Requirements for selection of conventional and innate T lymphocyte lineages

doi:10.1016/j.immuni.2007.09.012

Immunity. Author manuscript; available in PMC 2008 May 12.

Published in final edited form as:

Immunity. 2007 November; 27(5): 775–785.

doi: 10.1016/j.immuni.2007.09.012

PMCID: PMC2377064

NIHMSID: NIHMS35168

Requirements for selection of conventional and innate T lymphocyte lineages

Reiko Horai,^1,² Kristen L. Mueller,¹ Robin A. Handon,¹ Jennifer L. Cannons,¹ Stacie M. Anderson,¹ Martha R. Kirby,¹ and Pamela L. Schwartzberg¹^*

¹National Human Genome Research Institute National Institutes of Health, Bethesda, MD 20892

²Current address: National Eye Institute, National Institutes of Health, Bethesda, MD 20892

^*Correspondence: Email: pams/at/mail.nih.gov

The publisher's final edited version of this article is available at Immunity

See other articles in PMC that cite the published article.

Publisher's Disclaimer

Summary

Mice deficient in the Tec kinase Itk develop a large population of CD8 cells with properties resembling NKT and other innate T cell lineages, including expression of memory markers, rapid production of cytokines and dependence on IL-15. Like NKT cells, these CD8 cells can be selected on hematopoietic cells. We demonstrate here that these CD8 cell phenotypes result from selection on hematopoietic cells—forcing selection on the thymic stroma reduces the number and innate cell phenotypes of mature CD8 SP cells associated with Itk-deficiency. We further demonstrate that, similar to NKT cells, selection of innate-type CD8 cells in Itk-deficient mice strictly requires the adaptor molecule SAP, whereas, acquisition of the innate characteristics requires CD28. Our results suggest that SAP and Itk reciprocally regulate selection of innate and conventional CD8 cells on hematopoietic cells and the thymic epithelium, respectively, whereas CD28 regulates development of innate phenotypes resulting from selection on hematopoietic cells.

The development of immune responses requires the coordinated activation of multiple cells of the innate and adaptive immune system. Although lymphocytes are generally thought to provide the adaptive arm of the immune system, there is now a growing appreciation that certain T cell lineages exhibit properties of innate cell lineages and contribute to immediate, early responses to pathogens. In particular, among the earliest lymphocyte responses are those of NKT cells, H2-M3-restricted cells and other lymphocytes selected by MHC class Ib molecules, which exhibit properties of innate immune cells (Behar and Porcelli, 2007; Kerksiek et al., 1999; Seaman et al., 2000; Urdahl et al., 2002). These properties include the expression of memory markers, the rapid production of cytokines (within hours of stimulation) and dependence on IL-15 for their homeostasis or expansion (Das et al., 2001; Ohteki, 2002; Urdahl et al., 2002; Yoshimoto and Paul, 1994). Unlike memory T cells, which also exhibit these properties after initial activation in peripheral lymphoid tissues, innate lymphocyte lineages develop these properties within the thymus (reviewed in (Baldwin et al., 2004).

The development of T cells with these distinct innate properties within the thymus suggests that the requirements for their selection and differentiation may differ from those required for conventional T cell populations. Recent data support this idea. In particular, NKT cells and other MHC class Ib-restricted cells that arise in K^bD^b-deficient animals can be selected on hematopoietic cells within the thymus, unlike conventional T cells that are selected by recognition of peptide-MHC molecule ligands present on the thymic stroma (Bendelac, 1995; Bix et al., 1993; Ohteki and MacDonald, 1994; Urdahl et al., 2002). Moreover, a number of signaling molecules have been implicated in the development of NKT cells that are not required for the development of conventional T cells. These include the adaptor molecule SAP (SH2D1A), the tyrosine kinase Fyn, protein kinase C–theta (PKC-θ) and members of the nuclear factor of kappa B (NFκB) transcription factors (Chung et al., 2005; Eberl et al., 1999; Gadue et al., 1999; Nichols et al., 2005; Pasquier et al., 2005; Schmidt-Supprian et al., 2004; Sivakumar et al., 2003; Stanic et al., 2004). Intriguingly, all of these molecules have been implicated in signaling pathways downstream from the SLAM family of receptors, a class of cell surface molecules with complex roles in multiple hematopoietic cell lineages (Cannons et al., 2004; Ma et al., 2007). Nonetheless, several of these molecules, particularly Fyn, PKC-θ and NF-κB are also activated in mature T cells by TCR engagement, limiting our understanding of the specific roles they play in development of innate lymphocytes.

Itk is a Tec family tyrosine kinase that plays important roles downstream of the TCR in the phosphorylation of PLC-γ, the regulation of Ca⁺⁺ mobilization and the activation of ERK (Berg et al., 2005). Itk also contributes to TCR- and chemokine-induced actin reorganization and “inside-out signaling” to integrins (Finkelstein and Schwartzberg, 2004; Gomez-Rodriguez et al., 2007). Itk-deficient mice show impaired positive selection of both MHC class I and class II specific TCR transgenes (Liao and Littman, 1995; Lucas et al., 2002; Schaeffer et al., 2000). Surprisingly, however, Itk-deficient mice with polyclonal repertoires only show reduced numbers of CD4 SP thymocytes, while CD8 SP thymocytes are relatively normal in number, giving rise to decreased CD4:CD8 cell ratios. Further analyses of CD8 SP thymocytes in these animals demonstrated that most of the CD8 SP cells exhibit properties of innate T lymphocyte lineages; these CD8 cells express activation/memory markers including CD44 and CD122, rapidly produce cytokines ex vivo, and are IL-15 dependent (Atherly et al., 2006; Berg, 2007; Broussard et al., 2006; Dubois et al., 2006). Data from fetal thymic organ cultures demonstrated that these properties developed within the thymus and were not the result of activation in the periphery (Broussard et al., 2006). Strikingly, bone marrow transfers into B2m- and B2m/MHC class II-deficient mice revealed that the CD8 cells in Itk^−/− mice, like NKT cells and CD8 cells that are selected by MHC class Ib molecules, can be selected on hematopoietic cells, independent of the presence of selecting MHC class I molecules on the thymic stroma (Broussard et al., 2006). Together, these characteristics argue that the CD8 cells that develop in Itk-deficient mice resemble innate lymphocyte lineages. Moreover, since the majority of CD8 cells in Itk^−/− mice exhibit these innate characteristics, Itk appears to differentially affect the development of conventional and non-conventional innate-type lineages.

