SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF) of receptors, whose expression is essential for T, NK, and B-cell responses. Additionally, the expression of SAP in double-positive thymocytes is mandatory for natural killer T (NKT) cells and, in mouse, for innate CD8+ T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1 + 6]−/− and Slamf[1 + 5 + 6]−/−B6 mice have ~70% reduction of NKT cells compared to wild-type B6 mice. Unexpectedly, the proportion of innate CD8+ T cells slightly increased in the Slamf[1 + 5 + 6]−/−, but not in the Slamf[1 + 6]−/− strain, suggesting that Slamf5 may function as a negative regulator of innate CD8+ T cell development. Accordingly, Slamf5−/− B6 mice showed an exclusive expansion of innate CD8+ T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7−/− strain showed an expansion of both splenic innate CD8+ T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3−/− BALB/c mice, the proportions of thymic promyelocytic leukemia zinc finger (PLZFhi) NKT cells and innate CD8+ T cells significantly increased in the SAP-independent Slamf8−/− BALB/c strain. In summary, these results show that NKT and innate CD8+ T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8+ T cells.


INTRODUCTION
During mainstream T lymphocyte development, some of the key factors that drive the transition from double-positive (DP) CD4 + CD8 + precursors to single positive (SP) T cells involve antigen specificity and TCR strength (1)(2)(3). During this process, thymic epithelial cells interact with DP cells to determine the fate of immature thymocytes by orchestrating positive and negative selection (4)(5)(6)(7). By contrast, homotypic DP-DP interactions drive development and selection of the so-called innate αβ T lymphocytes, which include natural killer T (NKT) and innate CD8 + T cells (8)(9)(10). During development in the thymus, as well as in the effector phase in the periphery, NKT and innate CD8 + T cells, which have a restricted TCR repertoire, interact with different nonclassical MHC class I molecules (11)(12)(13)(14). These cells are thought to be the first responder cells that can rapidly release various cytokines and control both viral and bacterial infections (15).
Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) (encoded by Sh2d1a in mouse) and several SLAM family (SLAMF) receptors provide DP thymocytes with positive signals that favor their maturation in the thymus (16)(17)(18)(19)(20). Cognate activation of NKT cells is restricted to CD1d-lipid complexes and is modulated by SAP and at least three members of the SLAMF of receptors (20)(21)(22). The homophilic interactions of Slamf1 and Slamf6 between DP thymocytes are particularly important for the development of the NKT cell lineage (20). Binding of SAP to the immunoreceptor tyrosine-based switch motifs (ITSMs) present in the cytoplasmic tail of several SLAMF receptors (23)(24)(25) promotes a unique interaction between the active configuration of the Src tyrosine kinase Fyn and the SLAMF receptor (26,27), while at the same time blocking the recruitment of the protein phosphatases SHP-1 and SHP-2 (28)(29)(30), leading to efficient T cell activation and survival (31,32). Recent evidence now demonstrates that Slamf3, another SAP-associated SLAMF receptor, acts as an inhibitory receptor for NKT and innate CD8 + T cell development (22). This suggests that differential SLAMF receptor expression can positively or negatively influence innate T cell development.
Mutations in the human SH2D1A gene lead to X-linked lymphoproliferative syndrome (XLP) (28). This rare inherited disorder is characterized by exaggerated T-and B-cell responses against Epstein-Barr virus (EBV), resulting in EBV-induced infectious mononucleosis, hypogammaglobulinemia, and a higher risk of developing various forms of lymphoma. Because patients with XLP lack NKT cells, the study of the regulation of their development and function by SLAMF receptors and SAP will shed light on the pathogenesis of this often-fatal disease. An important unanswered question in this field relates to how the coexpression of different arrays of SLAMF receptors favors the development of a particular innate T cell lineage. Therefore, in this study, we aimed to evaluate the relative contributions of three SAP-binding receptors, Slamf1, Slamf5, and Slamf6, and two SAP-independent receptors, Slamf7 and Slamf8, to the development of NKT and innate CD8 + T cells in the mouse.

