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The Journal of Immunology, 2007, 179, 3480 -3487
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Y Chromosome-Linked Factor Impairs NK T Development1

Johnna D. Wesley2,*, Marlowe S. Tessmer2,*, Christophe Paget{dagger}, François Trottein{dagger} and Laurent Brossay3,*

* Department of Molecular Microbiology and Immunology and Graduate Program in Pathobiology, Division of Biology and Medicine, Brown University, Providence, RI 02912; and {dagger} Centre d’Immunologie et Biologie Parasitaire, Institut Pasteur de Lille, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
V{alpha}14 invariant (V{alpha}14i) NK T cell development is unique from mainstream T cell selection, and the polygenic factors that influence NK T cell ontogeny are still unclear. In this study, we report the absence of V{alpha}14i NK T cells in B6.IFN-{alpha}betaR1–/– male mice, whereas both the conventional T and NK cell populations are relatively unaffected. The lack of V{alpha}14i NK T cells in the B6.IFN-{alpha}betaR1–/– males is not due to an insufficient level of CD1d1 or a defect in CD1d1-Ag presentation, but it is intrinsic to the male V{alpha}14i NK T cells. This surprising defect displays ≥99% penetrance in the male population, whereas female mice remain unaffected, indicating the deficiency is not X linked. Analysis of the V{alpha}14i NK T cell compartment in B6.Tyk2–/–, B6.STAT1–/–, 129.IFN-{alpha}betaR1–/–, and B6.IFN-{alpha}betaR1–/+ mice demonstrate that the deficiency is linked to the Y chromosome, but independent of IFN-{alpha}beta. This is the first study demonstrating that Y-linked genes can exclusively impact V{alpha}14i NK T development and further highlight the unique ontogeny of these innate T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The V{alpha}14 invariant (V{alpha}14i)4 NK T cells are a unique subset of T lymphocytes (1). These lymphocytes are thymus dependent, but depend on bone marrow-derived cells for selection (2). In mice, they are found in the spleen, bone marrow, thymus, and liver at a concentration of ~106 cells/organ (3). In addition to activated T cell markers, such as CD69 and CD44, they also express NK cell markers such as NK1.1 and members of the Ly-49 family. A self glycosphingolipid, isoglobotrihexosylceramide, can specifically stimulate NK T cells when presented by CD1d on activated dendritic cells (4). In addition to the well-described {alpha}-galactosylceramide ({alpha}-GalCer), microbial glycosylceramides and microbial diacylglycerol Ag, which strongly activate NK T cells, have been recently characterized (5, 6, 7, 8, 9, 10).

V{alpha}14i NK T cells develop within the thymus, arising from the same common lymphoid precursor pool from which mainstream T cells develop (11, 12). Once the {alpha}beta T cell lineage commitment is made and CD4+CD8+ (double-positive (DP)) thymocytes are generated, the V{alpha}14i NK T and conventional T cell selection pathways diverge (13, 14). V{alpha}14i NK T cell precursors are selected following {alpha}-chain rearrangement and expression of the V{alpha}14-J{alpha}18 invariant {alpha}-chain, which preferentially associates with specific beta-chain, Vbeta8.2, Vbeta7, or Vbeta2, to produce the characteristic TCR (13, 15, 16, 17, 18). In contrast to mainstream T cells that are selected by antagonist/partial ligands, V{alpha}14i NK T cells are selected and allowed to progress only if they recognize agonist glycolipids (19, 20, 21). One V{alpha}14i NK T cell-selecting ligand candidate, isoglobotrihexosylceramide, has been proposed (2, 4), although it has been recently challenged (22, 23). The functional consequences of this agonistic selection are robust proliferation of the V{alpha}14J{alpha}18+ thymocytes and up-regulation of early activation markers, CD44 and CD69 (24, 25). As a result of their unique selection process, V{alpha}14i NK T cells exit the thymus functionally competent with an activated phenotype, resembling effector memory T cells, poised to respond rapidly to a given stimulus (26, 27, 28).