The large number of “innate-like” CD8 cells that develop in Itk^−/− mice provides an opportunity to analyze the requirements for the development of innate T cell phenotypes. We demonstrate here that the innate cell characteristics of CD8 cells that develop in Itk-deficient mice are the result of selection on hematopoietic cells—forcing selection on MHC molecules expressed by the thymic stroma completely rescued the innate CD8 cell phenotypes associated with Itk-deficiency. To determine how selection by hematopoietic cells leads to development of cells with innate-cell characteristics, we evaluated the requirements for co-stimulatory molecules expressed on hematopoietic cells in the thymus. We found that, similar to findings with NKT cells, selection of the innate-type CD8 cells on hematopoietic cells in Itk-deficient mice strictly required the adaptor molecule SAP, which is required for signaling from SLAM family receptors on T lymphocytes. We further show that development of the innate characteristics of CD8 cells in Itk-deficient mice also required costimulation through CD28. However, SAP and CD28 appear to have distinct roles in the development of these innate cells: CD28 deficiency did not prevent selection on hematopoietic cells, but prevented the full acquisition of the innate-type phenotypes. Our results argue that costimulatory signals from hematopoietic cells in the thymus are required for the development of the innate CD8 T cell population in Itk^−/− mice. Together, these data suggest that Itk, SAP, and CD28 play distinct roles in innate T cell development, where Itk and SAP-mediated pathways differentially regulate selection of conventional T cells on the thymic stroma versus innate T cell lineages on hematopoietic cells, respectively and CD28 influences the acquisition of innate-like phenotypes.

Results

Itk-deficiency increases selection of MHC class Ib-restricted cells on hematopoietic cells

It has recently been recognized that Itk-deficiency leads to the development of a large population of CD8 cells that resemble innate lymphocyte populations (Atherly et al., 2006; Broussard et al., 2006; Dubois et al., 2006). Mice lacking MHC class Ia molecules (K^b−/−D^b−/−) select a small population of CD8 cells on non-conventional MHC class Ib that also exhibit these properties. We have further shown that Itk deficiency increased the number of CD8 cells developing in K^b−/−D^b−/− mice, suggesting that Itk deficiency increases selection of MHC class Ib-restricted cells (Broussard et al., 2006).

To further evaluate the CD8 cells in Itk-deficient mice, we compared their phenotypes with CD8 cells developing in K^b−/−D^b−/− mice. As we have previously demonstrated, CD8 SP thymocytes in Itk^−/− mice appear more mature (HSA^lo) than cells in WT mice and show high levels of CD44, CD122 and low levels of the β7 integrin, characteristics of innate CD8 cells (Figure 1A, second row). Notably, a larger proportion of the mature (HSA^lo) CD8 SP cells in Itk^−/− and Itk^−/−K^b−/−D^b−/− mice consistently exhibited markers of innate cell phenotypes than the CD8 SP cells in K^b−/−D^b−/− mice, which were more variable in their phenotype (Figure 1A, third row and Supplemental Figure 1A). Similar results were obtained in analyses of mature peripheral CD8 cells (Supplemental Figure 1B). Moreover, although Itk deficiency increased the number of CD8 SP thymocytes in K^bD^b-deficient mice (Figure 1A and (Broussard et al., 2006)), the increased number of mature (HSA^lo) CD8 SP cells in Itk^−/−K^b−/−D^b−/− mice only partially accounted for the size of the population of mature CD8 SP cells in Itk^−/− mice (Figure 1B). Thus, many of the non-conventional CD8⁺ T cells in Itk^−/− mice appear to be selected by MHC class Ia molecules (Broussard et al., 2006). These results support the idea that selection on MHC class Ib alone does not account for the altered development of CD8 cells in Itk^−/− mice--rather, it is the loss of Itk that predisposes CD8 cells to have these phenotypes irrespective of the selecting MHC ligand.

Figure 1

Itk^−/− and Itk^−/−K^b−/−D^b−/− CD8 cells show innate-like phenotypes

We have previously demonstrated that development of CD8 cells in Itk^−/− mice requires MHC class I, but like MHC class Ib-restricted cells, these cells can be selected on hematopoietic cells: CD8 cells fail to develop in Itk^−/−B2m^−/− mice, yet develop when Itk^−/− bone marrow was transferred into B2m^−/− mice in which MHC class I expression is restricted to the transferred hematopoietic cells (Broussard et al., 2006) and Figure 1C and and4).4). To further evaluate how Itk affects selection of MHC class Ib-restricted cells, we performed bone marrow transfers into B2m-deficient mice. Bone marrow transfers from Itk^−/−K^b−/−D^b−/− mice into B2m^−/− recipients gave rise to more CD8 cells than K^b−/−D^b−/− → B2m^−/− recipients (Figure 1C), demonstrating that Itk-deficiency specifically increased selection of MHC class Ib-restricted cells on hematopoietic cells. These observations raise the possibility that Itk-deficiency increases the numbers of innate-like cells by promoting selection on hematopoietic cells.

