Gut-associated lymphoid tissues contain 2 distinct lineages of NK cells. (A) Representative gating strategy to identify NKG2A⁺ and NKp44⁺ NK cells among live mononuclear cells in tissues in rectal mucosa specimens. (B) Flow cytometry plots demonstrating disparate expression of CD56 and CD16 on NKG2A⁺ and NKp44⁺ NK cells. (C) Representative histogram overlays depicting expression of NKp46, CD8α, CD117, CD127, CCR6, and CXCR3 on the 2 NK-cell subpopulations. Isotype-matched controls are shown in gray. RT-PCR analysis was used to quantify mRNA transcripts of transcription factors (D) and regulatory cytokines IL-22 and CCL20 (E) in NKG2A⁺ and NKp44⁺ NK cells sorted from rectal mucosa of normal macaques. Mann-Whitney U test (*P < .05). (F) Flow cytometry plots depicting CD107a and intracellular cytokine responses in the 2 NK-cell subpopulations after mitogen stimulation. Polychromatic flow cytometry and ICS experiments are representative of 8-16 animals per group; molecular analyses show geometric means ± SEM of 4 animals per group. (G) Flow cytometry plots comparing distribution of NKG2A⁺ and NKp44⁺ NK cells in blood and various tissues of rhesus macaques using the gating strategy shown in panel A; data are representative of > 12 animals. MLN indicates mesenteric lymph node; and PLN, peripheral lymph node.