MICE
Slamf [1 + 6] −/− and Slamf[1 + 5 + 6] −/− mice on a C57BL/6 (B6) are described by Wang et al. (manuscript submitted for publication). Jα18 −/− mice on a B6 background, originally from Dr. Taniguchi (Riken, Yokohama, Japan), were provided by Dr. Exley (Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School, Boston, MA, USA). Slamf7 −/− B6 mice (B6N.129S5-Slamf7 tm1Lex /Mmucd, identification number 032574-UCD) were obtained from the Mutant Mouse Regional Resource Center (MMRRC), an NIH-funded strain repository, and was donated to the MMRRC by Genentech, Inc., Sh2d1a −/− mice on B6 and BALB/c backgrounds have been previously described (47). Slamf8 −/− mice on a BALB/c background have been previously described (48). Age-and sex-matched controls on the B6 and BALB/c backgrounds were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) or from Charles River Laboratories (Wilmington, MA, USA). All animals were housed in the Center for Life Science animal facility of the BIDMC. The experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee at BIDMC.

GENERATION OF SLAMF5 −/− MICE
A B6 background murine bacterial artificial chromosome (BAC) clone (BAC RP23-77A8) was used as the PCR template for cloning the 5 , 3 arm and middle homologous fragments of the targeting vector. The PCR primers for the 6-kb 5 targeting arm were designed from the upstream sequence of exon 1. The 2.1-kb middle arm that contains the promoter region plus exon 1, and the 4-kb 3 arm intron 1 fragments were generated by PCR. The PCR products were cloned into a targeting vector containing a LoxP-FRT-Neo-FRT-LoxP cassette (Figure 2A). The resulting targeting construct was sequenced to confirm the correct sequences and orientation of the inserted PCR fragments. The targeting vector was then incorporated into Bruce 4 ES cells by electroporation. Southern blot hybridization was performed with a 5 external probe to detect the homologous recombination event, and the positive clones were confirmed using a 3 external probe. Four independent positive ES clones were microinjected into blastocysts from BALB/c mice and gave rise to germline transmission of the Slamf5 mutant DNA. The resulting mice were bred with FLP transgenic B6 mice. FLP-mediated recombination resulted in the deletion of the FRT-flanked Neo gene. Subsequently, Neo-free mice were bred with CreTg mice to generate Slamf5 −/− mice (Figures 2A,B).

INTRACELLULAR STAINING
After surface staining (including the Fixable Viability Dye eFluor® 455UV), cells were fixed in IC/Fixation buffer (eBioscience) and then incubated for 30 min on ice with anti-PLZF plus anti-Eomes antibodies in 1× permeabilization buffer (eBioscience). After several washes, the cells were acquired on a BD™ LSR II flow cytometer (BD Biosciences).

EX VIVO CELL ACTIVATION AND INTRACELLULAR CYTOKINE STAINING
Total thymocytes from wild-type (WT) or Slamf8 −/− BALB/c mice were incubated overnight at 37°C in the presence of the Cell Stimulation Cocktail (plus protein transport inhibitors) reagent (eBioscience). After surface staining (including the Fixable Viability Dye eFluor® 455UV), cells were fixed/permeabilized and incubated for 30 min with anti-IFN-γ antibody on ice. Samples were acquired on a BD™ LSR II flow cytometer. Data analysis was performed using FlowJo software.

STATISTICAL ANALYSIS
Statistical significance was determined by unpaired t -test (twotailed with equal SD) using Prism software (GraphPad, San Diego, CA, USA). The p value <0.05 was considered statistically significant.