Figure 4

SAP is required for thymic selection on hematopoietic cells

Development of innate-like CD8 cells requires selection on hematopoietic cells

Although adoptive transfer experiments show that the innate-like CD8 cells in Itk^−/− mice can be selected on hematopoietic cells (Broussard et al., 2006), it remained unclear as to whether these CD8 cells are only selected on hematopoietic cells or can also be selected on the thymic stroma. Furthermore, it was unclear how these potential selection pathways may affect the development of these cells.

To address these questions, we generated bone marrow chimeras that express MHC class I only on thymic stromal cells by transferring bone marrow cells from Itk^−/−B2m^−/− or B2m^−/− controls into WT mice. In this situation, MHC class I is only expressed on the radioresistant thymic stroma, not on hematopoietic cells. Analyses of these chimeras revealed that CD8 cells could develop in mice that had received bone marrow from either B2m^−/− or Itk^−/−B2m^−/− mice (Figure 2A–B). However, while intact Itk^−/− mice or chimeras in which Itk^−/− bone marrow is transfered into WT mice exhibit elevated numbers of CD8 SP cells compared to WT mice (Figure 1A, C and (Broussard et al., 2006)), fewer mature (HSA^loTCR^hi) CD8 cells arose in the Itk^−/−B2m^−/− → WT bone marrow chimeras compared to the B2m^−/− → WT chimeras (Figure 2A–B). Thus, Itk-deficient CD8 cells can be selected on the thymic stroma, although their selection is less efficient. Notably, the Itk-deficient CD8 SP cells that developed via recognition of MHC class I molecules on radioresistant stromal cells had reduced surface levels of CD44 and CD122, similar to conventional and not innate CD8 T cells (Figure 2A). These results argue that Itk is required for efficient selection on the thymic stroma, and moreover, that selection on hematopoietic cells is required for development of the innate-type CD8 cells in Itk-deficient mice.

Figure 2

Selection on thymic stroma prevents development of innate phenotypes of Itk^−/− CD8⁺ T cells

SAP deficiency prevents selection of innate-like CD8 cells in Itk^−/− mice

To further understand the requirements for development of the innate-like CD8 cells in Itk^−/− mice, we considered what signals provided by hematopoietic cells may affect development of innate cell lineages. Hematopoietic cells express a variety of costimulatory molecules known to contribute to T cell activation. The SLAM family receptors are one such family of immunomodulatory receptors expressed on hematopoietic cells that mediate cellular signals through homotypic interactions (Ma et al., 2007). Recent studies have revealed that SAP, a small SH2 domain-containing adaptor that mediates signaling through SLAM family receptors, is strictly required for development of NKT cells, which are also selected on hematopoietic cells (Chung et al., 2005; Nichols et al., 2005; Pasquier et al., 2005). We therefore asked whether SAP is required for the development of the innate-like CD8 T cells in Itk^−/− mice.

To address this question, we interbred Itk-deficient and SAP-deficient mice. Remarkably, SAP deficiency decreased both the percentage and number of CD8 SP thymocytes in Itk^−/− mice and completely prevented the innate-like phenotypes of the CD8 cells as determined by expression of CD44 and CD122 (Figure 3A fourth row). The percentage of CD8 SP cells producing IFN-γ upon ex vivo stimulation was also dramatically reduced in Itk^−/−SAP^−/− CD8⁺ T cells compared with Itk^−/− CD8⁺ T cells (Figure 3A, right panel).

Figure 3

SAP is required for the development of innate-like CD8 T cells in Itk^−/− mice

Development of the innate cell properties in CD8 cells in Itk-deficient mice has been reported to correlate with expression of the T-box transcription factor Eomesodermin (Atherly et al., 2006), which induces expression of memory cell markers including CD122 and CD44 (Intlekofer et al., 2005). CD8 cells in Itk-deficient mice have high levels of Eomesodermin mRNA. To evaluate whether SAP deficiency affects expression of Eomesodermin, we sorted thymic cell populations from Itk^−/−, Itk^−/−SAP^−/− and control mice. SAP deficiency strongly reduced expression of Eomesodermin in Itk^−/− CD8 SP cells (Figure 3C), suggesting that SAP-mediated signals participate in pathways leading to the induction of Eomesodermin. Together, these data suggest that SAP-mediated signaling is required for the development of the innate-like phenotypes of CD8 T cells in Itk^−/− mice.

SAP is required for selection on hematopoietic cells

SAP deficiency could prevent development of the innate-type CD8 cells in Itk-deficient mice either by affecting selection on hematopoietic cells or by preventing the development of the innate characteristics of cells selected on hematopoietic cells. Analyses of thymic cell populations in Itk^−/−SAP^−/− mice revealed decreased percentages of SP CD8 cells in the thymus compared to Itk^−/− mice (Figure 3A), similar to what we observed in the Itk^−/−B2m^−/− → WT bone marrow chimeras (Figure 2). Moreover, CD8 SP cells in Itk^−/−SAP^−/− mice included more immature (HSA^hiTCR^lo) cells that were CD5^lo (Figure 3A) resulting in lower percentages and numbers of mature CD8 cells compared to WT mice (Figure 3B). Thus, the thymic profiles in Itk^−/−SAP^−/− mice resemble those of chimeras in which selection was fixed on the thymic stroma. These results suggested that SAP-deficiency may prevent the selection of the innate CD8 cell population on hematopoietic cells in Itk^−/− mice.