Because of this similarity to effector memory T cells and the known influence of IFN-{alpha}beta on the survival of the conventional T cell memory pool (29, 30, 31), we initially sought to examine the contribution of IFN-{alpha}beta to V{alpha}14i NK T cell development and response to Ag. Surprisingly, examination of naive IFN-{alpha}betaR1-deficient animals revealed that male, but not female, IFN-{alpha}betaR1–/– mice lack V{alpha}14i NK T cells in the thymus and peripheral tissues. This deficiency is not due to the absence or decreased levels of key survival cytokines, such as IL-15, because conventional T cells and NK cells were comparable in both male and female mice. Furthermore, the lack of V{alpha}14i NK T cells in these mice is not the result of inadequate CD1d1 expression or function, but is intrinsic to the male V{alpha}14i NK T cells. However, analysis of the V{alpha}14i NK T cell compartment in B6.Tyk2–/–, B6.STAT1–/–, 129. IFN-{alpha}betaR1–/–, and IFN-{alpha}betaR1–/+ mice demonstrates that the V{alpha}14i NK T cell defect is independent of IFN-{alpha}beta, but instead linked to the Y chromosome. Collectively, our findings highlight the unique developmental program that V{alpha}14i NK T cells undergo and demonstrate a previously unknown role of a Y chromosome-linked factor during NK T development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Inbred 129Sv/Ev, C57BL/6, and B6.SJL-Ptprca/BoAiTac mice were purchased from Taconic Farms Laboratory Animals and Services. B6.CD1d1–/– mice were a gift from L. Van Kaer (Vanderbilt University, Nashville, TN). IFN-{alpha}betaR1–/– (generated by M. Auguet, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland (32) and provided by both M. Aguet and C. Biron, Brown University, Providence, RI), Tyk2–/– (generated in the laboratory of M. Muller (33) and provided by G. Yap, Brown University, Providence, RI), and STAT1–/– mice (generated in the laboratory of J. Durbin (34) and provided by C. Biron) were generated as described and backcrossed to the C57BL/6 background. The 129.IFN-{alpha}betaR1–/– mice were generated, as described (32), and were a gift from C. Biron. Additional backcrossing to generate B6.IFN-{alpha}betaR1–/+ and 129/B6.IFN-{alpha}betaR1–/– animals were performed in our breeding facility. All mice, except controls, were bred in pathogen-free breeding facilities at Brown University. All animals were age and sex matched, and all experiments were conducted in accordance with institutional guidelines for animal care.

Lymphocyte isolation and flow cytometric analysis

Hepatic lymphocytes were isolated by mincing and passage through a 70-µm nylon cell strainer (Falcon), followed by layering onto a two-step discontinuous Percoll gradient (Pharmacia Fine Chemicals) and centrifugation. Splenic lymphocytes were obtained by mincing and passage through nylon mesh (Tetko), followed by layering on Lympholyte-M (Cedarlane Laboratories) and centrifugation. Thymocytes were isolated by mincing and passing through a 70-µm nylon cell strainer.

For flow cytometry, cells were incubated with 2.4G2 anti-FcR mAb and stained with indicated Abs. Depending on the experiment and the tissue, 2.5 x 105-2 x 106, events were collected on a FACSCalibur. The data were acquired and analyzed using CellQuest software (BD Biosciences).

Abs and reagents

CD19 FITC, TCRbeta FITC, CD11b FITC, CD11c FITC, HSA FITC, NK1.1 PE, CD1d1 PE, TCRbeta PE Cy7, B220 PerCP Cy5, CD44 allophycocyanin, CD25 allophycocyanin, CD62L allophycocyanin, CD127 allophycocyanin, and TCRbeta allophycocyanin were all purchased from eBioscience. TCR{gamma}{delta} FITC, CD122 PE, CD25 PE, TCR{gamma}{delta} PE, CD69 PerCP, NK1.1 PerCP Cy5.5, CD11b PerCP, CD4 PerCP, CD8 PerCP, CD11c allophycocyanin, and B220 allophycocyanin were purchased from BD Pharmingen. For NK T cell identification, CD1d tetramers were obtained from the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility at Emory University. Additionally, the following mAbs were purchased from BD Pharmingen and used for ELISA: IFN-{gamma} mAbs (clones R4-6A2 and XMG1.2), IL-2 mAbs (clone JES6-1A12), IL-4 mAbs (clones 4B11 and BVD6-24G2), and streptavidin peroxidase.

Lymphopenia-induced proliferation in vivo assay

Performed as described by Matsuda et al. and Elewaut et al. (26, 35), thymocytes were isolated from naive wild-type, B6, or IFN-{alpha}betaR1–/– female mice. Before labeling with 5 µM CFSE, CD8+ cells were depleted using anti-CD8 magnetic beads (Miltenyi Biotec) and the AutoMACS, following manufacturer’s instructions. A total of 5–8 x 106 CFSE-labeled, CD8-depleted thymocytes was injected, i.v., into wild-type B6.SJL naive male and female mice, sublethally irradiated (750 rad) 3 days before transfer. Seven days posttransfer, all animals were sacrificed, and donor NK T cell proliferation was assessed in multiple tissues.

Bone marrow chimeras

Bone marrow cells were isolated from the femurs of male and female IFN-{alpha}betaR1–/– animals, as described (36). Mature T cells were depleted using anti-CD5 magnetic beads and the AutoMACS (Miltenyi Biotec). Male and female B6.SJL naive mice were lethally irradiated (950 rad) 1 day before i.v. injection of 5–8 x 106 T cell-depleted bone marrow cells from either male or female IFN-{alpha}betaR1–/– mice. In additional experiments, wild-type B6 or IFN-{alpha}betaR1–/– male-derived bone marrow was mixed with male congenic B6.SJL bone marrow at a ratio of 70:30 or 50:50. All animals were allowed to reconstitute for 8–9 wk before sacrifice and the NK T cell compartment in liver, spleen, bone marrow, and thymus was analyzed by flow cytometry.