DIFFERENTIAL EXPRESSION OF SLAMF RECEPTORS BY MOUSE THYMOCYTE SUBSETS
Since both NKT and innate CD8 + T cells are selected from homotypic interactions between DP thymocytes, we thought that it was relevant to determine the specific SLAMF receptor expression pattern of these cells ( Figure S1 in Supplementary Material). As previously reported (20,22), we found that the homotypic SAPdependent receptors Slamf1, Slamf3, Slamf5, and Slamf6 are moderately to highly expressed by DP TCRβ low thymocytes. Although Slamf2 is highly expressed by these cells, its receptor, Slamf4, is virtually absent, suggesting that this heterophilic interaction does not play a role during homotypic DP-DP contacts ( Figure S1D in Supplementary Material). On the other hand, CD44 hi NK1.1 + mature thymic NKT cells showed less Slamf1 and Slamf6 expression than their DP progenitors, while Slamf2, Slamf3, Slamf4, and Slamf5 were upregulated ( Figure S1D in Supplementary Material). Thymic innate CD8 + T cells maintained the levels of Slamf1, Slamf4, and Slamf6 expression compared to DP TCRβ low thymocytes, but slightly upregulated Slamf2, Slamf3, and Slamf5 ( Figure  S1D in Supplementary Material). Once in the spleen, mature NKT cells expressed Slamf1, Slamf5, and Slamf6 at a higher level than in the thymus, but lacked Slamf4 expression. Slamf2 and Slamf3 levels did not change on these cells. With the exception of Slamf1 and Slamf6, which were downregulated, the other SLAMF receptors showed a similar level of expression in splenic innate CD8 + T cells compared to their thymic counterparts ( Figure S1D in Supplementary Material). Together, these data suggest that the SAP-dependent receptors Slamf1, Slamf3, Slamf5, and Slamf6 may participate in early homotypic cell-cell interactions leading to NKT and/or innate CD8 + T cell differentiation.

SAP-INDEPENDENT RECEPTORS SLAMF7 AND SLAMF8 ARE HIGHLY EXPRESSED IN THYMIC-RESIDENT DENDRITIC CELLS
We used the Gene Skyline tool from the Immunological Genome Project's website [Immgen.org; (52)] to determine Slamf7, Slamf8, and Slamf9 mRNA expression levels by different thymic populations ( Table 1). Slamf7 mRNA was differentially expressed by mature NKT cells, dendritic cells (DC), and medullary epithelial cells, while Slamf8 mRNA was only present in DC and thymic fibroblasts, but not in DP thymocytes. Slamf9 mRNA was moderately expressed by DP thymocytes, DC, and medullary epithelial cells only. Furthermore, Sh2d1a mRNA (encoding SAP) was only detected in DP and mature NKT cells, while Sh2d1b1 mRNA (encoding EAT-2A) was not expressed by any of the cell subsets analyzed. These data suggest that the SAP-independent SLAMF receptors, Slamf8 and Slamf9, may also modulate innate T cell differentiation in mice.
marrow chimera experiments (20), NKT cell development was severely impaired as judged by a 70% reduced frequency and absolute cell counts (data not shown) of PBS-57 CD1d-tetramer (CD1d-Tet) reactive cells (NKT) both in the thymus and spleen of Figures 1A,B). Importantly, these results further confirm the significance of Slamf1 and Slamf6 coexpression in DP thymic precursors for NKT cell development. Since the additional disruption of the Slamf5 gene in the triple-knockout strain did not further reduce the NKT cell compartment (Figures 1A,B), we conclude that in the absence of Slamf1 and Slamf6, Slamf5 does not play a major role in NKT cell differentiation. As NKT cells have been shown to support innate CD8 + T cell development (10,43), we hypothesized that both Slamf[1 + 6] −/− and Slamf[1 + 5 + 6] −/− B6 mice would present a marked reduction in the proportions of innate CD8 + T cells due to the dramatic loss of NKT cells (Figures 1A,B). Unexpectedly, we did not find a significant impairment of the innate CD8 + T cell (Figures 1C,D). Instead, we found a slight increase of splenic innate-like CD8 + T cells in the absence of Slamf1, Slamf5, and Slamf6 (Figures 1C,D). These results raised the possibility that Slamf5 could be acting as a SAPdependent negative regulator of innate CD8 + T cell expansion in the periphery.
The above findings also suggest that the requirement of NKT cells during innate CD8 + T cell development and/or expansion is not absolute. To test this, we looked for the presence of innatelike CD8 + T cells in Jα18 −/− B6 mice, which, like Sh2d1a −/− B6 mice, completely lack NKT cells (59). Similar to what we found in Slamf[1 + 5 + 6] −/− mice (Figures 1C,D), Jα18 −/− B6 mice did not present significant alterations in the innate CD8 + T cell compartment, showing similar percentages of these cells both in the thymus and spleen compared to WT (Figures 1E,F). Intriguingly, although SAP was shown to be required for the selection of innate-like CD8 + T cells in the Itk −/− background (40), we found a significant increase of these cells in the thymus of Sh2d1a −/− B6 mice (Figures 1E,F). Together, these results show that in the absence of NKT cells, innate-like CD8 + T cells can develop relatively normally in the thymus and spleen of B6 mice.

