Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor β (IL-2Rβ CD122) and the polymorphic antigen NK1.1. These “NK-like” T cells are distinguished by their highly skewed Vα and Vβ repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1⁺ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2Rβ⁺ T cells with a skewed Vβ repertoire characteristic of “NK-like” T cells. Furthermore lack of NK1.1 expression on IL-2Rβ⁺ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2Rβ⁺ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that “NK-like” T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.     

The effect of in vivo depletion of NKR-P1+ or CD8+ lymphocytes on the acute rejection of allogeneic lymphocytes (ALC) in the rat

We have depleted lymphocyte subsets in PVG and AO rats with MoAbs 3.2.3 (against NKR-P1 on NK and NK/T cells) and OX-8 (against CD8 on CTL and NK cells), and examined the effect on the killing of YAC-1 target cells in vitro and the effect on the acute rejection of small allogeneic lymphocytes in vivo (allogeneic lymphocyte cytotoxicity, ALC). While 3.2.3 treatment led to only a partial depletion of 3.2.3-positive cells in PVG rats, this treatment drastically reduced the number of NKR-P1+ cells in AO rats, abolished splenic NK activity against the NK-sensitive tumour target YAC-1, and markedly diminished the ALC response. Rats treated with OX-8 for 1 day showed a similar loss of NK cell function in vivo and in vitro. However, in rats treated with OX-8 for 3 days a 3.2.3+ and OX-8- population consisting of NK cells appeared, restoring ALC. The results demonstrate that NK cell responses can be greatly diminished after in vivo treatment with these MoAbs. Furthermore, they demonstrate that ALC is not necessarily linked to expression of the CD8 molecule.     

In Vivo CD3+CD25+ Lymphocyte Subpopulation Is Down-regulated Without Increased Serum-Soluble Interleukin-2 Receptor (sIL-2R) by Gonadotropin Releasing Hormone Agonist (GnRH-a)

PROBLEM: To test further whether the suppression of the CD3⁺CD25⁺ lymphocyte sub-population by gonadotropin-releasing hormone agonist (GnRH-a) is related to the change in levels of cytokines and soluble interleukin-2 receptor (sIL-2R). METHOD: Twenty-seven infertile patients were enrolled under the long protocol of GnRH-a agonist (buserelin acetate) and superovulation with gonadotropin from our IVF-ET program. Peripheral B cells, NK cells, CD4⁺ and CD8⁺ T cells and the expression of CD69, CD25, HLA-DR, and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. Serum levels of cytokines and sIL-2R were measured. RESULTS: The B cells, NK cells, T cells, CD4⁺, CD8⁺ T cells, CD3⁺DR⁺, and CD3⁺CD71⁺ lymphocyte subpopulations were not changed after the use of GnRH-a. The CD25⁺ T cell subpopulation was significantly down-regulated, but the CD69⁺ T cell subpopulation was increased when the GnRH-a was used for approximately 2 wk. The serum levels of interleukin-lp (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), and sIL-2R were not changed. CONCLUSION: The GnRH-a had a transiently suppressive effect on CD25⁺ T cells, but a stimulatory effect on CD69⁺ T cells. However, the serum level of cytokines or sIL-2R did not change. These immunological modulations seems to be the result of interaction between GnRH-a and estrogen.     

Profiles of NK, NKT cell activation and cytokine production following vaccination against hepatitis B

Human natural killer (NK) cells (CD56+ CD3-) represent crucial components of the innate immune system especially against viral infections and because their activation can modulate the outcome of the adaptive immune response. NKT cells (CD56+CD3+), a lymphocyte T population characterized by expression of surface markers of NK cells, are known to be abundant in the liver and their activation could be associated with hepatic injury. Using three-color flow cytometry to measure surface receptors and intracellular cytokines, we have explored early activation signals and cytokine production in NK and NKT cells within a group of hepatitis B vaccinated and non-vaccinated individuals. A specific increase of the CD56bright cell population, the activation receptor CD69 and IFN-gamma, was observed in NK cells following incubation with recombinant HBsAg in responders to vaccination. Comparable results were observed in NKT cells showing an increment of CD69, CD25, IL-2 and IFN-gamma expression in responder subjects. These parameters were statistically diminished in non-responder individuals (p<0.05) in both groups of cells. These results demonstrate a diminished activation of these cells in non-responders to the vaccine, suggesting that NK and NKT cells play an important role in the immune response following hepatitis B vaccination.     