In vitro {alpha}-GalCer stimulation

Following isolation of splenic lymphocytes from naive IFN-{alpha}betaR1–/– and wild-type B6 mice, both male and female, 5 x 105 unfractionated splenocytes were plated per well on a 96-well, flat-bottom plate in the presence of either 100 ng/ml {alpha}-GalCer or 1 µl/ml DMSO and cultured for 5 days. IL-4 and IFN-{gamma} levels in the supernatant were measured by ELISA. In additional experiments, 2.5 x 105 unfractionated splenocytes from naive IFN-{alpha}betaR1–/–, CD1d1–/–, and wild-type B6 were pulsed with 100 ng/ml {alpha}-GalCer or 1 µl/ml DMSO for at least 1 h, washed, and cocultured with 5 x 104 V{alpha}14i NK T cell hybridomas, DNA34-1-2, and DNA34-1-4; IL-2 was measured in the supernatant by ELISA 20 h later.

Statistical analysis

Statistical significance, designated as p ≤ 0.05, was determined by paired, two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Male B6.IFN-{alpha}betaR1–/– mice lack V{alpha}14i NK T cells

IFN-{alpha}beta exerts direct influence over the survival of activated and effector memory T cells, promoting resistance to apoptosis during Ag-specific proliferation (29, 30, 31). We therefore assessed the involvement of this pleiotropic family of cytokines in the survival of V{alpha}14i NK T cells. Animals deficient in IFN-{alpha}betaR, specifically the R1 subunit, designated IFN-{alpha}betaR1, are unable to respond to type I IFNs (32). Initial characterization of the V{alpha}14i NK T cell compartment in the spleen and liver of naive male and female IFN-{alpha}betaR1–/– mice revealed the absence of V{alpha}14i NK T cells in number (data not shown) and frequency (Fig. 1A, bottom panels) in male, but not female animals. This defect was specific for the V{alpha}14i NK T cells because conventional T and NK cells were present in comparable levels to wild-type mice (Fig. 1, A and B). All lymphocyte populations, including the V{alpha}14i NK T cells, in female IFN-{alpha}betaR1–/– mice were similar to wild-type B6 mice (Fig. 1B). V{alpha}14i NK T cells are detectable ~20 days post-birth and subsequently seed peripheral tissues, peaking at 6–8 wk of age (28). However, the peripheral compartment of V{alpha}14i NK T cells in the IFN-{alpha}betaR1–/– male mice is not corrected with age, and males remain deficient (Fig. 1C).


Figure 1
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  		FIGURE 1. Male B6.IFN-{alpha}betaR1–/– mice lack V{alpha}14i NK T cells. A, Splenic and hepatic lymphocytes were isolated from naive B6 and B6.IFN-{alpha}betaR1–/– male and female mice and stained with {alpha}-GalCer-loaded CD1d1 tetramer and anti-TCRbeta, anti-CD4, and anti-NK1.1 mAbs, and analyzed by FACS. B, Same as A. The percentages of hepatic V{alpha}14i NK T cells, NK cells, and CD4+ T cells from naive mice, average ± SD, are shown. Results shown are representative of at least 10 mice per strain. C, B6.IFN-{alpha}betaR1–/– hepatic V{alpha}14i NK T cells were examined at 3.2, 6, 8, and 12–14 wk of age, 2–4 mice per group; average ± SD is shown.

 
CD1d1 is functional and expressed at normal levels in the male B6.IFN-{alpha}betaR1–/– mice

Further confirmation of the absence of responsive V{alpha}14i NK T cells in the male IFN-{alpha}betaR1–/– mice was provided by in vitro stimulation of splenocytes from either male or female IFN-{alpha}betaR1–/– mice (Fig. 2A). Only cultures containing female-derived splenocytes produced high levels of IL-4 and IFN-{gamma}, hallmarks of {alpha}-GalCer-mediated activation of V{alpha}14i NK T cells. One explanation for the lack of detectable V{alpha}14i NK T cells and the corresponding lack of {alpha}-GalCer-mediated activation could be due to insufficient CD1d1 expression in the IFN-{alpha}betaR1–/– males. However, examination of CD1d1 levels revealed that in the IFN-{alpha}betaR1–/– mice, irrespective of sex, CD1d1 expression was comparable to wild-type animals (Fig. 2B).


Figure 2
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  		FIGURE 2. CD1d1 is functional and expressed at normal levels. A, Splenic leukocytes were isolated from naive wild-type B6, CD1d1–/–, and male and female B6.IFN-{alpha}betaR1–/– mice. A total of 5 x 105 cells was cultured per well in the presence of 100 ng/ml {alpha}-GalCer for 5 days. Supernatant was collected, and the level of IFN-{gamma} and IL-4 is shown as average ± SD. The results shown are representative of two independent experiments. B, Splenic and hepatic lymphocytes were isolated from naive B6 and B6.IFN-{alpha}betaR1–/– male and female mice and stained with anti-CD11b, anti-CD1d1, anti-B220, and anti-CD11c, and analyzed by flow cytometry. Total hepatic and splenic lymphocyte CD1d1 expression is shown. Results are representative of at least five mice per strain. C, Unfractionated splenocytes isolated from naive wild-type B6, CD1d1–/–, and male and female IFN-{alpha}betaR1–/– mice were pulsed with 100 ng/ml {alpha}-GalCer for 3 h, washed, and cocultured with DN3A4-1-2 and DN3A4-1-4 V{alpha}14i NK T cell hybridomas for 20 h. The level of IL-2 in the supernatant is shown as average ± SD. The results are representative of three independent experiments.

