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The lineage fate of developing thymocytes is determined by the persistence or cessation of TCR signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We now report that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during MHC-II specific positive selection and is especially critical for error-free lineage choice in TCR transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurs only for endogenously encoded CD4 coreceptors as CD4 transgenes are downregulated during positive selection, disrupting MHC-II specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC-II specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.
The fate of T cells developing in the thymus is determined by the specificity of their T cell antigen receptor (TCR). Thymocytes at the CD4+CD8+ (double positive, DP) stage of development are the first cells in the thymus to express fully assembled αβTCR complexes and either undergo positive selection or cell death (Starr et al., 2003). Positively selected DP thymocytes ultimately differentiate into either CD4+ helper or CD8+ cytolytic T cells, with thymocytes bearing TCR specific for MHC class II (MHC-II) ligands differentiating into CD4+ helper T cells and thymocytes bearing TCR specific for MHC class I (MHC-I) ligands differentiating into CD8+ cytolytic T cells (Starr et al., 2003). The cellular and molecular mechanisms by which positively selected thymocytes determine the ligand specificity of their TCR and their appropriate lineage fate have been the subject of intense investigation for years but have recently been clarified (Hedrick, 2008; Singer et al., 2008).
It is now understood that TCR mediated positive selection signals induce CD4+CD8+ double positive (DP) thymocytes to downregulate CD8 coreceptor expression and to convert into CD4+CD8lo intermediate thymocytes (Bosselut et al., 2003; Brugnera et al., 2000). Positively selected CD4+CD8lo intermediate thymocytes then differentiate into either CD4 or CD8 lineage T cells based on whether TCR signaling persists or ceases (Brugnera et al., 2000; Sarafova et al., 2005; Singer, 2002; Singer and Bosselut, 2004; Yasutomo et al., 2000). Persistent TCR signaling induces intermediate thymocytes to upregulate expression of ThPOK, the CD4 lineage-specifying transcription factor, and to differentiate into CD4+ helper T cells (He et al., 2005; He et al., 2008; Sun et al., 2005); whereas cessation of TCR signaling results in expression of RUNX3, a CD8 lineage-specifying transcription factor, and differentiation of intermediate thymocytes into CD8+ cytolytic T cells (Setoguchi et al., 2008; Taniuchi et al., 2004). Thus, CD4 lineage choice requires persistent TCR signaling, whereas disrupted TCR signaling results in CD8 lineage choice (Sarafova et al., 2005; Singer, 2002; Singer et al., 2008; Singer and Bosselut, 2004).
As CD4 and CD8 coreceptors stabilize and enhance MHC-specific TCR signals, quantitative changes in surface coreceptor expression during positive selection may affect TCR signaling duration and, consequently, CD4/CD8 lineage choice. Indeed, surface CD8 coreceptor expression declines on positively selected thymocytes as a result of decreased CD8 gene transcription, disrupting MHC-I specific TCR signaling and promoting CD8 lineage choice (Bosselut et al., 2003; Cibotti et al., 2000). In contrast to the kinetics of CD8 coreceptor expression during positive selection which is well understood, it is not known if surface CD4 coreceptor expression changes during positive selection and whether such changes affect the duration of MHC-II specific TCR signaling to contribute to CD4 lineage choice. Curiously, past experiments with transgenic CD4 coreceptors revealed that persistent CD4 coreceptor expression had a paradoxical impact on CD4/CD8 lineage choice in that it resulted in the aberrant generation of MHC-II specific CD8+ T cells (Davis et al., 1993). These studies were interpreted as supportive of the stochastic/selection model of CD4/CD8 lineage choice, but subsequent experimental data make the existence of a stochastic mechanism underlying CD4/CD8 lineage choice no longer tenable (Adoro et al., 2008; Itano and Robey, 2000; Singer et al., 2008). Consequently, the paradoxical observation that expression of transgenic CD4 coreceptors induces a number of MHC-II specific thymocytes to differentiate into CD8+ T cells remains unexplained.
