B cell adaptor for PI3-K (BCAP) was identified as a cytosolic adaptor protein that can recruit phosphatidylinositol 3-kinase (PI3-K). B-cells from mice deficient in BCAP have an impaired ability to reach full maturity, produce less immunoglobulin, have decreased proliferative capability, are more susceptible to apoptosis, and exhibit reduced calcium mobilization in response to antigen receptor crosslinking. We recently discovered that NK cells express BCAP and studied NK cells in BCAP-deficient mice. In sharp contrast to the studies of B cells, NK cells in mice lacking BCAP are more mature, more long-lived, more resistant to apoptosis, and exhibit enhanced functional activity compared to NK cells from normal mice. The paradoxical phenotype reveals inherent differences in the signals controlling the final maturation of B cells and NK cells, which depend on positive and negative signals, respectively. Since NK cells require inhibitory receptor signaling to become functional effector cells, we hypothesize that the loss of BCAP blunts activation in a manner that is similar to inhibitory signaling, thereby augmenting NK cell terminal maturation and function. NK cells that develop under conditions lacking inhibitory receptor signaling have been shown to become hypo-responsive, due to chronic activation signaling that is not adequately suppressed. Therefore, we propose that the increased function, accumulation, and survival of mature NK cells in BCAP-deficient mice is due to better damping of a desensitizing signal that is at least partially BCAP-mediated.Top

Growing evidence indicates that NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) through the Fc receptor, FcγRIIIA (CD16), is an important mechanism by which some therapeutic anti-tumor antibodies effectively reduce tumor burden in human patients. Some, but not all human tumors down-regulate expression of MHC class I (MHC-I) “self” molecules, and therefore, engagement of inhibitory KIR by MHC-I-bearing tumors has the potential to severely limit ADCC responses in the clinic. We collaborated with Dr. Louis Weiner (currently at Lombardi Cancer Center, Georgetown University) to test whether blocking KIR-mediated self-recognition with antibodies can diminish the inhibitory signal to boost in vitro ADCC responses. Indeed, our results demonstrated that KIR blockade potentiated ADCC responses by primary human NK cell toward autologous EBV-transformed MHC-I-expressing B cells treated with the anti-tumor antibody, rituximab. Maximal potentiation required a cocktail of multiple inhibitory receptor antibodies (anti-KIR, -ILT2, and -NKG2A/CD94) to block a wide spectrum of the available self-recognizing receptors. Importantly, inhibitory receptor blockade alone did not significantly increase killing of the autologous cells, indicating that additional physiologic checkpoints are in place. Adding rituximab to engage FcγRIIA, however, can break this tolerance. Furthermore, our results using the autologous target cell system also indicated that inhibitory receptor blockade could better potentiate ADCC responses by NK cells from donors homozygous for the low affinity allele of FcγRIIIA (158F). This is important, since follicular lymphoma patients expressing the low affinity FcγRIIIA (158F) have reduced clinical response rates to rituximab therapy, as compared to patients homozygous for the high affinity allele (158V). Our findings have significant translational relevance, since they are the first to establish the potential therapeutic benefit of blocking self-recognition by inhibitory KIR in combination with anti-tumor antibody treatments in human patients, which may be particularly beneficial in low-responding humans that express low affinity FcγRIIIA. Top

A structurally unique KIR, named 2DL4, activates potent cytokine and chemokine production, but only weak cytotoxicity responses in human NK cells. We have discovered two distinct activation signaling cassettes that are initiated from 2DL4: 1) physical linkage to the transmembrane adaptor protein, FcεRI--γ, which triggers calcium mobilization and weak cytotoxicity responses, and 2) FcεRI-γ-independent signaling that is likely mediated directly through the receptor’s cytoplasmic domain and is capable of stimulating production of the chemokine, MIP1α. Association with FcεRI--γ distinguishes 2DL4 from the DAP12-associated activating KIRs with short cytoplasmic domains (KIR2DS1-5 and KIR3DS1).