 
To further address the functionality of CD1d1 in the IFN-{alpha}betaR1–/– male mice, splenocytes and thymocytes from wild-type, IFN-{alpha}betaR1–/–, and CD1d1–/– mice were pulsed with {alpha}-GalCer and subsequently cocultured with V{alpha}14i NK T cell hybridoma cell lines, DNA3A4-1-2 and DNA3A4-1-4. Twenty hours post-culture, measurement of IL-2 in the supernatant revealed that IFN-{alpha}betaR1–/– splenocytes (Fig. 2C) and thymocytes (data not shown), irrespective of sex, presented {alpha}-GalCer to the NK T cell hybridomas as well (thymus) or better (spleen) than wild-type cells. These data suggest that the loss of V{alpha}14i NK T cells is not due to decreased CD1d1 expression, loss of CD1d1-mediated Ag presentation, or aberrant selection in the thymus.

V{alpha}14i NK T cells are absent in the thymus of B6.IFN-{alpha}betaR1–/– males

Aberrant sequestration of V{alpha}14i NK T cells in the thymus would result in the loss of this population in the periphery. However, examination of the thymus from both male and female IFN-{alpha}betaR1–/– also showed a clear sex-biased absence of the V{alpha}14i NK T cells (Fig. 3A). CD1d1 tetramer+ cells were readily detectable in the thymus from IFN-{alpha}betaR1–/– females, but thymocytes from male littermates were deficient in all CD1d1 tetramer+ populations (Fig. 3A and data not shown). Interestingly, the female mice generally had larger thymuses than their male counterparts, as assessed by visual size and cellularity, regardless of age (data not shown). A significant elevation in the percentage of CD4+ thymocytes within the HSAlow population was easily discernible in thymocytes from male IFN-{alpha}betaR1–/– mice (Fig. 3A, left panel). Additionally, a higher percentage of NK1.1+CD1d1 tetramer– cells was observed in the male IFN-{alpha}betaR1–/– thymus (Fig. 3A, middle panel). Also, a noteworthy increase in the percentage of {gamma}{delta} T cells was consistently observed in the thymus of IFN-{alpha}betaR1–/– males, but not females (Fig. 3B).


Figure 3
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  		FIGURE 3. V{alpha}14i NK T cells are absent in the thymus of male B6.IFN-{alpha}betaR1–/– mice. A, Thymocytes were isolated from male and female B6.IFN-{alpha}betaR1–/– mice and stained with {alpha}-GalCer-loaded CD1d1 tetramer, anti-HSA (CD24), anti-CD4, and anti-NK1.1, and analyzed by FACS. Results are representative of at least 10 mice per sex. B, Same as in A, but thymocytes were stained with anti-TCRbeta, anti-HSA, anti-NK1.1, and anti-TCR{gamma}{delta}. Results are representative of three independent experiments, two to five mice each strain/sex.

 
Thymic CD1d1 expression and function in B6.IFN-{alpha}betaR1–/– males are comparable to wild-type mice

The selection process of V{alpha}14i NK T cells is mediated by CD4+CD8+ (DP) thymocytes expressing the nonclassical, nonpolymorphic MHC class Ib-like molecule, CD1d1 (13). A defect in thymic expression of CD1d1 or in the selecting population could account for the lack of V{alpha}14i NK T cells. However, DP thymocytes from male IFN-{alpha}betaR1–/– mice expressed CD1d1 at a level comparable to wild-type B6 mice (Fig. 4, A and B). Furthermore, coculture of {alpha}-GalCer-pulsed thymocytes from IFN-{alpha}betaR1–/– mice with the V{alpha}14i NK T cell hybridomas demonstrated that both male- and female-derived thymocytes were capable of inducing IL-2 release from the hybridomas as well as wild-type thymocytes (data not shown). Therefore, neither insufficient expression of CD1d1 nor aberrant CD1d1-Ag presentation in the thymus was responsible for the profound deficiency of V{alpha}14i NK T cells in the IFN-{alpha}betaR1–/– males.