The present study was undertaken to determine if surface CD4 coreceptor expression undergoes changes during MHC-II specific positive selection and if such changes affect CD4/CD8 lineage choice. We now report that endogenous CD4 coreceptor expression is in fact upregulated on MHC-II signaled thymocytes during positive selection and that such CD4 upregulation is essential for error-free lineage choice, regardless of the absolute number or signaling strength of the CD4 coreceptors. In the absence of CD4 upregulation, MHC-II specific lineage choice is highly error-prone, a situation exacerbated in TCR transgenic mice whose thymocytes compete for a single selecting ligand. Thus, the kinetics of CD4 coreceptor expression during positive selection determines the integrity of CD4 lineage choice, complementing what is known about the kinetics of CD8 coreceptor expression to reveal an elegant symmetry between coreceptor kinetics and lineage choice.
To examine changes in CD4 coreceptor expression during MHC-II specific positive selection and their effect on MHC-II specific lineage choice, we compared MHC-II specific selection in mice that expressed CD4 coreceptor proteins under the control of either endogenous or transgenic transcriptional regulatory elements (Fig. 1). To generate experimental mice for this study, transgenes encoding CD4 protein under the control of the human CD2 promoter (CD4hCD2) (Van Laethem et al., 2007), the human CD3δ promoter (CD4T3) (Davis et al., 1993; Lee et al., 1992), or the murine E8III enhancer/CD8α promoter (8DP4) (Ellmeier et al., 1998; Sarafova et al., 2005) were introduced into β2moCD4o double knockout mice so that only MHC-II selecting elements and only transgenic CD4 coreceptor proteins were expressed in the thymus (Table 1).
In non-transgenic β2mo mice expressing endogenously encoded CD4 proteins (CD4endo), MHC-II specific selection resulted exclusively in CD4+ T cells, indicating that MHC-II specific lineage choice was error-free (Fig. 1A top left panel). In contrast, MHC-II specific lineage choice in CD4hCD2 and CD4T3 transgenic mice was error-prone as they contained substantial frequencies (13% and 5%) of CD8+ lymph node (LN) T cells (Fig. 1A, top middle panels). CD8+ LN T cells in CD4hCD2 and CD4T3 mice appeared as CD4+CD8+ because transgenic hCD2 and hCD3δ transcriptional control elements remained active in CD8+ T cells (Fig. 1A, top middle panels). Notably, lineage errors did not require persistent CD4 transgene expression as MHC-II specific selection was even more error-prone in a transgenic mouse (8DP4) in which transgenic CD4 expression was terminated early during positive selection (Fig. 1A, top right panel). Error-prone MHC-II specific lineage differentiation in 8DP4 mice resulted in mature T cells that were phenotypically CD4− and appeared as either CD4−CD8− or CD4−CD8+ cells (Sarafova et al., 2005). Thus, MHC-II specific lineage choice was error-free in non-transgenic mice but was error-prone in all three of the CD4 transgenic mice examined despite marked differences in duration of CD4 transgene expression. Similarly, MHC-II specific lineage choice by monoclonal AND thymocytes (Kaye et al., 1989) which are Vα11+Vβ3+ resulted only in CD4+ T cells when AND thymocytes expressed endogenously encoded CD4 coreceptor proteins, but was highly error-prone when AND thymocytes expressed transgene encoded CD4 coreceptor proteins (Fig. 1A bottom panels).
To understand the basis for error-free versus error-prone CD4 lineage choice, we considered that CD4 lineage choice requires MHC-II specific TCR signaling to persist during differentiation of signaled DP thymocytes into CD4+CD8lo intermediate thymocytes, whereas any disruption in TCR signaling during this developmental step results in CD8 lineage choice (Brugnera et al., 2000; Singer, 2002). Consequently, we quantified changes in surface CD4 expression during differentiation of signaled DP thymocytes into CD4+8lo intermediate cells. We identified signaled DP thymocytes and intermediate cells without relying on surface CD4 coreceptor expression by introducing into all mice a CD4 reporter (CD4r) transgene that reports endogenous Cd4 transcriptional activity by surface expression of hCD2 proteins (supplementary Fig. S1) (Sawada et al., 1994). Specifically, MHC-II-signaled DP thymocytes were identified as TCRhiCD4r+CD8+ cells; intermediate thymocytes were identified as TCRhiCD4r+CD8lo cells; and CD4SP thymocytes were identified as TCRhiCD4r+CD8− cells.