To address specifically whether SAP-deficiency affected selection of Itk^−/− CD8 cells on hematopoietic cells, we performed transfers of WT, Itk^−/− and Itk^−/−SAP^−/− bone marrow into B2m^−/− mice. While transfers of Itk-deficient bone marrow into B2m-deficient mice permitted development of a large population of CD8 cells, transfer of Itk^−/−SAP^−/− bone marrow gave rise to only low percentages and numbers of CD8 cells that did not appear to be mature based on TCR and HSA levels (Figure 4A, B and data not shown). Thus, in the absence of SAP, Itk^−/− cells cannot be selected efficiently on hematopoietic cells. These data suggest that SAP is required for the selection of the innate-like CD8 cells on hematopoietic cells in Itk-deficient mice.

CD28 deficiency also prevents development of innate cell lineages in Itk-deficient mice

One of the other key costimulatory molecules involved in T cell function is CD28, which is critical for full activation of mature T cells. Nonetheless, the role of CD28 in the thymus is relatively poorly understood. CD28’s interactions with B7-1 and B7-2 are required for development of CD4⁺CD25⁺FoxP3⁺ T cells, an important regulatory T cell lineage (Liston and Rudensky, 2007; Salomon et al., 2000; Tai et al., 2005; Tang et al., 2003). Other evidence suggests that signals from hematopoietic cells via CD28 provide distinct signals that may be associated with cell deletion and negative selection (Amsen and Kruisbeek, 1996; Buhlmann et al., 2003; Kishimoto et al., 1996; Lucas and Germain, 2000; Punt et al., 1994; Teh and Teh, 2001). In contrast, yet other data demonstrate that CD28 influences the efficiency of positive selection in the HY TCR transgenic system (Vacchio et al., 2005).

To determine whether CD28 contributes to the development of innate CD8 cells in Itk-deficient mice, we interbred Itk^−/− and CD28^−/− mice. Similar to Itk^−/−SAP^−/− mice, CD28 deficiency decreased the development of non-conventional CD8 cells in Itk^−/−CD28^−/− mice: Itk^−/−CD28^−/− SP CD8 cells exhibited reduced CD44 and CD122 levels and decreased IFN-γ production, although these phenotypes were more variable than in Itk^−/−SAP^−/− mice (Figure 5A, fourth row and Supplemental Figure 2). Similarly, Itk^−/−CD28^−/− CD8 SP cells had decreased expression of Eomesodermin (Figure 5C). However, CD28 deficiency did not decrease either the percentages or numbers of thymic CD8 SP cells (Figure 5A and B), in contrast to the phenotypes observed in Itk^−/−SAP^−/− mice. Instead, an increase in CD4 SP cells was observed. Thus, CD28 deficiency appeared to improve selection of both CD4 and CD8 conventional SP cells, while preventing the appearance of CD8 cells with innate characteristics. Nonetheless, CD28 deficiency did not increase expression of CD5, a marker that reflects TCR signaling strength (Azzam et al., 2001), suggesting that CD28 deficiency did not improve selection by affecting TCR signaling.

Figure 5

CD28 deficiency prevents innate CD8 T cell phenotype in Itk^−/− mice

To address whether the effects of CD28 were mediated by interactions with B7-1 and B7-2 expressed on the thymic stroma or on hematopoietic cells, B7-1^−/−B7-2^−/− mice were used as recipient mice for bone marrow transfers from Itk^−/− or WT donor mice. Transfer of Itk^−/− bone marrow cells into B7-1^−/−B7-2^−/− mice still gave rise to CD8 cells with innate phenotypes, as evidenced by high expression of CD44 and CD122 as well as low HSA levels (Figure 6A). These CD8 cell populations were similar to those seen in Itk^−/− mice or Itk^−/− → WT bone marrow chimeras (Figure 1 and data not shown). Similar results were obtained with bone marrow transfers into CD28-deficient hosts (data not shown). These results suggest that CD28 does not require B7-1 or B7-2 expressed on the thymic stroma to provide signals for the generation of innate-type lymphocytes.

Figure 6

CD28-B7 costimulation on hematopoeitic cells is required for development of innate CD8 cell phenotypes

To further evaluate how CD28 costimulation affects development of innate cell lineages, we performed bone marrow transfers into B2m^−/− recipients. Bone marrow transfer experiments using Itk^−/−CD28^−/− donors into B2m^−/− recipients revealed that CD28 deficiency on hematopoietic cells still permitted development of CD8 cells at numbers similar to that seen in Itk^−/− → B2m^−/− chimeras (Figure 6B–C). These findings contrast with those in Itk^−/−SAP^−/− → B2m^−/− bone marrow transfers, where very few CD8 cells developed (Figure 4). Nonetheless, the CD8 cells that developed in Itk^−/−CD28^−/− → B2m^−/− bone marrow transfers were CD44^loCD122^lo, ie they did not resemble innate cells, despite their selection on hematopoietic cells (Figure 6B). Thus, CD28 deficiency on hematopoietic cells was sufficient to prevent the innate phenotypes of the CD8 cells in Itk^−/− mice.

Together, these results indicate that CD28-B7 signals are not required for selection of CD8 cells on hematopoietic cells in Itk-deficient mice, but are important for the development of an innate cell program in cells selected on hematopoietic cells (Figure 6B). Thus, Itk, SAP, and CD28 influence the development of innate cells by distinct mechanisms: SAP and Itk reciprocally regulate selection of innate CD8 cells on hematopoietic cells versus the thymic stroma, whereas costimulation through CD28 promotes the acquisition of innate-type phenotypes in hematopoietically-selected cells (Figure 7).