SLAMF5 IS A NEGATIVE REGULATOR OF INNATE CD8 + T CELL EXPANSION
The enlarged population of CD44 hi CD122 + CD8 + T cells found in the spleen of Slamf[1 + 5 + 6] −/− mice but not in the spleen of Slamf[1 + 6] −/− mice (Figures 1C,D) prompts us to assess whether the Slamf5/SAP axis could be negatively regulating the development of these non-conventional lymphocytes. Therefore, we used the single-knockout Slamf5 −/− mouse to check this hypothesis (Figures 2A,B). Indeed, we found a significant increase in the percentage and absolute counts (data not shown) of innatelike CD8 + T cells in the spleen of Slamf5 −/− mice compared to WT B6 mice. In the thymus, we observed a similar trend, but this result did not reach statistical significance (Figures 2C,D). However, the NKT cell compartment of Slamf5 −/− mice appeared slightly reduced by the absence of this receptor (Figures 2E,F). Together, these data suggest that the SAP-binding receptor Slamf5 may be acting as a negative regulator of innate CD8 + T cell development and/or expansion in B6 mice ( Table 2).

SAP-INDEPENDENT RECEPTOR SLAMF7 NEGATIVELY CONTROLS THE DEVELOPMENT OF INNATE LYMPHOCYTES IN B6 MICE
Next, we evaluated whether Slamf7 deficiency (Figures 3A,B) could also alter the innate CD8 + and/or NKT cell compartment of B6 mice. We found a significant expansion of CD44 hi CD122 + innate-like CD8 + T cells in the spleen but not in the thymus of Slamf7 −/− mice as compared to WT B6 controls (Figures 3C,D). In contrast, and as shown in Figures 1E,F, Sh2d1a −/− B6 mice displayed a clear expansion of CD44 hi CD122 + innate-like CD8 + T cells in the thymus (Figures 3C,D). We also observed a significant increase of CD44 hi mature NKT cells in the thymus of Slamf7 −/− mice (Figures 3E,F). As expected, Sh2d1a −/− B6 mice almost completely lack mature NKT cells (Figures 3E,F). These results suggest that endogenous expression of Slamf7 negatively influences the fate of both innate CD8 + and NKT cells. Whether this occurs via EAT-2A, a second adaptor that was shown to bind the Slamf7 receptor (23,24) or via SAP recruitment to the cytoplasmic tail of Slamf7 remains to be determined.

EXPANSION OF INNATE-LIKE T CELLS IN SLAMF8 −/− BALB/c MICE
To date, nothing is currently known about the contribution of the SAP-independent receptor Slamf8 during T cell development. Thus, in order to test whether the lack of Slamf8 may influence this process, we systematically identified specific T cell subsets present in the thymus and spleen of Slamf8 −/− BALB/c mice by FACS (Figure 4E; Figure S2A in Supplementary Material). We also included SAP-deficient BALB/c mice (Sh2d1a −/− BALB/c) in these studies in order to evaluate the role of this SLAMF adaptor in a murine background other than B6. We readily identified an expansion of TCRβ hi CD8SP T cells in the thymus of Slamf8 −/− mice compared to WT BALB/c mice. This alteration appeared to be specific for this organ, as this was not observed in the spleen of Slamf8 −/− mice (expressed as a CD8:CD4 ratio; Figures 4A,B), a phenotype resembling Slamf3 −/− mice on the same background (22) ( Table 3). Because of the CD8-biased T cell expansion observed in Slamf8 −/− BALB/c mice, we analyzed the intracellular expression of the CD8-specific T-box transcription factor Eomes. Consistent with the above results, we found a twofold increase in the proportion of Eomes + CD8SP T cells in the thymus of Slamf8 −/− mice (Figures 4C,D). Interestingly, and despite the comparable ratio of CD8:CD4 T cells found in the spleen of Slamf8 −/− mice, Eomes + CD8SP T cells also increased in www.frontiersin.org  this organ compared to WT mice (Figures 4C,D). In contrast, Sh2d1a −/− mice, which displayed no change in CD8:CD4 ratios in thymus or spleen, showed a dramatic decrease in Eomes + CD8 + T cells (Figures 4C,D). On the other hand, the relative expression of Eomes and Irf4 (IFN regulatory factor 4) mRNA, whose expression was shown to inversely correlate with that of Eomes in recently activated CD8 + T cells (3,60), did not differ between Slamf8 −/− and WT resting thymocytes ( Figure 4F). In conclusion, the absence of Slamf8 expression favors the expansion of a CD8SP T cell population expressing Eomes in thymus and spleen. Moreover, although the absence of SAP expression does not alter the CD8:CD4 ratios in thymus or spleen, it dramatically decreases the frequency of Eomes + CD8SP T cells in these organs. www.frontiersin.org