We first examined thymocytes from 8DP4 mice because the 8DP4 transgene was transcriptionally regulated by E8III-CD8α enhancer elements so that TCR signaling downregulated CD4 surface expression and disrupted MHC-II signaling during differentiation of DP into intermediate thymocytes (Sarafova et al., 2005). As shown in a representative experiment, surface expression of 8DP4 encoded CD4 proteins was dramatically reduced (−88%) during differentiation of signaled DP into intermediate thymocytes as CD4 mean fluorescent intensity (MFI) declined from 132 to 16 (Fig. 1B, top panels), a change best appreciated in the expanded view that was generated by gating individually on each thymocyte subset and overlaying their profiles (Fig. 1B, top right panel). In marked contrast to 8DP4 encoded CD4 proteins, surface expression of endogenously encoded CD4 proteins substantially increased (+61%) during differentiation of signaled DP into intermediate thymocytes as CD4 MFI increased from 450 to 725 (Fig. 1B, middle panels).
To determine if such changes in CD4 expression during MHC-II specific positive selection affected the integrity of CD4 lineage choice, we assessed the frequency of MHC-II selected CD8+ SP thymocytes (Fig. 1C right). We found that β2mo mice expressing 8DP4 encoded CD4 proteins contained a high frequency of MHC-II selected CD8SP thymocytes, whereas β2mo mice expressing endogenously encoded CD4 proteins were essentially devoid of CD8SP thymocytes (Fig. 1C right). Thus endogenous CD4 surface expression increased during differentiation of MHC-II signaled DP into intermediate thymocytes and was associated with error-free CD4 lineage choice, whereas 8DP4 encoded CD4 surface expression decreased on MHC-II signaled thymocytes and was associated with error-prone CD4 lineage choice (Fig. 1C left and right panels).
To determine if CD4 lineage choice was error-prone in 8DP4 mice because CD4 expression was downregulated on MHC-II signaled thymocytes and not because of 8DP4 transgene expression per se, we generated β2mo mice that expressed both the 8DP4 transgene and endogenously encoded CD4 proteins (referred to as 8DP4.CD4endo mice). Examination of 8DP4.CD4endo thymocytes mice revealed that surface CD4 expression increased (+52%) during differentiation of MHC-II signaled DP into intermediate thymocytes (Fig. 1B last row) because upregulation of endogenous CD4 proteins more than compensated for decreased expression of 8DP4 encoded CD4 proteins during MHC-II specific positive selection as CD4 MFI increased from 614 to 933 (Fig. 1B last row). Importantly, we found that 8DP4.CD4endo mice were essentially devoid of CD8SP thymocytes (Fig. 1C), revealing that CD4 lineage choice in 8DP4.CD4endo mice was error-free despite expression of the 8DP4 transgene.
Based on our findings in 8DP4 mice that the kinetics of CD4 expression during MHC-II specific positive selection affects CD4 lineage choice, we predicted that error-prone CD4 lineage choice in CD4 transgenic mice might be due to a failure of transgenic CD4 proteins to be upregulated during MHC-II specific positive selection. To assess this prediction, we quantified changes in surface CD4 expression on DP and intermediate thymocytes from CD4hCD2 and CD4T3 transgenic mice, including an independently derived subline of CD4T3 (CD4T3L2) with lower overall CD4 protein expression (Fig. 2A, and supplementary Figs. S2 and S3). In fact, while surface expression of endogenously encoded CD4 proteins increased during differentiation of signaled DP into intermediate thymocytes, transgene encoded CD4 expression decreased in CD4hCD2, CD4T3, and CD4T3L2 mice (Fig. 2B). A summary of changes in CD4 expression during differentiation of MHC-II signaled DP thymocytes from the various mouse strains are displayed (Fig. 2B, upper left panel), along with frequencies of αβ LNT cells that had adopted the incorrect CD8 lineage fate (Fig. 2B, right panel). In fulfillment of our prediction, this analysis documented that, whereas surface expression of endogenously encoded CD4 proteins increased during differentiation of MHC-II signaled DP thymocytes into intermediate cells and resulted in error-free lineage choice, surface expression of transgene encoded CD4 proteins decreased and resulted in error-prone MHC-II specific lineage choices (Fig. 2B). Surprisingly, MHC-II specific lineage choice was error-prone in all CD4 transgenic mice even though overall CD4 levels on transgenic thymocytes varied widely, ranging from relatively high CD4 levels (CD4hCD2) to intermediate CD4 levels (CD4T3) to low CD4 levels (CD4T3L2) (Fig. 2B, supplementary Fig. S2B). Thus, regardless of the overall level of CD4 coreceptor expression on thymocytes, downregulation of surface CD4 expression during differentiation of signaled DP into intermediate thymocytes results in error-prone MHC-II specific lineage choice.