Figure 7

A model for the development of conventional and innate-like T cell lineages

DISCUSSION

We present here the results of experiments examining the requirements for selection of innate-like CD8 T cell populations in mice deficient in Itk. These animals develop a large population of innate-type CD8 cells that resemble NKT and other cells selected on MHC class Ib. Our findings demonstrate that development of these innate-type cells requires selection on hematopoietic cells and is dependent on the adaptor molecule SAP, suggesting that homotypic interactions between SLAM family members may be involved in this process. The similarities of these requirements with those observed for NKT cells suggest that these may be part of common characteristics of innate cell lineages selected on hematopoietic cells. Moreover, we find that CD28 is required for full development of these innate cell characteristics upon selection on hematopoietic cells, raising the possibility that CD28 costimulation plays a role in the maturation of other innate-cell lineages.

Our data provide insight into the requirements for selection of innate-cell lineages. Importantly, our data clearly demonstrate that selection on hematopoietic cells plays a fundamental role in determining the phenotypic characteristics of these cells, a finding that had been previously suggested by examination of the few CD8 cells selected in K^bD^b-deficient mice (Urdahl et al., 2002) and the CD4 cells selected in CIITA transgenic mice (Li et al., 2007). Our findings further argue that signaling pathways dependent on SAP are a critical part of this selection pathway directed by hematopoietic cells. It should be noted that not all innate lymphocyte lineages may follow these rules--the mechanisms driving selection of CD8αα lineages, which also exhibit characteristics of innate cells, remain unclear (Lambolez et al., 2007). Moreover, how these lineages relate to other regulatory T cell lineages in the thymus remains an open question.

Our results suggest that Itk may be one of the few molecules that specifically affects selection of conventional versus non-conventional or innate-type lymphocytes. Other molecules implicated in TCR signaling, including proximal tyrosine kinases and adaptors such as Lck, ZAP-70, LAT and SLP-76 seem to affect development of both conventional and innate lymphocyte lineages, perhaps due to their more profound effects on pre-TCR and TCR signaling (Starr et al., 2003). Yet other molecules, including SAP, Fyn, PKC-θ, NFκB1, Eomesodermin and T-bet help define pathways regulating non-conventional, innate-like lineages such as NKT cells (Chung et al., 2005; Eberl et al., 1999; Gadue et al., 1999; Godfrey and Berzins, 2007; Intlekofer et al., 2005; Nichols et al., 2005; Pasquier et al., 2005; Schmidt-Supprian et al., 2004; Sivakumar et al., 2003; Stanic et al., 2004). In contrast, Itk appears to be selectively required for effective differentiation of conventional T lineage cells (Atherly et al., 2006; Berg, 2007; Broussard et al., 2006).

In particular, our data suggest that Itk-deficiency specifically prevents efficient positive selection of mature SP T cells on the thymic stroma so that in the absence of this kinase, thymocytes are preferentially selected on hematopoietic cells. Indeed, when selection is forced to occur on the thymic stroma, as in Itk^−/−B2m^−/− bone marrow transfers into WT mice, fewer mature CD8 cells develop. However, in Itk^−/− mice, perhaps because selection on hematopoietic cells can still take place, development of non-conventional cells becomes a major route of CD8 cell development. The large numbers of innate-type CD8 cells that develop in Itk^−/− mice could result from expansion secondary to the lower numbers of mature conventional SP cells. However, the observation that large numbers of CD8 SP cells develop when Itk^−/− but not WT bone marrow is transferred into B2m^−/− and B2m^−/−MHC class II^−/− mice (Broussard et al., 2006) suggests that Itk-deficiency actually leads to increased selection on hematopoietic cells. These results raise the possibility that Itk may function as a negative regulator of signaling pathways that are required for development of innate cells, such as those downstream of SLAM family receptors. The differential effects of Itk on the selection of conventional CD8 cells on the thymic stroma versus innate T cells selected on hematopoietic cells, further suggests that Itk-dependent pathways may serve as a rheostat to determine the balance of adaptive versus innate T cell lineages. Why Itk deficiency specifically affects selection on thymic stroma remains an important question and may reflect the relative importance of TCR vs costimulatory signals for the development of these distinct T cell lineages. It is of interest that the development of innate CD8 cells in Itk^−/− mice can be prevented by either increasing TCR signal strength or by reducing costimulatory signals (Atherly et al., 2006; Broussard et al., 2006) and this manuscript).

One question that arises from this work is why CD8 cells appear to be the primary cells affected in Itk^−/− mice. Analyses of MHC class I and class II-restricted TCR transgenic mice demonstrate that Itk is required for efficient positive selection of both CD4 and CD8 T cells, yet only a large population of innate CD8 cells develop. These observations could imply that Itk is specifically required for the development of conventional CD8 cells (Berg, 2007). However, it is also possible that the specific development of innate CD8 (but not CD4) cells results from the lack of appreciable MHC class II expression on murine thymocytes. Although we do not know which hematopoietic cells are responsible for selection of the innate-type CD8 cells in Itk-deficient mice, double positive thymocytes are the major hematopoietic cells in the thymus and have been shown to mediate selection of NKT cells. Thus, although selection of conventional CD4 cells on the thymic stroma is also decreased, only a small population of CD4 cells with innate characteristics develop in Itk^−/− mice, which may be selected on cortical dendritic cells. If MHC class II molecules were expressed on thymocytes, as in the recently described CIITA transgenic mice (Choi et al., 2005; Li et al., 2005), a larger population of CD4⁺ cells with innate cell phenotypes might be observed. These observations also raise the possibility that Itk-dependent pathways might normally prevent efficient selection of T cells on hematopoietic cells, thereby guaranteeing that the majority of mature T cells represent the adaptive arm of the immune system.