CD44 hi CD122 + CD8SP T CELLS IN SLAMF8 −/− MICE EXPRESS THE TRANSCRIPTION FACTOR EOMES AND RAPIDLY PRODUCE IFN-γ UPON STIMULATION
Consistent with the enlarged Eomes + CD8 + T cell compartment in Slamf8 −/− mice, we found a larger proportion of CD44 hi CD122 + innate-like CD8 + T cells in these mice (Figures 5A,B), whereas Sh2d1a −/− mice showed a significant reduction of these cells in the thymus and spleen (Figures 5A,B). Interestingly, within the thymic innate-like CD8 + T cell population, Slamf8 −/− mice showed a higher proportion of Eomes + cells compared to WT mice (Figures 5C,D), supporting the idea that the expanded CD8 + T cell population documented in Figure 4 corresponds to truly bona fide innate CD8 + T cells. Consistent with a reduced CD44 hi CD122 + CD8SP T cell population, Sh2d1a −/− BALB/c mice displayed a dramatic reduction in Eomes + CD8SP T cells in the thymus and, to a lesser extent, in the spleen as well Frontiers in Immunology | T Cell Biology This work Unpublished observations (22) This work ↓, Reduction; ↑, expansion; ±, marginal/some effect; =, no effect.
( Figures 5C,D). These seemingly contrasting results, compared to Sh2d1a −/− B6 mice (Figures 1 and 3; Table 2), indicate that the consequences of a SAP mutation will be greatly influenced by the mouse background. In fact, this phenomenon was previously observed in Slamf3 −/− mice in which profound alterations of the innate CD8 + T cell compartment were only present on the BALB/c but not on the B6 background (22,58) (Table 3).
Finally, to confirm that the expanded population of innate-like CD8 + T cells found in Slamf8 −/− mice had the ability to produce effector cytokines upon activation, we stimulated total thymocytes ex vivo with PMA plus ionomycin. This treatment resulted in a higher percentage of IFN-γ producing Slamf8 −/− CD8SP T cells compared to WT cells (Figures 5E,F). Together, these data demonstrate that the lack of Slamf8 expression results in a preferential expansion of CD8 + T cells with an innate-like phenotype. These cells express the transcription factor Eomes and produce IFN-γ upon stimulation, suggesting that SAP-independent signals can also modulate the generation of non-conventional CD8 + T cells in mice.

SLAMF8 −/− MICE HAVE AN ENLARGED PROPORTION OF NKT PLZF hi CELLS IN THE THYMUS
Recent studies have strongly suggested that thymic NKT cells can change the size of the innate CD8 + T cell pool via the production of IL-4 (10,22,42,43,61). Therefore, we evaluated whether we could find an expansion of NKT cells in the thymus and spleen of Slamf8 −/− mice. Unlike Slamf3 −/− BALB/c mice, in which an expansion of NKT cells was reported (22), we did not observe significant differences in the percentage of total NKT cells in the thymus or spleen of Slamf8 −/− mice compared to WT mice (Figures 6A,B). Accordingly, and consistent with an essential role of SAP during NKT cell development on B6 mice (17)(18)(19), Sh2d1a −/− BALB/c mice were almost completely devoid of these cells (Figures 6A,B). These observations unmistakably demonstrate that SAP-derived signals contribute enormously to the NKT cell lineage commitment independently of the mouse genetic background. Moreover, the lack of Slamf8 does not greatly affect the proportions of CD1d-Tet reactive NKT cells.
Just as Eomes controls the effector program of innate CD8 + T cells (44), NKT cells in the thymus rely on the expression of PLZF to gain their phenotypical and functional properties (62)(63)(64)(65). Although we did not observe significant differences in the percentage of CD44 hi NKT cells between WT and Slamf8 −/− mice (Figures 6A,B), the proportion of PLZF hi CD1d-Tet reactive cells was slightly higher both in the thymus and spleen of Slamf8 −/− mice, as judged by intracellular PLZF staining (Figures 6C,D). In contrast, we found a significant reduction of the PLZF hi population in the few NKT cells that we could detect in Sh2d1a −/− BALB/c mice (Figures 6C,D). However, we did not detect differences in Zbtb16 mRNA expression (encoding PLZF) between WT and Slamf8 −/− resting thymocytes. On the other hand, an approximately threefold increase in IL-4 mRNA (encoding IL-4) was detected in Slamf8 −/− samples ( Figure 6E). Together, these data show that the absence of Slamf8 expression licenses the expansion of both Eomes + CD8SP and PLZF hi NKT cells preferentially in the thymus, and that those NKT cells are biased to produce IL-4, which in turn may contribute to the expansion of innate CD8 + T cells in Slamf8 −/− mice. This proposes an unprecedented role for the SAP-independent SLAMF receptor Slamf8 in regulating innate T cell development.