We also determined CD4 expression on signaled DP and intermediate thymocytes from CD4T3(β2mo) mice expressing both endogenous and transgenic CD4 proteins, since we thought that transcriptional upregulation of endogenous CD4 proteins during MHC-II specific positive selection might be counterbalanced by downregulation of transgenic CD4T3 proteins. In fact, in CD4T3(β2mo) mice overall CD4 surface expression remained essentially unchanged during differentiation of signaled DP into intermediate thymocytes, as well as during their subsequent differentiation into CD4SP cells (Fig. 2C, left panel). Interestingly, MHC-II specific lineage choice in these mice was also error-prone as revealed both by the frequency of αβ LNT cells that were CD8+ (Fig. 2C, right panel) and the presence in the thymus of HSA−TCRβhiCD8+ thymocytes (supplementary Fig. S3A). These results indicate that failure to upregulate surface CD4 expression during differentiation of signaled DP into intermediate thymocytes is sufficient for error-prone MHC-II specific lineage choice.
Since endogenous and transgenic CD4 proteins were transcriptionally regulated by different regulatory elements, the unique upregulation of endogenous CD4 proteins during MHC-II specific positive selection must have been the result of increased Cd4 gene transcription. To verify that the transcription of endogenous Cd4 genes in signaled thymocytes increased during MHC-II specific positive selection, we quantified surface expression of CD4r-encoded hCD2 proteins, as expression of the CD4r transgene tracks endogenous Cd4 gene activity. In fact, expression of CD4r-encoded hCD2 proteins was upregulated on signaled TCRhi thymocytes (Fig. 2B left lower panel), indicating that Cd4 gene transcription progressively increased in signaled thymocytes throughout MHC-II specific positive selection.
Because CD8 lineage choice is specifically associated with upregulation of the transcription factor RUNX3 (Egawa et al., 2007; Sato et al., 2005; Setoguchi et al., 2008; Taniuchi et al., 2002) while CD4 lineage choice is associated with upregulation of the transcription factors ThPOK (He et al., 2005; Sun et al., 2005), TOX (Aliahmad and Kaye, 2008; Wilkinson et al., 2002), and GATA3 (Hernandez-Hoyos et al., 2003; Wang et al., 2008), we quantified expression of these transcription factors in purified intermediate thymocytes during MHC-II specific positive selection (Fig. 3). As expected, expression of the CD4 lineage-associated factors ThPOK, TOX, and GATA3 were upregulated during differentiation of DP into intermediate thymocytes in all mice examined (Fig. 3), with the extent of upregulation greater in intermediate thymocytes expressing higher CD4 levels as a result of quantitatively stronger MHC-II specific positive selection signaling (Fig. 3). In contrast to induction of CD4 lineage-associated factors, MHC-II specific positive selection does not normally induce RUNX3 expression, and we confirmed this point in intermediate thymocytes from β2mo mice expressing only CD4endo coreceptors (Fig. 3). Importantly, however, RUNX3 expression was upregulated in MHC-II selected intermediate thymocytes from mice expressing CD4hCD2 or CD4endo+CD4T3 coreceptors (Fig. 3), i.e. only those mice in which MHC-II specific lineage choice was error-prone as revealed by the presence of HSA−TCRβhiCD8+ thymocytes (supplemental Fig. S3A,B) and CD8+ LNT cells (Fig. 2B,C; supplemental Fig. S3B). Notably, the relatively low expression of RUNX3 in intermediate thymocytes from mice expressing CD4hCD2 or CD4endo+CD4T3 coreceptors is consistent with induction of RUNX3 expression in the relatively few intermediate thymocytes making an erroneous lineage choice. Thus, failure of MHC-II specific positive selection to upregulate surface CD4 expression during differentiation of DP into intermediate thymocytes results in induction of the CD8 lineage-associated factor RUNX3 in intermediate thymocytes and generation of erroneous MHC-II specific CD8+ T cells.