Figure 4
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  		FIGURE 4. CD1d1 levels in B6.IFN-{alpha}betaR1–/– thymus are comparable to wild-type mice, but there are significantly more DN thymocytes. Thymocytes were isolated from male B6, B6.IFN-{alpha}betaR1–/–, and CD1d1–/–, stained with anti-HSA, anti-CD1d1, anti-CD4, and anti-CD8, and analyzed by FACS. A and B, A representative histogram of the mean fluorescent intensity of expression of CD1d1 on DP thymocytes is shown in A, and average mean fluorescence intensity ± SD is shown in B. C and D, The DP thymocyte population in the B6, IFN-{alpha}betaR1–/–, and CD1d1–/– mice is shown in C, and the average is provided in D. Results shown are representative of at least three mice per strain.

 
Further examination of the DP population clearly demonstrated that, although CD1d1 expression was similar, there were significantly fewer DP cells in the IFN-{alpha}betaR1–/– male mice compared with the wild-type animals (61.2 ± 2.7%, IFN-{alpha}betaR1–/–, vs 87.3 ± 2.7%, B6, p < 0.05; Fig. 4, C, left and middle panels, and D) and a correlative increase in CD4–CD8– (double-negative (DN)) thymocytes (17.9 ± 1.2%, IFN-{alpha}betaR1–/–, vs 3.8 ± 0.8%, B6, p < 0.05; Fig. 4, C, left and middle panels, and D). Notably, this accumulation of DN thymocytes is lacking in the female IFN-{alpha}betaR1–/– mice, which contain normal numbers of V{alpha}14i NK T cells.

The V{alpha}14i NK T cell defect is cell autonomous

To determine whether the defect was inherent to the male V{alpha}14i NK T cell, bone marrow chimeras were generated in which the irradiated male, congenic wild-type host was reconstituted with a 30:70 or 50:50 mixture of wild-type and IFN-{alpha}betaR1–/– male bone marrow (Fig. 5, A and B, and data not shown). Following reconstitution, analysis of the V{alpha}14i NK T cell compartment revealed that the only detectable V{alpha}14i NK T cells were derived from the wild-type (CD45.2–) bone marrow in all the tissues examined (Fig. 5, A and B). Furthermore, the distortion in the DN/DP ratio was recreated within the CD45.2+ (IFN-{alpha}betaR1–/–) population in the thymus (Fig. 5C, far left panel), whereas the CD45.2– (wild-type) thymic pool appeared completely normal (Fig. 5C). Taken together, these results demonstrate that the impaired development of V{alpha}14i NK T cells in IFN-{alpha}betaR1–/– males is cell autonomous.


Figure 5
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  		FIGURE 5. The V{alpha}14i NK T cell defect is cell autonomous. Bone marrow was isolated from the femur of male IFN-{alpha}betaR1–/–, wild-type congenic, and wild-type noncongenic mice and depleted of mature T cells. Host congenic B6 males were lethally irradiated 1 day before bone marrow transfer (5-8 x 106 cells/mouse) via tail vein injection. One group of hosts received a mixture of IFN-{alpha}betaR1–/– and wild-type congenic bone marrow (70:30), and the control group received a mixture of B6, noncongenic, and congenic marrow. Animals were allowed to rest for 7–9 wk before sacrifice. Liver, spleen, bone marrow, and thymus were collected, and lymphocytes were isolated. A, Hepatic lymphocytes were stained with {alpha}-GalCer-loaded CD1d1 tetramer, anti-TCRbeta, anti-NK1.1, and anti-CD45.2, and analyzed for reconstitution of the V{alpha}14i NK T cell population by flow cytometry. B and C, Thymocytes were stained with {alpha}-GalCer-loaded CD1d1 tetramer, anti-TCRbeta, anti-CD4, anti-CD8, anti-HSA, anti-NK1.1, and anti-CD45.2, and analyzed by flow cytometry. Representative dot plots showing the V{alpha}14i NK T cell compartment (B) and the DP/DN populations (C) are shown. D, Thymocytes were isolated from female B6 and IFN-{alpha}betaR1–/– mice and depleted of CD8+ T cells before labeling with 5 mM CFSE. A total of 8–10 x 106 CFSE-labeled thymocytes was injected via the tail vein into male or female congenic B6 mice that had been sublethally irradiated 3 days before transfer. All animals were sacrificed 7 days postthymocyte transfer, and proliferation of the donor population in the liver and spleen was analyzed by flow cytometry using {alpha}-GalCer-loaded CD1d1 tetramer, anti-CD45.1, anti-TCR, and anti-CD4. The results shown are representative of three independent experiments.

 
Additionally, the loss of the V{alpha}14i NK T cell compartment is not the result of an inability of the cells to survive following proliferation as demonstrated by lymphopenia-induced proliferation (Fig. 5D), in which both male and female irradiated wild-type mice received either female wild-type or IFN-{alpha}betaR1–/–-derived thymocytes. These experiments also indicate that the V{alpha}14i NK T cell deficiency was not the result of sex hormone-mediated immune effects because IFN-{alpha}betaR1–/– thymocytes proliferated and survived equally in both male and female hosts.