DISCUSSION
Inadequate TCR-related signaling during T cell development, including overly strong or weak MHC/TCR interactions, results in the deletion of up to 90% of developing thymocytes (66). SLAMF receptors and the adaptor SAP have been shown to play an essential modulatory role during selection of non-conventional T cells, including NKT and innate CD8 + T cells. These homophilic interactions provide developing NKT cells, and likely innate CD8 + T cells, with both positive and negative developmental cues. Interestingly, the SLAMF/SAP pathway does not seem to affect conventional αβ T cell development, suggesting the existence of a specialized mechanism by which innate lymphocytes are preferentially generated in the thymus. These observations could be partially explained by the particular modus operandi of the SLAMF receptors, in which, as self-ligands, homotypic cell-cell contacts can be specially favored by homophilic SLAMF-SLAMF interactions. Additionally, as the expression of SLAMF receptors is confined to the hematopoietic compartment, interactions of SLAMF-expressing DP thymocytes with thymic epithelial cells cannot lead to a productive activation of the SLAMF/SAP signaling cascade in those cells. On the other hand, if two interacting DP thymocytes display a similar SLAMF receptor expression pattern, then their homophilic SLAMF-SLAMF interactions will be particularly favored, leading to a successful expansion of these clones.
The generation of double and triple SLAMF-deficient mouse strains allowed us for the first time to study the simultaneous effects of a total abolition of Slamf1, Slamf5, and Slamf6 on NKT and innate CD8 + T cell development. Unlike our previous experiments using mixed-bone marrow chimeras (20), these mice provided us with the unique opportunity to avoid any possible unwanted effects derived from the sterile inflammation induced by the gamma radiation and/or the possible contribution of radioresistant cells inherent in these kinds of experiments. Since both Slamf [1 + 6] −/− and Slamf[1 + 5 + 6] −/− mice failed to recapitulate the total NKT cell developmental arrest found in Sh2d1a −/− mice (20) (Figure 1), we conclude that there must be yet another SLAMF receptor that is able to recruit SAP in the absence of Slamf1, Slamf5, and Slamf6. As we could not detect Slamf4 expression in DP thymocytes, we ruled out both Slamf2 and Slamf4 from the list of possible candidates. Since Slamf3 −/− B6 mice were reported not to have a decrease in NKT cell number (58), we can also rule www.frontiersin.org out this receptor. We then tested Slamf5 −/− and Slamf7 −/− B6 mice. Surprisingly, instead of a decrease in NKT and/or innate CD8 + T cells, we found a significant increase of both these cell populations in these mice (Figures 2 and 3), suggesting that these receptors act as negative regulators during innate T cell development. However, as it was shown for Slamf6 expression, in which this receptor was able to provide both positive and negative signals to NKT cell development in a SAP-dependent manner (53), it is still possible that in the presence of other SLAMF receptors, Slamf5 and Slamf7 play opposite roles to the signals coming from Slamf1 and Slamf6. Further experiments will hopefully shed light on this rather intricate but important relationship.
Interestingly, the reduction of NKT cells in Slamf[1 + 6] −/− and Slamf[1 + 5 + 6] −/− mice, or the complete absence of these cells in Sh2d1a −/− B6 mice, did not seem to impair the generation of innate-like CD8 + T cells (Figure 1; Table 2). As these results contradicted published evidence showing that NKT cells support the expansion of innate CD8 + T cells via IL-4 production (10), and that SAP expression is required for thymic selection of innate-like CD8 + T cells in Itk −/− mice (40), we sought to test if the absence of NKT cells in an otherwise normal SAP context would also affect innate CD8 + T cell development. Hence, we analyzed the presence of innate-like CD8 + T cells in Jα18 −/− mice (Figures 1E,F). Similar to our previous observations in Sh2d1a −/− B6 mice, we did not detect a reduction of CD44 hi CD122 + cells in this NKT-deficient strain (Figures 1E,F), suggesting that in the absence of NKT cells other cells can provide the factors needed to accomplish this task.
