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

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 CD56^bright, granzyme B^-, perforin^- NK cells (NK_reg) to be associated with a lack of cGvHD development. Transcriptome analysis of HSCT patient samples identified a unique transcriptome among the CD56^bright NK_reg 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 NK_reg cells to allow for functional analysis by the sorting of CD56^brightCD3^-CD16^- cells. Functional analysis suggested that the NK_reg 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 NK_reg cell suppressive mechanism may be more specific than the more widely studied regulatory cell population, T_reg cells, by suppressing CD4⁺ T cell subpopulations involved in inflammatory responses.

Methodology: To investigate the suppressive capacity of NK_reg cells, the cells were isolated from healthy donor peripheral blood and co-cultured with allogeneic CD4⁺ T cells, CD8⁺ T cells, T_reg cells, T_reg 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 (T_h) cell subset we stained the co-cultured cells with a T_h1/T_h2/T_h17 Phenotyping Kit. Further, PD-1 and LAG-3 neutralizing antibodies were added to the NK_reg/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: CD56^brightCD16^-perforin^- NK_reg cells strongly suppress CD4⁺ T cell proliferation (approximately 96% suppression of CD4⁺ T cell proliferation at the 1:1 ratio of NK_reg cells to CD4⁺ T cells). The contact-dependent mechanism of NK_reg 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 NK_reg 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 NK_reg 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, T_reg cell, B cell, NK cell, or a specific T_h cell subset proliferation (p>0.05). Further, though the average proliferation of the T_reg cells co-cultured with NK_reg cells and dendritic cells compared to just NK_reg cells was greater (127% proliferation compared to 100% proliferation, respectively), we did not observe NK_reg cells to induce a statistically significant proliferative effect towards the T_reg cells in the presence of dendritic cells.

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