The deficiency in V{alpha}14i NK T cells is independent of type I IFNs

The IFN-{alpha}betaR1–/– mice were originally generated on the 129Sv/Ev inbred mouse background (32), and two sources of IFN-{alpha}betaR1–/– mice were analyzed, one backcrossed 6 times and another >10 times onto the C57BL/6 background. Both sources lack V{alpha}14i NK T cells (data not shown). We further evaluated whether a similar sex-specific defect was present in the 129.IFN-{alpha}betaR1–/– mice. It is well known that the 129Sv/Ev mice have inherently fewer V{alpha}14i NK T cells than the inbred C57BL/6 strain. As expected, the average percentage (Fig. 6, A and B) and number (data not shown) of V{alpha}14i NK T cells in both the wild-type 129SvEv and 129.IFN-{alpha}betaR1–/– were substantially reduced compared with wild-type B6 mice (20–30%, Fig. 1B, compared with 3–6%, Fig. 6, A and B). However, neither the wild-type 129SvEv nor 129.IFN-{alpha}betaR1–/– mice display differences in the percentage of V{alpha}14i NK T cells in any of the tissues examined, irrespective of gender (Fig. 6, A and B).


Figure 6
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  		FIGURE 6. The V{alpha}14i NK T cell deficiency is male specific and independent of Tyk2 and STAT1. Splenic, hepatic, and thymic lymphocytes were isolated from naive wild-type and IFN-{alpha}betaR1–/– mice on the 129SvEv genetic background and from naive B6.Tyk2–/– and B6.STAT1–/– mice, male and female, and stained with {alpha}-GalCer-loaded CD1d1 tetramer and anti-TCRbeta, anti-CD4, anti-CD8, anti-CD1d1, anti-HSA, and anti-DX5 mAbs, and analyzed by FACS. A, The percentage of TCRbeta+CD1d1-tetramer+ cells is shown in the upper right of each representative dot plot showing hepatic V{alpha}14i NK T cells isolated from 129Sv/Ev wild-type and IFN-{alpha}betaR1–/– animals. B, Same as A; the percentages of hepatic V{alpha}14i NK T cells from naive male and female mice, average ± SD, are shown. Results shown are representative of at least three mice per strain. C, The percentage of TCRbeta+CD1d1-tetramer+ cells is shown in the upper right of each representative dot plot showing hepatic V{alpha}14i NK T cells isolated from B6.Tyk2–/– and B6.STAT1–/– mice. D, Same as in C. The average percentages of hepatic V{alpha}14i NK T cells from naive mice, male and female, ± SD, are shown. Results shown are representative of three to four mice per strain and sex.

 
The lack of a sex-biased loss of V{alpha}14i NK T cells in the 129.IFN-{alpha}betaR1–/– mice led us to more closely examine the requirement of IFN-{alpha}beta signaling in generating this phenotype in the IFN-{alpha}betaR1–/– males on the B6 background. IFN-{alpha}beta signals via a heterodimeric receptor composed of IFNAR1 and IFNAR2, with each subunit specifically associating with a distinct Janus kinase, Tyk2 and JAK1, respectively (37). These kinases primarily recruit and phosphorylate STAT1 and STAT2, inducing IFN-{alpha}beta-mediated activity (38, 39). The majority of IFN-{alpha}beta-driven action uses this conserved signaling pathway. To address the contribution of classical IFN-{alpha}beta signaling components in the sex-specific deficiency in V{alpha}14i NK T cells, we examined the NK T cell compartment in male and female animals deficient in either Tyk2 or STAT1, both on a C57BL/6 background (Fig. 6, C and D). Irrespective of sex, in both the Tyk2–/– and STAT1–/– mice the V{alpha}14i NK T cell compartment in the liver (Fig. 6, C and D) and spleen (data not shown) was comparable. Therefore, STAT1 and Tyk2 signaling are dispensable for the IFN-{alpha}betaR1-dependent development of V{alpha}14i NK T cells in male mice.

The lack of V{alpha}14i NK T cells is linked to the Y chromosome

The lack of a detectable loss of V{alpha}14i NK T cells in both the Tyk2–/– and STAT1–/– male mice suggested that the classical IFN-{alpha}beta signaling pathway was not involved in the observed phenotype. Therefore, IFN-{alpha}betaR1–/+ mice were generated by backcrossing IFN-{alpha}betaR1–/– mice with wild-type B6 animals. Two mating strategies were established, one in which the IFN-{alpha}betaR1–/– male was mated with a B6 female (B6IFN-{alpha}betaR1+/–F1) and another in which the male was wild type and the female was IFN-{alpha}betaR1–/– (IFN-{alpha}betaR1B6–/+F1). Surprisingly, the IFN-{alpha}betaR1B6–/+F1 males displayed a normal V{alpha}14i NK T cell compartment, but the B6IFN-{alpha}betaR1+/–F1 males were V{alpha}14i NK T cell deficient (Fig. 7). In addition, we crossed 129 females with IFN-{alpha}betaR1–/– males (B6 background) and vice versa and found that only the F1 mice carrying the IFN-{alpha}betaR1 Y chromosome had a V{alpha}14i NK T cell defect, arguing against a possible influence of the B6 genome (data not shown). Taken together, these results demonstrate that the observed phenotype is independent of IFN-{alpha}beta and linked to the Y chromosome of the original male founder.