Since all murine SLAMF receptors that have one or more cytoplasmic ITSM, with the exception of Slamf2, Slamf8, and Slamf9, can theoretically recruit SAP and/or EAT-2A, a redundancy in their function during innate cell development can be expected. Supporting this idea, none of the single SLAMF-deficient mice generated on the B6 background have shown substantial alterations in their innate T cell compartments ( Table 2). Remarkably, however, Slamf3 −/− (22) and now Slamf8 −/− mice (this work), both on the BALB/c background, show massive expansions of innate-like CD8 + T cells in the thymus ( Table 3), albeit to a far lesser extent than the Itk −/− or Rlk −/− Itk −/− B6 mice. These results strongly suggest that the mouse background can somehow act as a pressure selector of innate T cell development.
The recent identification of a specialized NKT cell subset present in BALB/c mice, but practically absent in B6 mice, capable of constantly producing IL-4 (termed NKT2), allowed Lee et al. to propose a model in which NKT2 cells are readily available in the thymus to secrete IL-4, resulting in intrinsically greater frequencies of innate CD8 + T cells (43) on this mouse background. Notably, these cells express the highest levels of PLZF among all mature NKT cells. Consequently, an involvement of NKT2 cells in supporting the expansion of innate-like CD8 + T cells in Slamf8 −/− mice can be anticipated, as we find an enlarged PLZF hi NKT population that correlates with a greater proportion of innate CD8 + T cells in these mice (Figures 5 and 6). However, the underlying mechanism by which the absence of Slamf8 favors the expansion of PLZF hi NKT cells remains undetermined. Since Slamf8 mRNA is expressed by DC and fibroblasts in the thymus, but not DP thymocytes (Table 1), we speculate that these cells may be providing some sort of signal to developing NKT and/or innate CD8 + T cells which is affected by the absence of Slamf8. In this context, IL-15 transpresentation has been shown to mediate survival of mature thymic NKT and innate CD8αα + intestinal intraepithelial T cells (67)(68)(69)(70). Thus, Slamf8 may be regulating the production and/or transpresentation of this cytokine.
By investigating the impact of two previously uncharacterized SLAMF receptors (Slamf7 and Slamf8) as well as the combined impact of multiple SLAMF receptor deletions (Slamf[1 + 6] −/− and Slamf[1 + 5 + 6] −/− mice), we are able to present new evidence that shall complement previous knowledge concerning the individual contributions of SLAMF receptors to NKT and innate CD8 + T cell biology. Importantly, the outcomes of our studies show the existence of positive and negative signals derived from different SLAMF receptors (Tables 2 and 3), which together orchestrate innate T cell development. The cellular and molecular mechanisms behind these observations certainly deserve more attention in the future, since these pathways could potentially be targeted in the aid of expanding innate T cell populations in situations where SAP is absent, such as in patients with XLP.   (DAPI negative), singlets, lymphocytes, and total thymocytes depicting the NKT (CD1d-Tet + TCRβ inter ), and the TCRβ hi mature lymphocyte gates. Bottom row from left to right: total live thymocytes depicting the CD8 single positive (SP), the CD4SP, and the CD4 + CD8 + DP TCRβ low immature thymocytes. (B) Representative FACS staining of innate CD8 + T cells (CD44 hi CD122 + TCRβ + ) showing the corresponding FMO (fluorescence minus one) negative control for the CD122 gate (left ), or Eomesodermin (Eomes, right ) in thymus and spleen from WT cells.