Cross-linking a mAb to NKR-P2/NKG2D on dendritic cells induces their activation and maturation leading to enhanced anti-tumor immune response

NKR-P2/NKG2D is the chief tumor recognition receptor of NK cells and some T cells, which recognizes stress inducible ligands on tumors and mediates cell activation. We have recently reported the involvement of NKR-P2 in rat dendritic cell (DC) activation. We demonstrate the potential of agonistic anti-NKR-P2 mAb (1A6), which mimics the NKR-P2 ligand and induces activation and maturation of DCs. Interaction of DCs with 1A6 enhances nitric oxide-mediated apoptosis in tumor cells. Cross-linking of NKR-P2 with mAb1A6 up-regulates MHC II, CD86, CD1a, antigen-presentation function and decreases endocytic activity of DC, thus drives DCs to play a pivotal role in adaptive immune responses. NKR-P2 cross-linking with 1A6 also induced the secretion of inflammatory cytokines, IL-1beta, tumor necrosis factor-alpha, IFN-gamma and IL-12 by DCs. Blocking of 1A6-mediated activation and maturation with inhibitors of PI3K, p38K and ERK1/2K suggests involvement of MAP kinase in signal transduction. 1A6 cross-linking activates nuclear factor kappa B, which acts as key executioner of DC activation. Administration of 1A6 antibody induces rapid regression and protective immune responses against transplantable tumors, suggesting mAb induced activation and maturation of DCs, leading to enhanced anti-tumor immune response.     

Foxp3(high) and Foxp3(low) Treg cells differentially correlate with T helper 1 and natural killer cells in peripheral blood

Regulatory T (Treg) cells interact with B, natural killer (NK), and dendritic cells in addition to other T cells. In this study, we aimed at determining whether Foxp3(+) T cells and subpopulations have any correlation with other lymphocyte subsets and their functions in a systemic immune environment. Peripheral blood was drawn from 22 nonpregnant healthy women. T, B, and NK cell subpopulations were measured by immunophenotype analysis. Intracellular Foxp3, cytokine expression (tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], and interleukin-10 [IL]-10), and NK-cell cytotoxicity were analyzed by flow cytometric analysis. Correlations between Foxp3(+) T cells and other immune variables were analyzed under control of age and menstrual phases. Foxp3(+), Foxp3(low), and CD4(+)Foxp3(+) cells significantly correlated with CD4(+)CD25(+), CD4(+)CD25(dim), and CD4(+)CD25(bright) cells. Foxp3(+), Foxp3(low), and CD4(+)Foxp3(+) cells positively correlated with CD3(+) and CD3(+)CD4(+) T cells, but negatively correlated with CD3(-)CD56(+) and CD3(-)CD56(dim) NK cells. CD4(+)Foxp3(high) Treg cells were positively correlated with CD3(+)CD4(+)TNF-α(+) (p = 0.014) and negatively correlated with CD3(+)CD8(+)IL-10(+) T cells (p = 0.001). The ratio of type 1/2 cytokine-producing CD3(+)CD8(+) cells demonstrated a positive correlation with CD4(+)Foxp3(high) cells (p ≤ 0.01). CD8(+)Foxp3(+) cells were positively correlated with CD3(+)CD4(+)IL-10(+) cells (p = 0.007) and negatively correlated with CD3(+)CD8(+)TNF-α(+) cells (p = 0.008). In conclusion, each Foxp3(+) Treg cell subpopulation has unique immune interaction, which controls particular subsets of lymphocytes.     

Lymphocyte subset patterns and cytokine production in Alzheimer's disease patients

To investigate the signs of inflammatory processes in Alzheimer's disease (AD), we examined peripheral blood mononuclear cells (PBMC) from 51 AD patients (29 with mild and 22 with moderately severe dementia) and 51 age-matched healthy controls (HC), using flow cytometry to analyse the absolute number and the percentage of T, B and NK cells. We also studied the surface expression of CD25, CD28, CD57, CD71, CD45RA and CD45RO markers on cells CD4+ and CD8+. In 30 AD patients and 20 HC the production of IL-2, IFN-gamma, IL-10 and TNF-alpha by PBMC after stimulation with [25-35], [1-40] and [1-16] beta-amyloid (betaA) fragments was also evaluated. A significant decrease in circulating B and CD8+CD28- cells, as well as an increase in CD8+ cells expressing CD71+ and CD28+, was observed in AD patients. A significant decrease in IL-10 production was also found after stimulation of PMBC with betaA [1-40]. The decreased IL-10 production was not related to disease severity. The observed imbalance of immune peripheral cell subpopulations and decreased IL-10 production point to a reduction of suppressor cell function in AD patients.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.     

Activation of the immune system of cancer patients by continuous i.v. recombinant IL-2 (rIL-2) therapy is dependent on dose and schedule of rIL-2.

The effect of dose and schedule of continuous i.v. rIL-2 infusions on leucocyte subset counts, activation status of CD56+CD3- natural killer (NK) and CD3+ T lymphocytes, and cytolytic activities of peripheral blood mononuclear cells (PBMC) was studied. A single 4-day course of rIL-2 in escalating doses (0.9-11.5 x 10(6) U/m2 per day) was given to 18 patients with various types of metastatic cancer. The serum IL-2 concentration during rIL-2 therapy ranged between 23 and 64 U/ml and was proportional to the administered rIL-2 dose, as was the rebound lymphocytosis following therapy. Before therapy, the CD56+CD3- NK cells expressed low levels of the p75 chain of the IL-2 receptor (IL-2R) and virtually no IL-2R(p55). Most CD3+ T cells were IL-2R(p55-,p75-). Between 2 and 4 days following therapy, i.e. at the time of lymphocytosis, the percentage of CD56+,CD3- NK cells among the lymphocytes had increased proportional to the administered rIL-2 dose. The levels of IL-2R(p75) expression by the CD56+,CD3- NK cells had increased. The percentages of CD3+ T cells expressing IL-2R(p55), HLA-DR and CD45RO had increased proportional to the administered rIL-2 dose. The level of lymphokine- activated killer (LAK) activity against Daudi cells was also positively correlated with rIL-2 dose. Subsequently, seven patients received 4-weekly cycles of rIL-2 (2.9-4.4 x 10(6) U/m2 per day) during 4 consecutive weeks. This schedule led to marked increments in lymphocyte and eosinophil counts, and to increased cytolytic activities compared with pretreatment. We conclude that CD56+,CD3- NK and CD3+ T cells are activated differentially by continuous i.v. rIL-2 proportional to dose and duration of treatment.     

Synergistic effect of interleukin-2 and a vaccine of irradiated melanoma cells transfected to secrete staphylococcal enterotoxin A

We have previously reported that immunization of mice with melanoma cells transfected to secrete the superantigen, Staphylococcal enterotoxin A (SEA), increased the production of antibodies to the B700 melanoma antigen, stimulated the production of endogenous interleukin 2 (IL-2), activated the expression of CD4, CD8 and CD25 T cell markers and enhanced NK cell activity. Now we show that immunization of mice with a vaccine of irradiated sea-transfected melanoma cells coupled with IL-2 therapy was even more effective in inhibiting the growth of primary melanoma tumors and the development of lung metastases than was the irradiated melanoma cell vaccine alone or IL-2 alone. The morphological and immunological effectiveness of the therapy was dose-dependent on IL-2.     

Down-regulation of interleukin-2 receptor expression on natural killer cells/large granular lymphocytes by interleukin-4.

It has recently been demonstrated that IL-4 inhibits IL-2 receptor expression on T cells. Studies have also shown that IL-4 can inhibit IL-2-induced natural killer cell (NK) cytotoxicity, and that IL-4 in combination with IL-2 and large granular lymphocyte (NK/LGL) cells suppresses Ig synthesis. Therefore, we examine whether IL-2 receptor expression on NK/LGL cells is affected with or without IL-4, using fluorescent receptor analysis. Our results demonstrate that IL-4 inhibits/down-regulates the expression of IL-2 receptors on either phytohemagglutinin or IL-2-stimulated NK/LGL cells.     

Interleukin-2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus-1 gp120

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)-infected patients marginally express interleukin-2 receptor (IL-2R)β and IL-2Rγ chains which are essential for IL-2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL-2Rβ and IL-2Rγ by purified CD4 lymphocytes in vitro. Cross-linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [³H]thymidine incorporation. It also induces the cell surface expression of IL-2Rβ and IL-2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV-1 gp120 before anti-CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [³H]thymidine incorporation. Furthermore, cell surface expression of IL-2Rβ and IL-2Rγ subunits, which form the functional intermediate-affinity IL-2R, are significantly inhibited. More importantly, addition of exogenous IL-2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL-2R are not functional. This is the first study of IL-2Rβ and IL-2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL-2R expression.     

Role of prolactin in the modulation of NK and lak cell activity after short- or long-term morphine administration in neoplastic patients

In a previous work we demonstrated that chronic in vivo antalgic therapy of cancer patients with morphine reduced the endogenous cytotoxic activity of natural killer (NK) cells, while increasing the development of lymphokine activated killer (LAK) cell cytotoxicity. In order to investigate the mechanisms by which morphine affects NK and LAK cell function further, we evaluated the modulation exerted by short- or long-term morphine administration on either NK/LAK cell cytotoxicities or plasma levels of prolactin (PRL) and other immunomodulating neurohormones. An intravenous morphine injection (10mg) significantly increased the plasma levels of PRL, reduced the cytotoxic activity of NK cells, and increased the development of LAK cell activity 30 min after drug injection in neoplastic patients. The administration of bromocriptine before the injection of morphine prevented both PRL augmentation and the increase in LAK cell activation, although it did not prevent the inhibition of NK cytotoxicity. The chronic oral administration of morphine (90±30mg/day for 1 month) also resulted in higher PRL levels; the NK and LAK cell activities were, respectively, lower than or higher than those found in neoplastic patients untreated with morphine. The plasma levels of thyrotropin (TSH), adrenocorticotropic hormone (ACTH) and cortisol were not significantly modified in either short- or long-term experiments. The absolute number and the percentages of lymphocyte populations, as well as the percentage of IL-2 receptors, were not modified after short-term morphine administration whereas little changes of T lymphocyte populations and NK cell number were observed after oral treatment with morphine. In vitro morphine did not affect the development of LAK cell activity. In conclusion, our findings indicate that morphine reduces NK cytotoxicity and increases the development of LAK cell cytotoxicity after short- and long-term administration. The effect of morphine on LAK cell activation but not on NK cell reduction is related to the modulation of PRL levels determined by the opioid drug.     

Cell surface phenotyping and cytokine production of Epstein-Barr Virus (EBV)-transformed lymphoblastoid cell lines (LCLs)

Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBV-LCLs) are routinely used for the in vitro expansion of T cells. However, these cell lines are reported to produce the cytokine IL-10, which is inhibitory for T cells. We, therefore, characterized a panel of 37 EBV-LCLs for a variety of cell surface markers, for secretion of various cytokines including IL-10 and for immunoglobulin production. These cell lines were derived from normal donors or patients with nonsmall cell lung cancer, acute myelogenous leukemia, melanoma or colon cancer. Overall, 26 lines were positive for CD19 and CD20, and 11 were negative for both. All of the lines were strongly HLA-DR+, while CD40 expression was variable. Twenty-four (65%) were both CD23+ and secreted immunoglobulin, and 33 expressed kappa and/or lambda light chains. Additionally, all of the EBV-LCLs were negative for T cell (CD3), NK cell (CD16, CD56), monocyte (CD14) and granulocyte (CD66b) surface markers. Some level of IL-10, IL-6, IL-12p40 and TNF-alpha cytokine production was detected in 33, 18, 19 and 12 EBV-LCLs, respectively. Together, these data reflect the heterogeneity of EBV-LCLs, which cautions their use nondiscriminately in various immunologic assays.     

Expression of complement regulatory proteins on human natural killer cell subsets

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely distributed on human leucocytes and protect against complement-mediated damage. To investigate heterogeneity in CReg protein expression by human natural killer (NK) cells, levels were assessed on resting and activated NK cell subsets identified phenotypically on the basis of expression of CD56 and CD158 markers. Levels of all three CReg proteins on CD56+ cells were lower than on T cells (p<0.05). Freshly isolated CD56(bright) cells expressed higher levels of CD55 than CD56dim cells (p<0.05). CD158a+ cells expressed significantly lower levels of both CD46 and CD59, and CD158e+ cells expressed significantly lower levels of CD46, than CD158a(-) CD158e(-) cells, respectively (both p<0.05). Stimulation with PHA did not significantly alter NK cell surface CReg protein levels whereas, following culture with IL-2, CD46 and CD59 were decreased on both CD56bright and CD56dim subsets (p<0.05). In the case of CD59, this was independent of T cells. Only CD46 was significantly downregulated on CD158b+ (GL183+) and CD158e (NKB1+) subsets (p<0.05). However, culture in IL-15 significantly increased levels of all three CReg proteins. These observations that CReg proteins are downregulated on certain NK cell subsets following activation with IL-2 are opposite to previous findings for other leucocyte subpopulations. Activated NK cells may instead use other strategies for protection against complement-mediated damage in a local inflammatory response.     

gammadelta T cells express activation markers in the central nervous system of mice with chronic-relapsing experimental autoimmune encephalomyelitis.

In this study, we assessed the expression of activation markers on gammadelta T cells in central nervous system (CNS) lesions of SJL mice adoptively sensitized to develop experimental autoimmune encephalomyelitis (EAE) using myelin basic protein-reactive T cells. Although disease expression is known to be dependent upon T cells that express the alphabeta T cell receptor (TCR), a role for gammadelta T cells has been implicated in some studies but not in others. Using three-color flow cytometric analysis of both total and gammadelta T cells in spleen and CNS, the data showed that expression of CD69 (early activation marker), CD62L (lymphocyte homing receptor), CD25 (IL-2Ralpha), CD122 (IL-2Rbeta) and CD95/CD95L (Fas/FasL), fluctuated on gammadelta T cells in EAE lesions in a disease-related fashion. Furthermore, the pattern of expression for these markers on gammadelta T cells was distinct from that found on the total lymphocyte population. Cytokine analysis of gammadelta T cells in the CNS demonstrated a bias towards a Th1-like cytokine profile. From these data, we conclude that gammadelta T cells in EAE lesions display an activated phenotype and form a dynamic component of the total lymphocyte population in the CNS, supporting a contributory role for these cells.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15.

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

The JAK3 inhibitor CP-690550 selectively reduces NK and CD8+ cell numbers in cynomolgus monkey blood following chronic oral dosing

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase associated with the common gamma chain, an integral component of cytokine receptors of the interleukin (IL)-2 family, including IL-4, -7, -9, -15, and -21. CP-690550 is a JAK3 inhibitor with immunosuppressive properties under development for transplantation. We evaluated alterations in circulating lymphocyte subsets in cynomolgus monkey blood following chronic (3-week), oral CP-690550 administration. Natural killer (NK) and CD8+ T cell numbers were reduced in a dose- and time-dependent manner; the latter was a primary effect on memory subsets. CD4+ T and B cell numbers were unaffected or slightly increased, respectively. NK cell numbers were reduced approximately 80% (vs. 35% in vehicle-treated animals) and returned to baseline levels within 3 weeks following treatment cessation. CD8+ T cells declined by a maximum 43% (vs. 25% for vehicle-treated animals) but rebounded significantly (300%) within 2 weeks after the last dose. Although CP-690550 did not result in reduction of CD4+ T cell number, these cells also increased (225%) within 2 weeks of treatment cessation. IL-15 is important for maintaining homeostasis of these cell types, and CP-690550 inhibited IL-15-induced CD69 expression in NK cells [inhibitory concentration 50% (IC50)=48.0+/-8.4 nM] and CD8+ T cells (IC50=16.2+/-1.5 nM).