Figure 7
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  		FIGURE 7. The lack of V{alpha}14i NK T cells is linked to the Y chromosome. Hepatic, thymic, and splenic lymphocytes were isolated from IFN-{alpha}betaR1–/+ generated by two different mating schemes to yield F1 progeny carrying a Y chromosome from the IFN-{alpha}betaR1–/– male or from a wild-type B6 male. The percentage of hepatic TCRbeta+CD1d1 tetramer+ cells is shown in the upper right corner of each representative dot plot. Results are representative of two independent experiments, two to three mice each strain/sex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is widely accepted that V{alpha}14i NK T cells, although developing from the same common lymphoid precursor as conventional T cells, are a separate lineage (1, 28, 40, 41). The selection program that the V{alpha}14i NK T cells undergo is distinct from mainstream thymic selection and is uniquely mediated by cells that eventually give rise to the V{alpha}14i NK T cell pool (13, 14, 25, 42). NK T cell precursors are chosen based on their ability to bind the agonistic self-lipid-CD1d1 complexes with appropriate affinity (19, 20, 21, 43). Before emigrating to the periphery, full effector function is acquired and NK T cells are, characteristically, effector memory T cells (3). The dissimilarities between V{alpha}14i NK T cell development and mainstream T cells are highlighted by a number of recently identified genetic defects that negatively influence V{alpha}14i NK T cell development and function while leaving the conventional T cell population relatively unaffected (44, 45, 46, 47, 48). The findings presented in this study further highlight the unique development that V{alpha}14i NK T cells undergo and provide evidence for a novel, Y-linked influence over the ontogeny of this T cell population.

Our initial finding of a peculiar deficiency of V{alpha}14i NK T cells in male IFN-{alpha}betaR1–/– mice prompted us to first determine whether additional lymphocyte subsets were similarly affected. However, examination of NK and CD4+ T cells revealed that these populations were comparable in IFN-{alpha}betaR1–/– and wild-type mice, irrespective of sex. The CD8+ T cell compartment in the IFN-{alpha}betaR1–/– males was only slightly decreased in comparison with female IFN-{alpha}betaR1–/– and wild-type animals (data not shown), unlike the complete loss of the V{alpha}14i NK T cell subset in the periphery. Additional evidence of the lack of this T cell population was provided by coculture of splenocytes from male or female IFN-{alpha}betaR1–/– mice with the prototypical ligand, {alpha}-GalCer, because IL-4 and IFN-{gamma} were only detectable in the supernatant from female-derived splenocyte cultures. The fact that other lymphoid subsets, such as NK and mainstream T cells, were relatively unaffected, whereas NK T cells were lacking, indicated that the underlying mechanism does not involve factors critical to general lymphocyte development or survival, but was unique to the V{alpha}14i NK T cell lineage. Furthermore, this block in NK T cell development is not overcome as the mice age because males remain deficient in the V{alpha}14i NK T cell compartment exclusively.

The specific deficiency of V{alpha}14i NK T cells in the IFN-{alpha}betaR1–/– male mice led us to investigate whether an insufficient expression of CD1d1 was the primary mechanism behind the loss of V{alpha}14i NK T cells. Although peripheral levels of CD1d1 are normal in the IFN-{alpha}betaR1–/– male mice, V{alpha}14i NK T cells do not require transient peripheral contact with CD1d1 for homeostatic maintenance and survival (26, 49, 50). Insufficient CD1d1 expression in the thymus or a defect in the main selecting population, DP thymocytes (13, 14), would severely impact V{alpha}14i NK T cell development. However, CD1d1 expression was comparable in the thymus of male IFN-{alpha}betaR1–/– and wild-type mice. Additionally, CD1d1-mediated Ag presentation was similar and, thus, not mediating the loss of this innate T cell subset.

Closer examination of the selecting population in the thymus revealed that the DP thymocyte population was significantly reduced in the IFN-{alpha}betaR1–/– males in comparison with both wild-type males and IFN-{alpha}betaR1–/– females. Furthermore, there was a correlative increase in the DN population. In wild-type animals, the DP thymocytes are the major thymic subset, constituting >90% of all thymocytes, whereas the immature, DN population comprises ~1–5%. In the IFN-{alpha}betaR1–/– male mice, but not female littermates, this distribution is skewed, with ≤65–70% of the cells expressing both CD4 and CD8 and >15% of the thymocytes existing as DN cells. This disruption of the DN to DP ratio does not significantly impair the development of mainstream T cells, although alterations in thymic populations were consistently observed. Notably, a small decrease in CD8 single-positive (SP), correlative increase in CD4 SP cells, and a surprising increase in the {gamma}{delta} T cell population were seen in only the male IFN-{alpha}betaR1–/–, suggesting that the defect mediating the loss of the V{alpha}14i NK T cells was inherent to the T cell precursor pool. Interestingly, in mixed bone marrow chimeric mice, only bone marrow from the wild-type animals was capable of establishing the V{alpha}14i NK T cell compartment. The IFN-{alpha}betaR1–/– population clearly showed a distorted DN/DP ratio and lack of V{alpha}14i NK T cells. The consistent appearance of the DN/DP distortion and increase in CD4 SP cells suggest a very early developmental block in V{alpha}14i NK T cell selection, most likely before the DP stage, impacting the committed NK T cell precursor.