Our results also highlight the role of SAP and SLAM family members in the development of innate cell lineages. The SLAM family receptors are emerging as important immunoregulatory receptors that mediate interactions between hematopoeitic cells to regulate T helper cell polarization, humoral immunity and the development of autoantibodies, host responses to pathogens, and NKT cell development (Ma et al., 2007). Our results provide further evidence that signaling pathways involving SAP are required not only for selection of NKT cells but also for other innate cells selected on hematopoietic cells. Our results suggest that Itk and SAP play complementary roles as determinants of the balance of conventional and innate T cell lineages, respectively. Which SLAM family members participate in hematopoietic selection and whether they are the same for all innate cell lineages remains an important question. Furthermore, whether SAP and SLAM family receptors are required for later stages of development of innate cell lineages remains unknown.

Nonetheless, SAP-associated receptors do not appear to be the only costimulatory pathways affecting development of these innate cell lineages. While the role of CD28 in thymic development has been controversial, a growing body of data indicates that CD28 does participate in thymic development, particularly of CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Liston and Rudensky, 2007; Salomon et al., 2000; Tai et al., 2005; Tang et al., 2003). In this respect it is of interest that CD8⁺CD122⁺ cells have also been described to have regulatory T cell function (Rifa'i et al., 2004), suggesting parallels between these two lineages. It is also of interest that some data suggest that CD28 and B7’s may not be required for selection of CD4⁺CD25⁺ cells but rather their maturation and acquisition of FoxP3 expression and regulatory function (Liston and Rudensky, 2007). Similarly, our data demonstrate that CD28 is not required for selection of CD8 cells on hematopoietic cells, but rather for the full acquisition of the innate cell program, including the induction of high levels of Eomesodermin. Indeed, our data raise the possibility that maturation of other innate cell lineages may also be partially dependent on CD28. Whether there are similar requirements for the expression of Eomesodermin and the related transcription factor T-bet in other cell lineages remains to be seen. The role of CD28 may be more complex, however, because CD28 deficiency appears to increase the selection of both CD4 and CD8 conventional-appearing T cells in Itk-deficient mice (Figure 5A and data not shown), perhaps reflecting its possible role in negative selection. Indeed, one potential reason why Itk-deficient mice may develop this large population of innate CD8 cells is that cells that would normally undergo negative selection from agonist peptides in the thymus may not be efficiently deleted (Schaeffer et al., 2000). Nonetheless, neither CD28 nor SAP appear to affect TCR signaling since CD8 cells in Itk^−/−CD28^−/− and Itk^−/−SAP^−/− mice still exhibit low levels of CD5 (Figure 3A and Figure 5A). It should also be noted that neither SAP- nor CD28-deficiency increased the low numbers of total thymocytes in Itk^−/− mice, which are likely to result from Itk’s effects on pre-TCR signaling (Figure 3B, Figure 5B, data not shown and (Lucas et al., 2003).

Together our data help define pathways that are differentially required for T cell selection on thymic stroma versus hematopoietic cells, and the generation of conventional and innate T cell lineages that are required for proper immune homeostasis and responses to infections. In particular, our results suggest that costimulation is critical for the development of innate T cell phenotypes in the thymus: whereas SAP and Itk reciprocally regulate selection of innate CD8 cells on hematopoietic cells, costimulation through CD28 influences the maturation and acquisition of innate-type phenotypes on cells selected on hematopoietic cells. The similarities of these observations with those seen for CD-1d-restricted NKT cells suggests these findings may help define common requirements for the development of innate T lymphocyte lineages.

Experimental Procedures

Mice

Itk^−/− and SAP^−/− mice on the C57BL/6 background are previously described (Liao and Littman, 1995; Czar et al, 1998). K^b−/−D^b−/− and B2m^−/− mice were obtained from Taconic, and C57BL/6, CD28^−/− and B7-1^−/−B7-2^−/− mice were obtained from Jackson Laboratory. All animals were bred and maintained under specific pathogen-free conditions in the National Human Genome Research Institute (NHGRI) Animal Facility and experiments were performed according to NHGRI animal care and use committee guidelines.

Thymocyte preparation and sorting

Single cell suspensions of thymocytes were prepared from age-matched mice between 6–10 weeks old. For total thymocyte sorting, cells were stained with PerCP-Cy5.5-anti-CD4 (RM4-5) and APC-anti-CD8 (53-6.7) antibodies (BD Biosciences, San Diego, CA), and sorted for CD4⁻CD8⁻, CD4⁺CD8⁺, CD4⁺CD8⁻, and CD4⁻CD8⁺ populations.

Flow cytometry

Antibodies used for the staining are as follow: FITC-TCRβ (H57–597), PE-β₇ integrin (M293), PE-CD62L (MEL-14), PE-Cy5-HSA/CD24, Biotin-CD122 (TM-β1), Streptavidin PE-Cy7, APC-CD5 (53-7.3), APC-CD44 (IM7), APC-Alexa Fluoro 750-CD8, Pacific Blue-CD4. Cells were acquired by FACSCalibur or LSRII (BD Biosciences), and data analyzed using Flowjo software (Tree Star, Inc. Ashland, OR).

Ex vivo stimulation and intracellular cytokine staining

Thymocytes and splenocytes were stimulated ex vivo with PMA and Ionomycin in the presence of Brefeldin A (BD Biosciences) as previously described (Broussard et al., 2006). After 4 h, cells were harvested and stained with FITC-anti-CD44 (IM7), Pacific Blue-anti-CD4 and PerCP-Cy5.5-anti-CD8 antibodies. Cells were fixed with 4% PFA and permiabilized with PBS containing 0.1% BSA and 0.05% Triton X-100. Intracellular cytokines were stained with PE-anti-IL-4 (11B11) and APC-anti-IFN-γ (XMG1.2) antibodies.