Our studies have also shed new light on the unique signaling mechanisms triggered through 2DL4. NF-κB, ERK, and p38 are stimulated very early and transiently after receptor engagement (0.5-2 minutes), while JNK is activated much later (10-30 minutes). All of these signaling events occur independent of receptor association with FcεRI-γ. Pharmacological inhibitors revealed that all three MAP kinases are essential for triggering downstream cytokine and chemokine production.

The only well-characterized ligand for 2DL4 is a soluble form of the non-classical MHC class I molecule, HLA-G, which is mainly produced by fetal-derived trophoblast cells in pregnant women. This has led to a hypothesis that 2DL4 may play an important role in stimulating NK cells to secrete cytokines that support placental development. Importantly, HLA-G is reportedly also expressed by numerous tumor cells, indicating that this receptor may also play important roles in tumor surveillance. We further hypothesize that additional ligand(s) may exist. Our studies defining the unique signaling pathways and surface regulation of 2DL4 should establish molecular mechanisms by which the receptor may play important roles in regulating pregnancy and preventing cancer.Top

Recently, we have characterized serine and threonine phosphorylation sites on the cytoplasmic domain of inhibitory KIR, three of which are conserved in all human inhibitory KIR. We identified four serine/threonine (S/T) phosphorylation sites on the KIR3DL1 cytoplasmic domain (S364, S367, S394, and T399). Our studies concluded that casein kinase I (CKI) phosphorylates S364, casein kinase II (CKII) phosphorylates S367, and PKC phosphorylates S394. The kinase phosphorylating T399 has not yet been identified. Pharmacological inhibitors indicated that most constitutive KIR phosphorylation is mediated by CKI, and primary phosphorylation of either S364 or S367 by CKI or CKII, respectively, promotes secondary phosphorylation of the other site.

The functional impacts of the CK and PKC phosphorylation sites were tested by mutation of the individual serines to non-phosphorylatable alanine (A) or to the phosphomimetic aspartic acid (D), which mimics the charge introduced by a phosphate at that serine within the protein sequence. We found that non-phosphorylatable mutation of the PKC phosphorylation site (S394A) or both CK phosphorylation sites (S364/367A) increases the amount of receptor expressed on the cell surface. Turnover or internalization rates of the S364/367A mutant were unchanged, but a non-phosphorylatable PKC site mutant (S394A) showed enhanced surface turnover, while a phosphomimetic mutant (S394D) demonstrated reduced turnover capacity. Finally, the phosphomimetic mutant was internalized at a slower rate and exhibited slightly reduced inhibitory function, as compared to the wild type receptor. Together, these results indicate that serine phosphorylation of KIR by PKC stabilizes the receptor on the NK cell surface. Importantly, stable surface expression of inhibitory KIR is essential to maintain self tolerance by NK cells, implying that normal phosphorylation of the serine by PKC is necessary to prevent autoimmune responses by human NK cells. Top

Prostaglandin D2 (PGD2) has been shown to be increased in bronchiolar lavage fluid after allergen challenge and has been implicated as an important mediator of asthma, since it can cause bronchioconstriction and chemotaxis of Th2 cells. This prostaglandin is produced by mast cells and alveolar macrophages. We tested the hypothesis that PGD2 can influence the function of NK cells at sites of allergic responses. Treatment of NK cells with PGD2 was found to inhibit cytotoxicity, cytokine production (IFN-γ, TNF-α, and GM-CSF), and chemotaxis, but did not affect proliferation or differentiation. Pharmacologic studies indicated that the PGD2 effect was mediated through binding to the D-prostanoid (DP) receptor on the surface of NK cells. PGD2 binding to DP stimulated the production of cAMP, which was responsible for the inhibitory effects. Therefore, we hypothesize that PGD2 plays a critical role in suppressing inflammatory responses by NK cells at sites of allergic insult, such as at mucosal surfaces.Top