Next, we examined if CD4 coreceptor signal strength affected the integrity of CD4 lineage choice, as it has been suggested that CD4 lineage choice is driven by strong coreceptor signals transduced by the CD4 cytosolic tail (Itano et al., 1996). Consequently, we assessed whether modifications to the cytosolic tail of transgenic CD4 coreceptor proteins affected CD4/CD8 lineage choice. We compared β2moCD4o transgenic mice expressing hCD2-driven CD4 transgenes that encoded re-engineered CD4 proteins containing the cytosolic tail of CD4 (CD4hCD2), CD8α (4aahCD2), CD8β (4bbhCD2), or no tail at all (44thCD2) (supplementary Fig. S4A). Surface CD4 expression was roughly comparable to that of endogenous CD4, with the exception of the 44thCD2 transgene which displayed very high CD4 expression levels because tailless CD4 proteins cannot be internalized from the cell surface (supplementary Fig. S4A). As we previously reported (Van Laethem et al., 2007), the re-engineered CD4 proteins displayed a distinct hierarchy of Lck binding such that coreceptors bearing the CD4 cytosolic tail bound the most Lck, coreceptors bearing the CD8α cytosolic tail bound less Lck, while coreceptors bearing CD8β or no cytosolic tail bound no detectable Lck (supplementary Fig. S4B). Notably, Lck binding determines CD4 signaling strength and had a significant quantitative effect on MHC-II specific positive selection, as the frequency of signaled TCRβhi thymocytes in each strain paralleled their CD4-Lck associations (CD4hCD2>4aahCD2>4bbhCD2=44thCD2) (Fig. 4A). But, regardless of CD4 signal strength or overall CD4 expression levels, MHC-II specific lineage choice was error-prone in all of these CD4 transgenic mice, as each contained CD8+ LN T cells (Fig. 4A, right panels). Contrary to the strength-of-signal perspective (Itano et al., 1996), CD4 lineage choice was not less error-prone with stronger signaling CD4 coreceptors and was not more error-prone with weaker signaling CD4 coreceptors (Fig. 4A right panels as an example of one experiment). In fact, the full course of all our experiments revealed that lineage choice was error-prone in CD4 transgenic mice regardless of CD4 signal strength (Fig. 4B right panel). We then examined whether CD4 coreceptor expression was up- or downregulated on MHC-II signaled thymocytes in these mice (Fig. 4B). In fact, in each of these transgenic strains we found that CD4 expression declined during differentiation of MHC-II signaled DP into intermediate thymocytes (Fig. 4B left) and was associated with CD8 lineage choice errors (Fig. 4B right). Thus, failure to upregulate CD4 during positive selection caused lineage choice to be error-prone, regardless of CD4 signal strength or overall CD4 expression levels.