Bone marrow cell transfers

Bone marrow cells were prepared from femur and tibiae as previously described (Broussard et al., 2006). Recipient mice were sublethally irradiated at 950–1000 rad, depending on the age and body size, and 1.5–2 × 10⁷ cells were transferred intravenously. Thymocytes and splenocytes for flow cytometric analyses were harvested 7 weeks after the transfer and thymocytes for real time PCR analyses were harvested 5 weeks after the transfer.

RNA isolation and real time PCR

Total and sorted thymocytes were lysed in TRIzol (Invitrogen) and total RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was synthesized from total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative real time PCR was performed using a 7900 sequence detection system (Applied Biosystems). TaqMan Endogenous Control for Eukaryotic 18S rRNA (VIC-MGB probe) and the primer and probe sets for murine Eomesodermin (Mm01351988_m1, FAM-MGB probe) were from Applied Biosystems. Results were normalized to 18S rRNA and expressed relative to WT levels (WT=1).

Supplementary Material

Supplemental Figure 1. (A) Variability of the thymic profiles in K^b−/−D^b−/− mice in one representative experiment.

(B) Profiles of peripheral lymph nodes from WT, Itk^−/−, K^b−/−D^b−/−, and Itk^−/−K^b−/−D^b−/− mice. WT profiles are in gray.

Supplemental Figure 2. Variability of the thymic profiles in Itk^−/−CD28^−/− mice.

Click here to view.^{(124K, pdf)}

Acknowledgements

The authors thank Drs. R. Germain and R. Bosselut for helpful comments and A. Venegas for technical assistance.

Footnotes

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

Amsen D, Kruisbeek AM. CD28-B7 interactions function to costimulate clonal deletion of double-positive thymocytes. Int Immunol. 1996;8:1927–1936. [PubMed]
Atherly LO, Lucas JA, Felices M, Yin CC, Reiner SL, Berg LJ. The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells. Immunity. 2006;25:79–91. [PubMed]
Azzam HS, DeJarnette JB, Huang K, Emmons R, Park CS, Sommers CL, El-Khoury D, Shores EW, Love PE. Fine tuning of TCR signaling by CD5. J Immunol. 2001;166:5464–5472. [PubMed]
Baldwin TA, Hogquist KA, Jameson SC. The fourth way? Harnessing aggressive tendencies in the thymus. J Immunol. 2004;173:6515–6520. [PubMed]
Behar SM, Porcelli SA. CD1-restricted T cells in host defense to infectious diseases. Curr Top Microbiol Immunol. 2007;314:215–250. [PubMed]
Bendelac A. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med. 1995;182:2091–2096. [PMC free article] [PubMed]
Berg LJ. Signalling through TEC kinases regulates conventional versus innate CD8(+) T-cell development. Nat Rev Immunol. 2007;7:479–485. [PubMed]
Berg LJ, Finkelstein LD, Lucas JA, Schwartzberg PL. Tec family kinases in T lymphocyte development and function. Annu Rev Immunol. 2005;23:549–600. [PubMed]
Bix M, Coles M, Raulet D. Positive selection of V beta 8+ CD4-8- thymocytes by class I molecules expressed by hematopoietic cells. J Exp Med. 1993;178:901–908. [PMC free article] [PubMed]
Broussard C, Fleischecker C, Horai R, Chetana M, Venegas AM, Sharp LL, Hedrick SM, Fowlkes BJ, Schwartzberg PL. Altered development of CD8+ T cell lineages in mice deficient for the tec kinases Itk and Rlk. Immunity. 2006;25:93–104. [PubMed]
Buhlmann JE, Elkin SK, Sharpe AH. A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection. J Immunol. 2003;170:5421–5428. [PubMed]
Cannons JL, Yu LJ, Hill B, Mijares LA, Dombroski D, Nichols KE, Antonellis A, Koretzky GA, Gardner K, Schwartzberg PL. SAP regulates TH2 differentiation and PKCθ-mediated activation of NF-κB1. Immunity. 2004;21:693–706. [PubMed]
Choi EY, Jung KC, Park HJ, Chung DH, Song JS, Yang SD, Simpson E, Park SH. Thymocyte-thymocyte interaction for efficient positive selection and maturation of CD4 T cells. Immunity. 2005;23:387–396. [PubMed]
Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R. Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J Immunol. 2005;174:3153–3157. [PubMed]
Das G, Sheridan S, Janeway CA., Jr The source of early IFN-gamma that plays a role in Th1 priming. J Immunol. 2001;167:2004–2010. [PubMed]
Dubois S, Waldmann TA, Muller JR. ITK and IL-15 support two distinct subsets of CD8+ T cells. Proc Natl Acad Sci U S A. 2006;103:12075–12080. [PubMed]
Eberl G, Lowin-Kropf B, MacDonald HR. Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice. J Immunol. 1999;163:4091–4094. [PubMed]
Finkelstein LD, Schwartzberg PL. Tec kinases: shaping T-cell activation through actin. Trends Cell Biol. 2004;14:443–451. [PubMed]
Gadue P, Morton N, Stein PL. The Src family tyrosine kinase Fyn regulates natural killer T cell development. J Exp Med. 1999;190:1189–1196. [PMC free article] [PubMed]
Godfrey DI, Berzins SP. Control points in NKT-cell development. Nat Rev Immunol. 2007;7:505–518. [PubMed]
Gomez-Rodriguez J, Readinger JA, Viorritto IC, Mueller KL, Houghtling RA, Schwartzberg PL. Tec kinases, actin, and cell adhesion. Immunol Rev. 2007;218:45–64. [PubMed]
Intlekofer AM, Takemoto N, Wherry EJ, Longworth SA, Northrup JT, Palanivel VR, Mullen AC, Gasink CR, Kaech SM, Miller JD, et al. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol. 2005;6:1236–1244. [PubMed]
Kerksiek KM, Busch DH, Pilip IM, Allen SE, Pamer EG. H2- M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp Med. 1999;190:195–204. [PMC free article] [PubMed]
Kishimoto H, Cai Z, Brunmark A, Jackson MR, Peterson PA, Sprent J. Differing roles for B7 and intercellular adhesion molecule-1 in negative selection of thymocytes. J Exp Med. 1996;184:531–537. [PMC free article] [PubMed]
Lambolez F, Kronenberg M, Cheroutre H. Thymic differentiation of TCR alpha beta(+) CD8 alpha alpha(+) IELs. Immunol Rev. 2007;215:178–188. [PubMed]
Li W, Kim MG, Gourley TS, McCarthy BP, Sant'Angelo DB, Chang CH. An alternate pathway for CD4 T cell development: thymocyte-expressed MHC class II selects a distinct T cell population. Immunity. 2005;23:375–386. [PubMed]
Li W, Sofi MH, Yeh N, Sehra S, McCarthy BP, Patel DR, Brutkiewicz RR, Kaplan MH, Chang CH. Thymic selection pathway regulates the effector function of CD4 T cells. J Exp Med. 2007;204:2145–2157. [PMC free article] [PubMed]
Liao XC, Littman DR. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity. 1995;3:757–769. [PubMed]
Liston A, Rudensky AY. Thymic development and peripheral homeostasis of regulatory T cells. Curr Opin Immunol. 2007;19:176–185. [PubMed]
Lucas B, Germain RN. Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement. J Immunol. 2000;165:1889–1895. [PubMed]
Lucas JA, Atherly LO, Berg LJ. The absence of Itk inhibits positive selection without changing lineage commitment. J Immunol. 2002;168:6142–6151. [PubMed]
Lucas JA, Miller AT, Atherly LO, Berg LJ. The role of Tec family kinases in T cell development and function. Immunol Rev. 2003;191:119–138. [PubMed]
Ma CS, Nichols KE, Tangye SG. Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annu Rev Immunol. 2007;25:337–379. [PubMed]
Nichols KE, Hom J, Gong SY, Ganguly A, Ma CS, Cannons JL, Tangye SG, Schwartzberg PL, Koretzky GA, Stein PL. Regulation of NKT cell development by SAP, the protein defective in XLP. Nat Med. 2005;11:340–345. [PubMed]
Ohteki T. Critical role for IL-15 in innate immunity. Curr Mol Med. 2002;2:371–380. [PubMed]
Ohteki T, MacDonald HR. Major histocompatibility complex class I related molecules control the development of CD4+8- and CD4-8- subsets of natural killer 1.1+ T cell receptor-alpha/beta+ cells in the liver of mice. J Exp Med. 1994;180:699–704. [PMC free article] [PubMed]
Pasquier B, Yin L, Fondaneche MC, Relouzat F, Bloch-Queyrat C, Lambert N, Fischer A, de Saint-Basile G, Latour S. Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J Exp Med. 2005;201:695–701. [PMC free article] [PubMed]
Punt JA, Osborne BA, Takahama Y, Sharrow SO, Singer A. Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28. J Exp Med. 1994;179:709–713. [PMC free article] [PubMed]
Rifa'i M, Kawamoto Y, Nakashima I, Suzuki H. Essential roles of CD8+CD122+ regulatory T cells in the maintenance of T cell homeostasis. J Exp Med. 2004;200:1123–1134. [PMC free article] [PubMed]
Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity. 2000;12:431–440. [PubMed]
Schaeffer EM, Broussard C, Debnath J, Anderson S, McVicar DW, Schwartzberg PL. Tec family kinases modulate thresholds for thymocyte development and selection. J Exp Med. 2000;192:987–1000. [PMC free article] [PubMed]
Schmidt-Supprian M, Tian J, Grant EP, Pasparakis M, Maehr R, Ovaa H, Ploegh HL, Coyle AJ, Rajewsky K. Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-kappaB activation. Proc Natl Acad Sci U S A. 2004;101:4566–4571. [PubMed]
Seaman MS, Wang CR, Forman J. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J Immunol. 2000;165:5192–5201. [PubMed]
Sivakumar V, Hammond KJ, Howells N, Pfeffer K, Weih F. Differential requirement for Rel/nuclear factor kappa B family members in natural killer T cell development. J Exp Med. 2003;197:1613–1621. [PMC free article] [PubMed]
Stanic AK, Bezbradica JS, Park J-J, Kaer LV, Boothby MR, Joyce S. Cutting Edge: The ontogeny and function of Vα14Jα18 natural T lymphocytes require signal processing by protein kinase C θ and NF-κB. J Immunol. 2004;172:4667–4671. [PubMed]
Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu Rev Immunol. 2003;21:139–176. [PubMed]
Tai X, Cowan M, Feigenbaum L, Singer A. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat Immunol. 2005;6:152–162. [PubMed]
Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK, Zheng XX, Strom TB, Bluestone JA. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol. 2003;171:3348–3352. [PubMed]
Teh HS, Teh SJ. The affinity/avidity and length of exposure to the deleting ligand determine dependence on CD28 for the efficient deletion of self-specific CD4+CD8+ thymocytes. Cell Immunol. 2001;207:100–109. [PubMed]
Urdahl KB, Sun JC, Bevan MJ. Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol. 2002;3:772–779. [PMC free article] [PubMed]
Vacchio MS, Williams JA, Hodes RJ. A novel role for CD28 in thymic selection: elimination of CD28/B7 interactions increases positive selection. Eur J Immunol. 2005;35:418–427. [PubMed]
Yoshimoto T, Paul WE. CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J Exp Med. 1994;179:1285–1295. [PMC free article] [PubMed]