Elucidation of Human Invariant NKT Cell Immunophenotypes to Provide Insights for Future Universal Donor Derived Cellular Therapeutics

Invariant Natural Killer T (iNKT) cells are innate lymphocytes capable of mounting responses to lipid antigen stimulation. iNKT cells use a unique, invariant, T cell receptor (TCR) to recognize lipid antigens in the context of an MHC-like molecule CD1d. Unlike MHC molecules, CD1d molecules are monomorphic giving iNKT cells the potential to be sourced from universal donors. This unique property allows for off-the shelf cellular therapies in multiple disease scenarios without the risk of MHC mismatch and graft-versus-host disease. Murine iNKT cell research has provided substantial yet incomplete insights on the multifunctional and transcriptionally distinct subsets of these cells harboring varying pro- and anti-inflammatory capabilities. Studies in humans have yet to fully elucidate functionally distinct subsets within the iNKT cell population. Our objective was to further characterize and validate our previous work describing the transcriptional heterogeneity of human iNKT cells to better leverage their capabilities for use as therapeutics drawing on distinct functional capacities for different disease conditions.

We previously performed single cell RNA-seq analysis of Vα24⁺Vβ11⁺ iNKT cells enriched and purified from peripheral blood (PB), cord blood (CB), thymus, and bone marrow (BM) of healthy donors. We described transcriptionally distinct clusters of varying tissue predominance with 3 primary transcriptional signatures: Th1/17/NK-like, Th2-like, and naive precursors. We had also incorporated oligomer- conjugated antibodies which allowed us to identify iNKT cells with surface protein expression of CD45RA that lacked CCR7 at the transcript level. These cells demonstrated the highest expression of cytotoxicity genes, suggesting a similar immunophenotype to T effector memory CD45RA⁺ (TEMRA) cells. In the present study, we have now verified the existence of this population by flow cytometry on peripheral blood-derived iNKT cells from healthy donors. Our data shows that CD45RA⁺CCR7^- (TEMRA-like) iNKT cells account for 2.62% (SEM 0.47, n=11) of the total iNKT cell population in peripheral blood. Our data also showed that 75.57% (SEM 6.02, n=11) of iNKT cells are CD45RA^-CCR7^- (effector memory-like), 17.94% (SEM 3.75, n=11) are CD45RA^-CCR7⁺ (central memory-like), and 3.88% are CD45RA⁺CCR7⁺ (naïve-like).

While only CD4⁺ or double negative (DN, CD4^-CD8^-) iNKT cells arise in mice, human iNKT cells can be CD4⁺, DN, or CD8⁺. Our previous data captures transcriptional evidence of CD8⁺ iNKT cells and suggests a CD8αα⁺ subset based on relative transcript levels of CD8A and CD8B. In the present study, we have verified that both CD8αβ and CD8αα populations exist in human peripheral blood-derived iNKT cells at the protein level using flow cytometry. Furthermore, we have confirmed CD8αα⁺ iNKT cells constitute 87.20% (SEM 2.17, n=7) of the total CD8⁺ iNKT cell compartment in human peripheral blood.

Our previous transcriptional findings suggested novel putative populations of iNKT cells, including TEMRA-like iNKT cells and CD8aa-expressing iNKT cells. We have now validated the existence of these unique populations in human peripheral blood. Currently, we are assessing the functional implications of these populations. Investigations are also underway to identify surface markers that would allow the separation of the Th1/17/NK-like, Th2-like, and naive precursor subsets. These data will pave the way for developing the optimal iNKT cell therapeutics for different disease scenarios.