Finally, we wished to understand our early observation that lineage choice was especially error-prone in CD4hCD2 transgenic mice that expressed the AND TCR transgene (AND.CD4hCD2 mice) (Fig. 1A bottom panels). Indeed, as compared to CD4hCD2 mice with polyclonal TCR, MHC-II specific lineage choice in AND.CD4hCD2 mice was excessively error-prone, as nearly 50% of AND T cells in thymus and LN of AND.CD4hCD2 mice were mature CD8+ T cells (Fig. 5, compare rows 2 and 4). Note that the erroneous differentiation of AND.CD4hCD2 thymocytes into CD8+ T cells occurred despite their relatively homogeneous expression of Vα11hiVβ3hi transgenic AND TCR (Fig. 5 lower panels). Consequently, we considered that, unlike thymocytes with polyclonal TCR, AND TCR transgenic thymocytes compete with one another for binding to the identical peptide/MHC-II selecting ligand (Huesmann et al., 1991; Wong et al., 2000). But ligand competition among AND thymocytes, in and of itself, was not an explanation for error-prone lineage choice, as AND thymocytes in mice expressing wildtype CD4endo coreceptors (AND.CD4endo) contained no mature CD8+ AND T cells in either thymus or LN (Fig. 5 row 2). Nevertheless, we reasoned that AND thymocyte competition for limiting ligand would exacerbate TCR signaling disruptions caused by downregulation of transgenic CD4 coreceptors, increasing lineage choice errors. If our reasoning were correct, it would then be predicted that decreasing the frequency of cells in an individual thymus that expressed the AND TCR would decrease ligand competition and make AND lineage choice less error-prone.
To test this prediction, we constructed radiation bone marrow (bm) chimeras in which lethally irradiated β2mo (CD45.1) host mice were reconstituted with donor bm stem cells from β2moCD4o AND.CD4hCD2 (CD45.2) and non-transgenic β2mo (CD45.1) mice in varying ratios so that AND.CD4hCD2 (CD45.2) bm cells constituted 100%, 10%, or 1% of the mixed donor bm inoculum (Fig. 6A). Ten weeks after bm reconstitution, LN T cells from ‘high frequency AND chimeras’ were 80-95% AND (CD45.2), LN T cells from medium frequency AND chimeras were 20-45% AND, and LN T cells from low frequency AND chimeras were 1-5% AND (Fig. 6A and data not shown). Remarkably, the relative frequency of AND T cells that erroneously differentiated into CD8+ T cells was not constant, as the frequency of AND T cells that were CD8+ was relatively high (35%) in chimeras with many AND (CD45.2) T cells, but was relatively low (7%) in chimeras with few AND (CD45.2) T cells (Fig. 6A). In fact the lineage error rate among AND.CD4hCD2 T cells was relatively low in chimeras in which fewer than 30% of T cells expressed the AND TCR transgene, but sharply increased in chimeras in which more than 30% of T cells expressed the AND TCR transgene (Fig. 6B). That is, the frequency of lineage choice errors among developing AND.CD4hCD2 T cells was quantitatively affected by the number of AND.CD4hCD2 thymocytes that simultaneously competed for the same intra-thymic selecting ligand. We conclude that lineage choice is strikingly error-prone in AND.CD4hCD2 mice because ligand competition exacerbates AND TCR signaling disruptions in thymocytes that downregulate transgenic CD4 coreceptors during MHC-II specific selection.
Kinetic changes in CD8 coreceptor expression during MHC-I specific positive selection are important for CD8 lineage choice, but the importance of CD4 kinetics for CD4 lineage choice has not previously been appreciated. The present study now reveals that transcriptional upregulation of CD4 coreceptor expression during MHC-II specific positive selection is essential for error-free CD4 lineage choice, especially in TCR transgenic mice whose thymocytes compete for the identical selecting ligand, and that it is important for CD4 upregulation to occur during differentiation of signaled DP into CD4+CD8lo intermediate thymocytes when lineage choice is determined. Curiously, such CD4 upregulation is a feature unique to endogenous Cd4 genes, as the CD4 transgenes that we examined all downregulated CD4 coreceptor expression during MHC-II specific positive selection, causing MHC-II specific lineage choice to be error-prone regardless of the number or signaling strength of the transgenic CD4 coreceptors. Thus, it is the kinetics of CD4 coreceptor expression, not CD4 coreceptor number or signaling strength, that determines the integrity of CD4 lineage choice during MHC-II specific positive selection. Together with current knowledge about the kinetics of CD8 coreceptor expression during positive selection (Kioussis and Ellmeier, 2002; Singer et al., 2008), the present study reveals that TCR mediated positive selection signals downregulate CD8 but upregulate CD4 coreceptor expression, disrupting MHC-I specific TCR signaling to promote CD8 lineage choice and prolonging MHC-II specific TCR signaling to promote CD4 lineage choice.
