-7 optimally balanced T cell expansion, polyfunctionality, and CD8+ T cell skewing of a final therapeutic T cell product. Additionally, the transcriptomes of CD4+ and CD8+ T cells cultured with IL-15 + IL-7 displayed the strongest induction of antiviral type I interferon (IFN) response genes. Subsequently, microexpansion results were successfully translated to a Good Manufacturing Practice (GMP)-applicable format where IL-15 + IL-7 outperformed IL-4 + IL-7 in specificity and expansion, especially in the desirable CD8+ T cell compartment. These results demonstrate the functional implications of IL-15-, IL-4-, and IL-7-containing cocktails for therapeutic T cell expansion, which could have broad implication for cellular therapy, and pioneer the use of RNA sequencing (RNA-seq) to guide viral-specific T cell (VST) product manufacturing.

Keywords: COVID-19; RNA-seq; SARS-CoV-2; cytokines; immunocompromised; manufacturing; therapeutic viral-specific T cells.

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Conflict of interest statement

P.J.H. is a cofounder and is on the board of directors of Mana Therapeutics. P.J.H. is on a scientific advisory board for Cellevolve. M.D.K. is on a scientific advisory panel for Gilead Sciences. The remaining authors declare no competing financial interests.

Figures

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Graphical abstract
Figure 1
Figure 1
PBMCs from 9 donors were stimulated with SARS-CoV-2 pepmixes and then seeded at 200,000 cells per well in a 96-well plate Cells were split on day 7 and then proceeded directly to intracellular flow cytometry on day 10. Expansion, phenotype, and anti-viral T cell specificity were evaluated in 5 cytokine conditions: IL-4 + IL-7, IL-15, IL-15 + IL-4, IL-15 + IL-6, and IL-15 + IL7. (A) Absolute counts of CD3+ T, CD4+ T, CD8+ T, and CD3-CD56+ NK cells on day 10. An approximation of fold expansion is provided in Figure S4. (B) Proportions of naive, central memory, effector memory, terminal effector, T cell receptor (TCR)αβ, and TCRγδ CD3+ T cells on day 10. (C) CD4+ T cell specificity as measured by absolute count of CD4+IFNγTNF-α+ cells on day 10. (D) CD8+ T cell specificity as measured by absolute count of CD8+IFNγTNF-α+ cells on day 10. (E) CD3+ T cell specificity as measured by absolute count of CD3+CD107a+ cells on day 10. ∗Statistically significant, p < 0.0125 corrected for multiple comparisons. Error bars represent 95% confidence intervals.
Figure 2
Figure 2
Transcriptomic analysis of CSTs shows that IL-15 + IL-7 increases stimulation of important antiviral interferon response genes (A) Heatmap displays 450 differentially expressed genes (DEGs) in CD4+ T cells cultured in at least one cytokine-containing condition versus CD4+ T cells cultured without cytokines (fold change >2 or <-2, false discovery rate [FDR] <0.05, ANOVA, Partek). Colors denote average log2(fold change in gene expression) for cells cultured in cytokine-containing conditions versus cells cultured without cytokines. Selected enriched pathways and representative genes (metascape) are shown for each module of DEGs. (B) Heatmap displays 851 DEGs in CD8+ T cells cultured in at least one cytokine-containing condition versus CD4+ T cells cultured without cytokines (fold change >2 or <-2, FDR<0.05, ANOVA, Partek). Colors denote average log2 fold change in gene expression for cells cultured in cytokine-containing conditions versus cells cultured without cytokines. Selected enriched pathways and representative genes (metascape) are shown for each module of DEGs. (C) Scatterplot displays normalized enrichment score (NES) and FDR for an antiviral interferon-stimulated gene cassette (Immunological Genome Project, GEO: GSE75306) in CD4+ T cells. Dashed line denotes an FDR of 10%. NESs are shown for each condition versus all other conditions. Conditions where antiviral gene cassette is significantly induced are marked in red; conditions where antiviral gene cassette is significantly repressed are marked in blue. Representative GSEA plot for CD4+ T cells cultured in IL-15 + IL-7 versus other conditions. (D) Scatterplot displays NES and FDR for an antiviral interferon-stimulated gene cassette (Immunological Genome Project, GEO: GSE75306) in CD8+ T cells. Dashed line denotes an FDR of 10%. NESs are shown for each condition versus all other conditions. Conditions where antiviral gene cassette is significantly induced are marked in red; conditions where antiviral gene cassette is significantly repressed are marked in blue. Representative GSEA plot for CD8+ T cells cultured in IL-15 + IL-7 versus other conditions.
Figure 3
Figure 3
Microexpansion of donor PBMCs identifies optimal cytokine conditions for production of CD4+ and CD8+ CSTs (A) Fold expansion of live cells following 10 day expansion. (B) Proportions of total T cells (CD3+), CD4+ T cells, CD8+ T cells, TCRαβ T cells, TCRγδ T cells, CD56+CD3- NK cells, CD19+ B cells, and CD14+ monocytes after expansion. (C) Proportion of CD3+ total T cells identified as naive (CCR7+, CD45RO-), central memory (CCR7+, CD45RO+), effector memory (CCR7-, CD45RO+), and terminal effector (CCR7-, CD45RO-) cells after expansion. (D) CD4+ T cell specificity with differing cytokine conditions (IL-4 + IL-7 versus IL-15 + IL-7) as measured by proportion of IFNγ+TNF-α+-secreting cells. (E) CD8+ T cell specificity with differing cytokine conditions (IL-4 + IL-7 versus IL-15 + IL-7) as measured by proportion of IFNγ+TNF-α+-secreting cells. (F) CD3+ T cell specificity with differing cytokine conditions (IL-4 + IL-7 versus IL-15 + IL-7) as measured by proportion of CD107a+-expressing cells. (G) Antiviral specificity of SARS-CoV-2-specific T cells (day 10) expanded with IL-4 + IL-7 or IL-7 + IL-15 to spike and membrane peptide libraries as measured by spot forming units (SFU) per 100,000 cells on IFNγ ELIspot assay. ∗Statistically significant, p < .05. ∗∗Statistically significant, p < .0125, corrected for multiple comparisons for (B). ∗∗∗Statistically significant, p < 0.017, corrected for multiple comparisons for (C). Error bars represent 95% confidence intervals.
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