Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8 alpha(+) and CD4(+)CD8 alpha(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8 alpha(+) and DN iNKT cells upon mitogen stimulation. CD8 alpha(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-gamma and TNF-alpha) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8 alpha>DN>CD4 pattern for Th1 and CD4>DN>CD8 alpha for Th2. All iNKT subsets could simultaneously produce IFN-gamma and IL-4, but single-positivity for IFN-gamma or IL-4 was strikingly rare in CD4(+) and CD8 alpha(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8 alpha(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.