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3441 CD56brightCD16-Perforin- regulatory Natural Killer Cells Associated with the Suppression of Chronic Graft-Versus-Host Disease Prevent CD4+ T Cell Proliferation through PD-1 and LAG-3 Dependent Pathways

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Translational Research, GVHD, Diseases, Immune Disorders
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Madeline Lauener, BSc1, Kirk R. Schultz2,3, Sayeh Abdossamadi4*, Elena Ostroumov5* and Megan Levings6*

1University of British Columbia, Vancouver, Canada
2University of British Columbia, Vancouver, BC, Canada
3B.C. Children's Hospital, Vancouver, BC, Canada
4BC Children's Hospital Research Institute, Vancouver, BC, CAN
5BC Children's Hospital Research Institute, Vancouver, Canada
6University of British Columbia, Canada, Vancouver, BC, CAN

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large cohorts of HSCT patients we identified increased numbers of CD56bright, granzyme B-, perforin- NK cells (NKreg) to be associated with a lack of cGvHD development. Transcriptome analysis of HSCT patient samples identified a unique transcriptome among the CD56bright NKreg cells which associated with patients who failed to develop cGvHD, including overexpression of genes IL7R, GPR183, and Granzyme K, and low expression of genes such as perforin, granzyme B, and CD16. We then utilized the transcriptome information and further cell phenotyping to determine the optimal sorting approach for viable NKreg cells to allow for functional analysis by the sorting of CD56brightCD3-CD16- cells. Functional analysis suggested that the NKreg population suppresses CD4+ T cells through a non-cytolytic, and direct cell-cell contact-dependent mechanism, though the exact receptor and ligand interaction of which CD4+ T cell populations are impacted is unknown. We hypothesized that the NKreg cell suppressive mechanism may be more specific than the more widely studied regulatory cell population, Treg cells, by suppressing CD4+ T cell subpopulations involved in inflammatory responses.

Methodology: To investigate the suppressive capacity of NKreg cells, the cells were isolated from healthy donor peripheral blood and co-cultured with allogeneic CD4+ T cells, CD8+ T cells, Treg cells, Treg cells and dendritic cells, B cells, or NK cells for 96hrs. After 96hrs, the proliferation and viability of the responder cells were evaluated via proliferation dye dilution, and 7-AAD dye, respectively. To determine if the CD4+ T cell suppression is specific to a T helper (Th) cell subset we stained the co-cultured cells with a Th1/Th2/Th17 Phenotyping Kit. Further, PD-1 and LAG-3 neutralizing antibodies were added to the NKreg/CD4+ T cell co-culture to determine the receptor dependence. All samples were acquired with the BD FACSymphony Flow Cytometer and the data was analyzed via Kaluza software.

Results: CD56brightCD16-perforin- NKreg cells strongly suppress CD4+ T cell proliferation (approximately 96% suppression of CD4+ T cell proliferation at the 1:1 ratio of NKreg cells to CD4+ T cells). The contact-dependent mechanism of NKreg suppression of CD4+ T cell proliferation was significantly decreased when blocking either the PD-1 (15% decrease in suppression, p=0.03), or LAG-3 (29% decrease in suppression, p=0.04) receptors, at the 1:2 ratio of NKreg cells to CD4+ T cells. When both receptors are blocked the inhibition of suppressive effect is comparable to that of the LAG-3 blocking antibody being added individually (30% decrease in suppression, p=0.03). The suppressive mechanism of NKreg cells was observed to be selective in that they strongly suppress CD4+ T cell proliferation, but do not result in statistically significant suppression of CD8+ T cell, Treg cell, B cell, NK cell, or a specific Th cell subset proliferation (p>0.05). Further, though the average proliferation of the Treg cells co-cultured with NKreg cells and dendritic cells compared to just NKreg cells was greater (127% proliferation compared to 100% proliferation, respectively), we did not observe NKreg cells to induce a statistically significant proliferative effect towards the Treg cells in the presence of dendritic cells.

Conclusion: As a result of our studies, we have confirmed the NKreg cell immune suppressive function towards CD4+ T cell proliferation, a main contributor to cGvHD development. Further, we demonstrated a PD-1/LAG-3-dependent direct contact mechanism of NKreg cell suppression, which is selective of total CD4+ T cells, with a lack of suppressive effect towards CD8+ T cells, Treg cells, B cells, and NK cells. The results of these studies contribute to our better understanding of how NKreg cells may induce a cell-specific suppressive function to promote immune tolerance, providing potential cell therapeutic applications for enhancing NKreg cell suppressive function through increasing PD-1/LAG-3 ligand or receptor expression on NKreg cells.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH