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Originally published online as doi:10.1189/jlb.0907626 on February 13, 2008

Published online before print February 13, 2008
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(Journal of Leukocyte Biology. 2008;83:1128-1135.)
© 2008 by Society for Leukocyte Biology

Characterization of a subset of bone marrow-derived natural killer cells that regulates T cell activation in rats

Taba Kheradmand1, Prachi P. Trivedi1,2, Norbert A. Wolf, Paul C. Roberts3 and Robert H. Swanborg4

Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan, USA

4Correspondence: Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 East Canfield Ave., Detroit, MI 48201, USA. E-mail: rswanbo{at}med.wayne.edu


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ABSTRACT
 
We report that bone marrow-derived natural killer (BMNK) cells from DA or F344 rats inhibit PMA/ionomycin-induced T cell proliferation. These NK-regulatory cells are NKR-P1Adim, whereas a minor subpopulation is NKR-P1Abright. Only the NKR-P1Adim BMNK cells inhibit T cell proliferation. If activated with rat Con A supernatant, the NKR-P1Adim cells become NKR-P1Abright and lose the ability to inhibit T cell proliferation. In contrast to BMNK cells, all DA and F344 rat NK cells isolated from the blood, spleen, cervical, or mesenteric lymph nodes or Peyer’s patches are NKR-P1Abright and lack the ability to inhibit T cell proliferation. Inhibition of T cell proliferation correlates with significant down-regulation of CD3, suggesting that this may be the mechanism through which the NKR-P1Adim cells mediate suppression. The nitric oxide synthase inhibitor NG-monomethyl-arginine acetate-abrogated NKR-P1Adim cell inhibition of T cell proliferation. We conclude that rat bone marrow NKR-P1Adim cells represent a unique population that may play a role in maintaining immune homeostasis by regulating the clonal expansion of activated T cells.

Key Words: suppression • tolerance • cytokines


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INTRODUCTION
 
Natural killer (NK) cells are large, granular, bone marrow-derived lymphocytes comprising ~10-20% of the circulating lymphocyte population. They are primarily confined to peripheral blood, bone marrow, and spleen but can migrate to tissues such as lymph nodes in response to chemoattractants during an inflammatory response [1 ]. Unlike T and B lymphocytes, the NK cells do not express clonal, antigen-specific receptors [2 ]; nevertheless, they are still capable of discriminating normal cells from target cells. NK cells express a repertoire of activating and inhibiting receptors that allow them to identify target cells, e.g., tumor cells, that lack expression of self-MHC class I molecules and/or express ligands for the activating receptors.

Because of their ability to identify target cells without prior sensitization, NK cells are one of the early responders during an infection and hence are an important part of the innate immune system. They also play an important role in shaping the adaptive immune responses that follow. It has long been known that NK cells can influence the adaptive immune microenvironment by secreting cytokines and chemokines [3 , 4 ]. However, in recent studies, it is becoming evident that NK cells can also influence adaptive immune responses by direct interaction with cells of the adaptive immune branch such as T cells, B cells, and the antigen-presenting dendritic cells (DCs). Interestingly, NK cells have the ability to both positively and negatively regulate the activity of these immune cells.

The ability of NK cells to act as effectors, as well as to differentially regulate the cells of the adaptive immune system may stem in part from the existence of functionally and phenotypically distinct subsets of NK cells. For example, human CD56bright and CD56dim peripheral blood NK cell subsets have been shown to have distinct phenotypes and functional capabilities, including cytotoxicity and cytokine secretion [3 ]. Recently, murine CD27 was identified as a marker to dissect mature NK cells into two phenotypically and functionally distinct subsets, the CD27low and CD27high NK cells [5 ]. Also, NK cells derived from distinct sites in the body may have differential properties. Human decidual NK cells, implicated in regulation of maternal-fetal tolerance, were shown to be both phenotypically and functionally different from the peripheral blood NK cell subsets [6 ]. Similarly, in mice, differences in expression of several cell-surface proteins, including integrins, chemokine, and cytokine receptors have been noted between mature bone marrow NK cells and mature peripheral NK cells [7 ].

Previous studies from our laboratory revealed that highly purified bone marrow-derived NKR-P1A+CD3– NK cells (BMNK) from DA rats could inhibit T cell proliferation in response to the self-antigen, myelin basic protein (MBP) [8 ], as well as to Con A and PMA/ionomycin [9 ]. Lewis rat NK cells also exhibited this activity on syngeneic autoreactive T cells [8 ]. This suggests that NK cells could play a role in the maintenance of immune homeostasis.

NK cell-mediated inhibition of T cell proliferation was antigen non-specific, contact-dependent, and reversible and was associated with up-regulation of the cell cycle inhibitor, p21, resulting in cell cycle arrest at the G0/G1 stage [9 ]. These findings suggested that rat BMNK cells might play an important role in maintaining immune homeostasis by regulating clonal expansion of activated T cells. In the present study, we compared the phenotypic and functional characteristics of rat bone marrow derived vs. splenic NK cells (SpNK), in order to determine whether the ability to regulate T cell function was unique to the bone marrow NK cell subset. Our results show that indeed the rat BMNK cells have distinct functional and phenotypic characteristics and that the ability to regulate T cell proliferation is unique to the BMNK cell subset.


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MATERIALS AND METHODS
 
Animals
DA rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN, USA), and maintained in our Association for the Assessment and Accreditation of Laboratory Animal Care-accredited animal facility. Naïve rats were used as a source of splenic T cells, thymocytes, and bone marrow-derived and splenic NK cells. The rat protocol was approved by the Institutional Animal Care and Use Committee at Wayne State University (Protocol # A 09-17-03). In some experiments, we employed F344 rat bone marrow, lymph nodes, and blood. These were kindly supplied by the laboratory of Dr. Paul C. Montgomery.

Antibodies and cytokines
NG-monomethyl-L-arginine acetate (MMA) was purchased from Calbiochem (San Diego, CA, USA). The following antibodies, fluorochromes, and recombinant (r) cytokines were used at dilutions recommended by the manufacturer. The following antibodies were specific for rat leukocytes. FITC-labeled anti-CD3 (clone G4.18), PE-labeled anti-NKR-P1A (clone 10/78), Biotin-labeled anti-NKR-PIA, and PE-labeled anti-rat IFN-{gamma} (DB-1), were obtained from BD Biosciences (San Diego, CA, USA). Alexa-647 mouse IgG1 (MOPC-21) and PE-conjugated mouse IgG1 (MOPC-31C) isotype controls were from BD. Recombinant mouse IL-15 (reactive with rat cells) was from Biosource International. As secondary reagent, we used streptavidin-PE-Cy5 (from BD). Con A supernatant (CAS) was derived from Lewis rat spleen cells cultured for 48 h at 37 C with 2 µg/ml concanavalin A. The Con A was removed by absorption with Sephadex G-10 for 2 h at 4 C.

Preparation of cell suspensions
T cells were isolated as described previously [10 , 11 ]. Briefly, spleens were homogenized, erythrocytes were lysed using Tris-NH4Cl (pH 7.2), and macrophages were depleted by adherence on plastic tissue culture flasks (Falcon). T cells were then enriched using T cell purification columns (Cedarlane).

Preparation of NK cells
Bone marrow was obtained from naïve DA or F344 rats by flushing the cavities of the femur and tibia bones with cold RPMI 1640 supplemented with 5% fetal calf serum (Gibco Life Technologies) using an 18-gauge needle. Single-cell suspensions were made using needles of increasingly smaller gauge [8 ]. Spleens were dissected from naïve DA or F344 rats, and mesenteric and cervical lymph nodes (MLN and CLN, respectively), as well as Peyer’s Patches (PP) and whole blood were obtained from F344 rats. The spleens, LN and PP were homogenized in cold RPMI 1640 supplemented with 5% fetal calf serum to obtain single cell suspensions, then treated with Tris-NH4Cl (pH 7.2) to remove erythrocytes and depleted of macrophages by adherence on plastic tissue culture flasks (175 cm2; Falcon) for 2 h at 37°C. NK cells were isolated from blood after removal of erythrocytes using lymphocyte isolation medium (Gibco), followed by treatment with Tris-NH4Cl and plastic adherence. Nonadherent cells were passed through nylon filters and collected in 50-ml conical tubes, washed with complete medium, and then layered onto Percoll (Pharmacia Biotech, Piscataway, NJ, USA) density gradients consisting of 75%, 65%, 55%, 45% Percoll and HBSS in 15-ml conical centrifuge tubes. The cells were centrifuged at 2000 rpm for 15 min, and cells in the 45% and 55% Percoll fractions (fraction 1) were pooled, washed twice with HBSS, and resuspended in RPMI with 5% fetal calf serum (FCS). This fraction, previously found to be enriched in NK+ cells [8 ], was further purified using the FACSVantage to obtain NKR-P1A+CD3– NK cells as given below.

Purification of NKR-P1A+CD3– cells
Fraction 1 cells were suspended at 1 x 107 cells/ml in PBS containing 1% bovine serum albumin (BSA), 0.02% sodium azide, and 5% normal rat serum and were incubated on ice for 20 min to block nonspecific binding sites. Cells were washed with PBS containing 1% BSA and 0.02% Na-Azide and then stained with PE-labeled anti-rat NKR-P1A (clone 10/78) and FITC-labeled anti-rat CD3 (clone G4.18) antibodies. PE-labeled anti-KLH antibody was used as isotype control antibody. Staining was done in the dark on ice for 30 min with intermittent shaking. The cells were washed and resuspended in PBS with 0.02% Na-Azide and sorted using the FACSVantage cell sorter (Becton Dickinson). The NKR-P1A+CD3– NK fraction was collected [11 ]. The purity of the NKR-P1A+CD3– NK cells exceeded 97%.

In some preliminary experiments, the NK cells were stained with PE-anti-NKR-P1A and isolated using the Easy Sep PE Selection Kit (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer’s instructions. These were confirmed by cell sorting experiments in the FACSVantage. The FACS sorted cell experiments are described in this manuscript. An exception is that the F344 LN and PP cell yields were extremely low, so those cells were analyzed without sorting.

Proliferation assays
T cells (0.5x106/well) were cultured in the absence or presence of 0.5 x 105 NK cells/well in 96-well plates. The cells were cultured in RPMI 1640 containing 5% FCS and were stimulated at 37 C, for 48 h with Phorbol-12-myristate-13 acetate (PMA) (10 ng/ml) and ionomycin (0.4 µg/ml) (Sigma). T cell proliferation was determined by incorporation of 3H-thymidine, as described previously [10 ]. The cells were pulsed with 3H-thymidine for the last 18–20 h of culture time. At the end of the incubation period cells were harvested using a Tomtec Harvester 96 and counted in a 1450 Microbeta Plus, liquid scintillation counter (Wallac, Wellesley, MA, USA). Samples were run in triplicate, and results are presented as mean cpm ± SD.

ELISA
Culture supernatants from T cell/NK cell cocultures were analyzed for the presence of cytokines using IL-4, IFN-{gamma}, and TGF-β ELISA kits (Biosource International, Camarillo, CA, USA), according to the manufacturer’s instructions.

Flow cytometry analysis
The expression of cell surface and cytoplasmic markers was determined by flow cytometry using either the BD FACScan or Dako CyAn flow cytometer, as described previously [9 ]. For intracellular cytokine staining, cells were first blocked with mouse anti-rat CD32 (Fc{gamma}II receptor mAb, PharMingen, San Diego, CA, USA), then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences), as per manufacturer’s instructions. They were then stained for intracellular IFN-{gamma} for 30 min on ice, washed and, examined by flow cytometry. Autofluorescence and isotype controls were included.

Statistical analysis
All experiments were repeated at least two times with similar results. Representative results are presented as mean ± SD. Statistical significance was calculated using Student’s t test.


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RESULTS
 
Differences between BMNK and SpNK cells
Studies in mice and humans have shown that SpNK cells enhance proliferation of T cells in vitro [12 ], which is in contrast to our observations with rat BMNK cells. To determine whether inhibition of T cell proliferation was a unique property of the BMNK cell subset, we isolated DA rat BMNK and SpNK cells by flow cytometry and studied their role in regulation of T cell proliferation.

As reported earlier [9 ], rat BMNK cells inhibit PMA-ionomycin-induced T cell proliferation. As shown in Fig. 1 , the postsort analysis of the two NKR-P1A+CD3– NK cell populations revealed that the SpNK cells were NKR-P1Abright, whereas the BMNK population was predominantly NKR-P1Adim, although a small subpopulation has the NKR-P1Abright phenotype.


Figure 1
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  		Figure 1. Sorted BMNK cells are predominantly NKR-P1AdimCD3–, with a minor NKR-P1AbrightCD3– component (left), whereas sorted SpNK cells are NKR-P1AbrightCD3– (right).

Then we isolated the NKR-P1Adim and NKR-P1Abright subsets of BMNK cells by flow cytometry, and determined which subset would inhibit T cell proliferation. As shown in Fig. 2A , the NKR-P1Adim BMNK cells inhibited the proliferation of autologous T cells to PMA-ionomycin, even at a ratio of 20:1 (T:NK), whereas the NKR-P1Abright BMNK cells did not inhibit T cell proliferation at the same T:NK ratio (Fig. 2A) . The sorted SpNK cells failed to inhibit T cell proliferation to PMA-ionomycin, even at ratios of 1:1 (NK:T) (Fig. 2B) . This suggests that the two BMNK subsets are functionally distinct and that the SpNK cells are unable to suppress T cell proliferation.


Figure 2
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  		Figure 2. (A) Sorted BMNKbright cells do not inhibit proliferation at 20:1=T:NK, whereas BMNKdim cells profoundly suppress T cell proliferation at 20:1 and 10:1 T:NK. (B) Sorted DA rat SpNK cells do not inhibit proliferation of syngeneic T cells even at NK:T cell ratios of 1:1 when cocultured for 48 h with PMA + ionomycin. Results in this and all subsequent proliferation experiments are presented as mean cpm of triplicate samples ± SD.

BMNK cell regulation is not strain-specific
We previously reported that Lewis rat BMNK cells also inhibit syngeneic T cell proliferation to Con A [8 ]. To further investigate possible strain differences, we tested whether F344 rat BMNK cells were capable of inhibiting F344 T cell proliferation to PMA + ionomycin. As shown in Fig. 3A , sorted F344 BMNK cells, like DA BMNK cells, had a significant inhibitory effect on T cell proliferation. However, F344 SpNK cells did not inhibit T cell proliferation (Fig. 3B) . We also isolated NK cells from F344 cervical LN, mesenteric LN, PP, and blood. The NK cells isolated from LN, blood and PP were NKR-P1Abright, whereas the BMNK cells were NKR-P1Adim (results not shown). The NK cells isolated from lymph nodes, PP (Fig. 4A ) and blood (Fig. 4B) failed to suppress F344 T cell proliferative responses to PMA + ionomycin. Thus, BMNK cells from F344, DA (this report) and Lewis rats [8 ] have a unique regulatory function.


Figure 3
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  		Figure 3. (A) Sorted BMNK cells from F344 rats inhibit PMA + ionomycin-induced proliferation of F344 T cells (5:1, T:NK). (B) F344 SpNK cells fail to inhibit T cell proliferation.


Figure 4
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  		Figure 4. (A) NK cells from cervical LN (CLN), mesenteric LN (MLN), and PP of F344 rats do not inhibit proliferation of PMA + ionomycin activated F344 T cells. T:NK = 5:1. (B) NK cells from blood of F344 rats do not inhibit proliferation of PMA + ionomycin activated F344 T cells. T:NK = 10:1.

One explanation is that rat BMNKR-P1Adim cells represent an immature population that differentiates to BMNKR-P1Abright cells that migrate to the periphery, e.g., the spleen. To investigate this possibility, we cultured sorted BMNK cells for 48 h with rat spleen cell-derived Con A supernatant (CAS) as a putative source of NK cell growth/differentiation factors. After 48 h, the NK cells were washed and cocultured with T cells in the presence of PMA and ionomycin. The CAS-activated NK cells failed to suppress T cell proliferation (Fig. 5 ). CD25, CD122, and NKR-P1A were up-regulated, suggesting that exposure to CAS activated the BMNK cells (Fig. 6 ).


Figure 5
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  		Figure 5. BMNK cells lose the capacity to inhibit T cell proliferation if activated for 48 h with Con A supernatant (CAS), followed by culture with PMA + ionomycin (PMA/I) for an additional 48 h. BMNK cells cultured in medium (Non-act. NK) for 48 h prior to addition to T cell cultures retain the ability to suppress T cell proliferation. Ratios of 10:1 T:NK are depicted in the figure. Con A was removed from CAS by absorption with Sephadex G25 for 2 h at 4 C.


Figure 6
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  		Figure 6. Con A supernatant up-regulates BMNK expression of NKR-P1A from 11% to 92% (A); CD25 from 14% to 76% (B); CD122 from 16% to 66% after 48-h activation (C). Each panel shows autofluorescence control (thin line), nonactivated BMNK cells (hatched histogram), and CAS-activated BMNK cells (bold line).

BMNK and SPNK cells differ with respect to IFN-{gamma} production
We subsequently carried out experiments to determine whether intracellular IFN-{gamma} was expressed by freshly isolated or IL-15-activated BMNK and SpNK cells. As shown in Fig. 7 , ~2% of untreated (ex vivo) SpNK cells expressed cytoplasmic IFN-{gamma}, and this increased to 63% when the SpNK cells were cultured for 48 h in the presence of 200 ng/ml IL-15. Intracellular IFN-{gamma} was also expressed by ~2% of untreated BMNK cells, but after activation for 48 h with IL-15 only 16% were positive (Fig. 7) . Thus, IFN-{gamma} production may reflect a more mature population of NK cells. In contrast, the regulatory NKR-P1Adim NK cells produce significantly less IFN-{gamma}.


Figure 7
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  		Figure 7. BMNK and SpNK cells were labeled with biotin-avidin-anti-NKR-P1A and anti-CD3-FITC, isolated, blocked, then fixed and permeabilized using the Cytofix/Cytoperm kit. They were then stained with anti-rat IFN-{gamma}-PE (black histograms), or PE-conjugated mouse IgG1 (MOPC-31C) isotype control (open histograms) and analyzed by flow cytometry for intracellular IFN-{gamma} expression. Samples were gated based on the isotype controls. Approximately 2% of untreated BMNK (A) and SpNK (C)cells expressed IFN-{gamma}. Following activation for 48 h with 200 ng/ml rIL-15, intracellular IFN-{gamma} expression is up-regulated to 63% of SpNK (D) cells but only to 16% in BMNK (B)cells.

To investigate whether NKR-P1Adim NK cells influence cytokine production in the presence of T cells, PMA, and ionomycin, 48-h culture supernatants were evaluated by ELISA. As shown in Fig. 8 , PMA + ionomycin-stimulated significant IFN-{gamma} production by T cells, and this was partially suppressed when bone marrow-derived NKR-P1Adim cells were added to the culture with PMA + ionomycin. We also evaluated IL-4 and TGF-β, but found no evidence that these immunomodulatory cytokines play a role in suppression of T cell proliferation (data not shown).


Figure 8
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  		Figure 8. PMA + ionomycin-stimulated T cells secrete 5592 ± 231 pg/ml IFN-{gamma}, which is inhibited (1184 ± 76 pg/ml) in the presence of BMNKR-P1Adim T cells.

NK cells down-modulate CD3 expression in T cells
When T cells were cultured with PMA + ionomycin, or with culture medium alone, and examined by flow cytometry, high levels of CD3 were expressed (Fig. 9 ). In contrast, when NKR-P1Adim cells were cocultured with the T cells and PMA + ionomycin, CD3 expression was markedly down-modulated (Fig. 9) . This is reminiscent of the report of Upham et al. [13 ] that rodent alveolar macrophages inhibit T cell proliferation by down-modulating CD3 through a nitric oxide (NO)-dependent mechanism.


Figure 9
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  		Figure 9. (A) Left: scatterplot of T cells cultured with medium. Center: scatterplot of T cells cocultured with BMNK cells in the presence of PMA + ionomycin. Right: histogram overlays showing control (thin line), cocultured T and NK cells with PMA + ionomycin (hatched histogram), and T cells cultured with medium alone (bold line). (B) Left: scatterplot of T cells cultured with PMA + ionomycin. Center: scatterplot of T cells cocultured with BMNK cells in the presence of PMA + ionomycin (same scatterplot shown in center panel of A). Right panel: histogram overlays showing control (thin line), cocultured T and NK cells with PMA + ionomycin (hatched histogram), and T cells cultured with PMA + ionomycin without BMNK cells (bold line). All cells were cultured for 36 h, then stained for flow cytometry. The data reveal that BMNK cells down-modulate CD3 expression on the T cells.

To ascertain whether NO plays a role in NK cell-mediated suppression of T cell proliferation, we added the nitric oxide synthase inhibitor, MMA [13 ] to the NK-T cell cocultures. As shown in Fig. 10 , MMA abrogated NKR-P1Adim cell mediated inhibition of T cell proliferation in a dosage-dependent fashion, suggesting that NO plays a role in the suppression of T cells by NK cells. Similar results were obtained in two additional experiments. A comparison of the left and center panels in Fig. 9 reveals that the fluorescence intensity, rather than the number of CD3+ T cells decreases.


Figure 10
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  		Figure 10. (A) The nitric oxide synthase inhibitor NG-monomethyl-arginine acetate (MMA) abrogates PMA + ionomycin-induced suppression of T cell proliferation by NKR-P1Adim BMNK cells in a dosage-dependent manner. The ratio of T cells to NK cells was 10:1. (B) MMA does not affect T cell proliferation to PMA + ionomycin in the absence of NKR-P1Adim BMNK cells.


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DISCUSSION
 
The major finding in the present investigation is that an NKR-P1Adim population of bone marrow-derived NKR-P1A+CD3– NK cells inhibits proliferation of T cells. The phenomenon does not appear to be strain-specific, since the effect is seen with DA, F344 (this report), and Lewis rats [8 ]. Nor do NK cells derived from other tissues exert an inhibitory effect on T cells. Thus, the BMNK cells are a unique population with regulatory function. The BMNK cells differ from NK cells derived from other tissues in that the former are NKR-P1Adim, whereas the latter, which do not inhibit T cell proliferation, express high levels of NKR-P1A. However, the inhibitory NKR-P1Adim BMNK cells lose the ability to suppress T cell proliferation if activated with CAS. Concomitantly, they become NKR-P1Abright. These are characteristics of the NK cells derived from spleen, LN, PP, and blood, which are also NKR-P1Abright and fail to regulate T cell proliferation. This suggests that upon exiting the bone marrow, NK cells may undergo differentiation to acquire unique functional and phenotypic characteristics suited to play distinct roles in the different environments. Similar observations have also been reported for the human decidual NK cell subset. It has been shown that the decidual NK cells acquire more immunomodulatory functions as compared with the peripheral blood NK cells. Moreover, decidual NK cells have a lower cytotoxicity potential, which can be restored upon activation [6 , 14 ]. Thus NK cells derived from different compartments in the body may represent different subsets that have gained specific functions in response to the need of the local environment.

Our previous work revealed that DA rat BMNK cells inhibit proliferation of syngeneic T cells by inducing G0/G1 stage cell cycle arrest via up-regulation of the cell cycle inhibitor protein, p21 [9 ]. Inhibition required cell-cell contact, was antigen nonspecific and reversible [9 ]. BMNK cells did not affect early events in T cell activation, including IL-2 secretion and IL-2R up-regulation, but specifically inhibited T cell division. Our study suggests a unique mechanism of T cell regulation by BMNK cells that could be of potential importance in understanding the interplay between innate and adaptive immunity in maintaining immune homeostasis.

Previous studies in both humans and mice revealed that NK cells have the capacity to inhibit proliferation of bone marrow erythroid and myeloid precursor cells and are implicated in the regulation of hematopoietic homeostasis [15 16 17 ]. On the other hand, recent studies in mice and humans [12 ] have shown that activated spleen and peripheral blood NK cells are capable of enhancing T cell proliferation via costimulatory receptors.

With respect to rats, it has been reported that depletion of NK cells results in exacerbation of experimental autoimmune encephalomyelitis (EAE) in Lewis rats [18 ]. Similar observations were reported in mice [19 ]. We previously found that sorted, highly purified NKR-P1A+CD3– NK cells inhibited MBP-specific proliferation of MBP-primed Lewis rat T cells [11 ]. Moreover, Todd et al. [20 ] reported that NKR-P1A+CD3– intraepithelial NK cells are deficient in diabetes-prone BB rats before onset of diabetes. The effect could be reversed, and diabetes was prevented by transplantation of bone marrow from histocompatible WF rats, which do not develop spontaneous diabetes [20 ]. These findings are consistent with the present report and support the hypothesis that BMNK cells exhibit regulatory function.

Regulatory NK cells have also been reported to have an immunomodulatory effect in human autoimmune diseases in patients treated with humanized mAb specific for the IL-2 receptor. mAb treatment reduces brain inflammation in patients with multiple sclerosis [21 ] and ameliorates ocular inflammation in patients with active uveitis [22 ], concomitant with expansion of CD56bright NK cells and contraction of circulating CD4+ and CD8+ T cells. It remains to be determined how the phenotypes of the rat and human NK cells compare.

The mechanism by which the NK cells inhibit T cell proliferation may also differ depending on the species under study. Xu et al. reported that NK cells exert a direct cytotoxic effect on encephalitogenic T cells in mice [23 ]. In contrast, our studies in rats suggest that the T cells remain viable, since the effect is reversible if the NK cells are removed [9 ].

With respect to the mechanism, by which NKR-P1Adim cells inhibit T cell proliferation, we propose that the NKR-P1Adim cells down-modulate CD3 on the PMA + ionomycin-activated T cells and that NO plays a role in suppression of T cell proliferation. This is consistent with the report that T cells stimulated in the presence of alveolar macrophages inhibit proliferation [13 ]. In that study, down-modulation of CD3 by alveolar macrophages was also associated with the production of nitric oxide [13 ]. Moreover, similar to our previous findings with NK cells [9 ], IL-2 R expression was up-regulated and IL-2 was produced, further suggesting that the mechanisms may be similar. In another report, it was shown that granulocytes mediate the down-regulation of CD3{zeta} in mitogen- or antigen-activated synovial fluid T cells from patients with rheumatoid arthritis, resulting in depressed proliferative responses and IFN-{gamma} production [24 ]. Moreover, it was recently reported that CD4+CD25+ T regulatory cells from diabetes-resistant nonobese-resistant mice that are specific for a glutamic acid decarboxylase peptide are able to inhibit diabetes in a contact-dependent manner [25 ]. These T regulatory cells secrete IFN-{gamma}, which stimulates antigen-presenting cells to produce NO. The NO then suppresses the proliferation of pathogenic T cells and inhibits diabetes [25 ].

We suggest that down-modulation of CD3 by NKR-P1Adim NK cells is involved in the inhibition of T cell proliferation and that NO production is involved in this phenomenon. Moreover, it is likely that this is not unique to NKR-P1Adim cells but is a property associated with alveolar macrophages [13 ] and granulocytes [24 ] and T regulatory cells [25 ]. In concert with our previous findings [9 ], the present report suggests that NKR-P1Adim cells secrete NO, which induces p21 production in the T cells, leading to cell cycle arrest at the Go/G1 phase, and culminating in suppression of T cell proliferation, through down-modulation of CD3. It has been reported that NK cells produce NO [reviewed in 26 ]. With respect to EAE, it has been reported that inhibition of nitric oxide synthase by administration of MMA to Lewis rats that have recovered from acute EAE results in chronic relapsing disease, suggesting that NO regulates this autoimmune response [27 ].

It has recently been reported that bone marrow is a major reservoir for memory T cells [28 , 29 ]. Since the regulatory NK cells that we have found appear to be restricted to bone marrow, it is possible that they may function to maintain these bone marrow memory T cells in a quiescent state until needed for recall responses. Since they have also been implicated in the regulation of autoimmune diseases, the NKR-P1Adim BMNK cells may play a broad role in maintaining general T cell homeostasis. This comparative study of the in vivo NK cell subsets may provide a key to understanding the complex, multiple and often contrasting immunomodulatory roles played by natural killer cells. This is currently under investigation.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants 5RO1NS048070-04 and 5RO1NS06985-37.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

2 Current address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. Back

3 Current address: Departments of Biomedical Sciences and Pathobiology, Virginia Tech College of Veterinary Medicine, Blacksburg, VA, USA. Back

Received September 12, 2007; revised January 11, 2008; accepted January 17, 2008.


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