Combinatorial Cytokine Secretion Signature of Donor-Derived T Cells Infused with the Graft: A New Potential Biomarker of Acute Graft-Versus-Host Disease in Αβt-Cell/CD19 B-Cell Depleted Hematopoietic Stem Cell Transplant Recipients

Background: Hematopoietic stem cell graft manipulation strategies, such as αβT-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT), address the lack of matched donors and reduce the incidence of severe acute graft-versus-host disease (aGvHD). However, grade II-IV aGvHD still occurs in 25-30% of αβhaplo-HSCT recipients . Studies aimed at understanding the pathogenesis underlying aGVHD in αβhaplo-HSCT are lacking. We hypothesized that αβT cells adoptively transferred with the HSCT (<1x10 ⁵/Kg) have unique combinatorial cytokine secretion signatures that may predict the occurrence of aGvHD. Here we used the IsoPlexis single-cell proteomics for CD4 ⁺ and CD8 ⁺ T cells to identify those putative signatures .

Methods: Six patients with hematologic malignancies receiving fully myeloablative αβhaplo-HSCT at Lucile Packard Children's Hospital, Stanford, between 08/2018 and 05/2020 were enrolled upon signing IRB approved informed consent. Three patients developed grade II-IV aGvHD, while three did not. Aliquot of the graft and of peripheral blood collected at the time of aGvHD onset or at corresponding time points for the patients who did not develop aGvHD, were analyzed. Single sorted CD4 ⁺ and CD8 ⁺ T cells were profiled by single-cell barcode chip assay from IsoPlexis system (IsoPlexis, Branford, CT) after stimulation with PMA (50 ng/mL) and Ionomycin (1mcg/mL). Following the Human Adaptive Immune Panel, cytokines from CD4 ⁺ and CD8 ⁺ single T cells were captured by fluorescence ELISA, which measured the numbers of cytokine-producing cells (secretion frequency) and numbers of cytokines produced by individual cells across five functional groups: effector, stimulatory, chemoattractive, regulatory and inflammatory (Table 1). Polyfunctionality was defined as the secretion of 2+ cytokines from each CD4 ⁺ and CD8 ⁺ T cell. The T cell polyfunctional strength Index (PSI) was defined as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Additional statistical analysis was performed using the Student's t test.

Results: We compared the combinatorial cytokine secretion signature of individual CD4 ⁺ and CD8 ⁺ T cells isolated from grafts infused into patients, who eventually did or didn't develop aGvHD. We are comparing the signature of post-HSCT CD4 ⁺ and CD8 ⁺ T cells isolated from patients who did or did not develop aGvHD. Collectively, we considered three variables: cytokine secretion frequency, numbers of cytokines produced by individual cells and characteristics of the cytokines secreted (functional group) upon stimulation.

Single-cell functional heterogeneity evaluated by t-Distributed Stochastic Neighbor Embedding (t-SNE), showed higher CD4 ⁺ and CD8 ⁺ T-cell polyfunctionality (up to 4+ cytokines) with effector and stimulatory dominant functions in the grafts of patients who developed aGvHD, compared to those who did not develop aGvHD (Fig1). The average PSI (driven by Granzyme B, TNF-α, IFN-γ, MIP-1β, IL2, and IL-8) was found to be higher in both CD4 ⁺ and CD8 ⁺ T cells from the grafts of patients who developed aGvHD (Fig 2). Combinatorial cytokine secretion analysis showed that T cells from grafts of patients who did not develop aGvHD had unique signatures with CD4 ⁺ T cells having the predominant cytokine secretion signature of IL2 and TNF-α, and CD8 ⁺ T cells having three predominant cytokine secretion signatures: IL2, IL8, TNF-α; MIP-1β, IL8; and MIP-1β, IFN-γ (Fig3).

Conclusions: Preliminary data from αβhaplo-HSCT pediatric recipients obtained using IsoPlexis single-cell functional proteomics for CD4 ⁺ and CD8 ⁺ T cells showed that an increased donor T-cell polyfunctionality with a Th1 dominant functional phenotype may be predictive of an increased risk of aGvHD, while CD4 ⁺ and CD8 ⁺ T cells infused into patients who didn't develop aGvHD, had combinations with limited cytokine secretion signatures. Ongoing analysis suggest that polyfunctional CD8 ⁺ T cells present in the graft of patients who developed aGvHD, are present at the time of aGvHD initiation, while the polyfunctional CD4 ⁺ T cell are not present at the onset of aGvHD.

Correlation with ongoing studies on circulating cytokines and clonotypic analysis of αβT cells infused with the graft will be crucial to elucidate the cross talking between the donor's immune system and recipient's inflammatory milieu.

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