It has been reported that steroidal sex hormones can have a dynamic affect on immune responses in general as well as on thymic development (51, 52, 53). The sex bias often seen in autoimmune disease onset, prevalence, and progression highlights the potential contribution of hormones to immunity. Therefore, the sex-specific characteristic of the observed phenotype prompted us to investigate the influence of a male vs female environment on V{alpha}14i NK T cell development and survival. However, in the context of lymphopenia-induced proliferation, female IFN-{alpha}betaR1–/– thymocytes, both V{alpha}14i NK T cells and conventional CD4+ T cells, survived and proliferated similar to the wild-type splenocytes in both male and female hosts. Furthermore, bone marrow chimeric mice revealed that regardless of the sex of the host, IFN-{alpha}betaR1–/– female bone marrow was sufficient to reconstitute all lymphocyte compartments, but male IFN-{alpha}betaR1–/– bone marrow was incapable of generating a normal V{alpha}14i NK T cell population. Collectively, these findings demonstrated that sex hormones were not mediating the loss of this T cell population in the male mice and that the defect was cell autonomous. Notably, the thymic alterations seen in the IFN-{alpha}betaR1–/– males and mixed bone marrow chimeras were also recapitulated in all wild-type animals, irrespective of gender, reconstituted with the male IFN-{alpha}betaR1–/– bone marrow.

Additional examination of the IFN-{alpha}betaR1–/– mice on the 129SvEv background clearly showed that, regardless of sex, the 129.IFN-{alpha}betaR1–/– mice are phenotypically normal in comparison with wild-type 129Sv/Ev mice. The lack of a sex-specific loss of the NK T cell population in the 129.IFN-{alpha}betaR1–/– indicated that the defect was specific to the B6 background and may be independent of IFN-{alpha}beta signaling. In an effort to delineate the potential downstream effect of the IFN-{alpha}betaR1 subunit, we examined the V{alpha}14i NK T cell compartment in animals deficient in key IFN-{alpha}beta signaling components, Tyk-2 and STAT1, both on a B6 background. No gender-specific deficiency was observed in either strain, and both Tyk-2–/– and STAT1–/– mice are V{alpha}14i NK T cell sufficient, irrespective of sex. Unlike the mutation in the gene encoding the signaling lymphocytic activation molecule-associated protein (54, 55, 56) and the X-linked inhibitor of apoptosis (57), which results in a deficiency in V{alpha}14i NK T cells and afflicts 50% of males carrying the mutation and occasionally females (54, 55), the V{alpha}14i NK T cell deficiency described in this study is exclusive to males with a ≥99% penetrance. This suggests that the V{alpha}14i NK T cell deficiency is independent of signaling lymphocytic activation molecule-associated protein or X-linked inhibitor of apoptosis, although epistatic interaction between X and Y chromosome-encoded products cannot be ruled out.

Our analysis of IFN-{alpha}betaR1–/+F1 mice revealed that the absence of V{alpha}14i NK T cells was uniquely dependent on the paternal background and dominantly influenced by the Y chromosome, specifically the B6.IFN-{alpha}betaR1–/–-derived Y chromosome. Therefore, it is likely that a natural germline mutation on the Y chromosome, associated with V{alpha}14i NK T cell development, occurred during the cross of the 129.IFN-{alpha}betaR1–/– mice to the B6 background. Experiments are currently underway to identify the affected gene(s) responsible for this phenotype. Notably, experiments using IFN-{alpha}betaR1–/– male mice on the B6 background may need to be revisited. Collectively, these findings contribute to the understanding of the unique polygenic factors mediating the developmental program of the V{alpha}14i NK T cells and demonstrate for the first time that the Y chromosome can influence lymphocyte development.


   Acknowledgments
 
We thank Stephanie Terrizzi and Céline Fugere for their excellent animal care, and Deanna Chaukos for technical assistance. We also thank Drs. Kronenberg and McKeown for critical analysis of the data.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Research Grants AI46709 and AI058181 to L.B. Back

2 J.D.W. and M.S.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Laurent Brossay, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Box G-B618, Brown University, Providence, RI 02912. Back

4 Abbreviations used in this paper: V{alpha}14i, V{alpha}14 invariant; {alpha}-GalCer, {alpha}-galactosylceramide; DN, double negative; DP, double positive; HSA, heat stable Ag; SP, single positive. Back

Received for publication May 31, 2007. Accepted for publication July 2, 2007.


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 Materials and Methods
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J. Immunol., February 15, 2009; 182(4): 1789 - 1793.
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