The present study provides a new dimension to the kinetic signaling perspective that CD4 lineage choice requires sustained TCR signaling during differentiation of DP into CD4+CD8lo intermediate thymocytes (schematized in Fig. 7A top panel). CD4 upregulation during this developmental step stabilizes MHC-II specific TCR/ligand interactions so that TCR signaling persists to drive CD4 lineage choice (schematized in Fig. 7A bottom panel). In contrast, CD4 downregulation makes it difficult to sustain MHC-II specific TCR/ligand interactions, so that TCR signaling disruptions occur resulting in lineage choice errors (Fig. 7A bottom panel). Our current observation that error-free CD4 lineage choice requires CD4 upregulation regardless of the absolute number or signaling strength of the CD4 coreceptors emphasizes the difference between the TCR signaling requirements for positive selection and the TCR signaling requirements for CD4 lineage choice: the ability of individual DP thymocytes to be signaled by their MHC-II specific TCR to undergo positive selection is affected by the absolute number and signaling strength of the CD4 coreceptors that they express; but, once signaled to undergo positive selection, error-free differentiation into CD4+ T cells requires that CD4 expression further increase to maintain MHC-II specific TCR signaling. In fact, because MHC-II specific TCR/ligand interactions would be especially difficult to sustain in the absence of CD4 coreceptor expression altogether, our present study readily explains why lineage choice among MHC-II signaled thymocytes is so highly error-prone in CD4-deficient mice (Matechak et al., 1996; Tyznik et al., 2004).
The present study also provides a new dimension to our understanding of ligand competition during thymic selection by demonstrating that ligand competition can affect the integrity of CD4 lineage choice by MHC-II specific TCR transgenic thymocytes. Indeed, coreceptor kinetics provides a straightforward explanation for why introduction of the AND TCR transgene caused CD4 lineage choice to be strikingly error-prone in CD4 transgenic mice, but did not cause CD4 lineage choice to be error-prone in CD4 wildtype mice (Wong et al., 2000). In both CD4 transgenic and CD4 wildtype mice, pre-selection AND thymocytes would compete with one another for the same peptide/MHC-II selecting ligand, and only pre-selection AND thymocytes that successfully bound its selecting ligand would be signaled to undergo positive selection. Importantly, in CD4 wildtype mice, endogenous CD4 expression is upregulated during positive selection so that signaled AND thymocytes express more endogenous CD4 than unsignaled AND thymocytes, with the result that unsignaled AND thymocytes cannot disrupt AND TCR signaling by competing signaled AND thymocytes away from the selecting ligand (supplementary Fig. S5). But in CD4 transgenic mice, transgenic CD4 expression is downregulated during positive selection so signaled AND thymocytes express less transgenic CD4 than unsignaled pre-selection AND thymocytes, with the result that unsignaled AND thymocytes can disrupt AND TCR signaling by competing signaled AND thymocytes away from the selection ligand (supplementary Fig. S5). Thus, in CD4 wildtype mice, endogenous CD4 upregulation prevents ligand competition among AND thymocytes from introducing lineage choice errors; but in CD4 transgenic mice, CD4 downregulation synergizes with ligand competition to make AND lineage choice strikingly error-prone.
The CD4T3 transgene used in this study was reported in the past to promote generation of MHC-II specific CD8+ T cells and to support the stochastic/selection model of lineage choice (Davis et al., 1993). It was thought that forced transgenic expression of CD4 coreceptors during positive selection rescued from cell death shortlived MHC-II signaled thymocytes that had stochastically made a CD8 lineage choice (Davis et al., 1993). However, this explanation and the presumptions underlying the stochastic/selection model have since been experimentally disproved (Adoro et al., 2008; Dave et al., 1998; Itano and Robey, 2000; Sarafova et al., 2005; Singer et al., 2008), leaving the generation of MHC-II specific CD8+ T cells in CD4T3 transgenic mice unexplained. The present study now indicates that MHC-II specific lineage choice is error-prone in CD4T3 and other CD4 transgenic mice because expression of transgenic CD4 coreceptors fails to be upregulated during MHC-II specific positive selection which leads to TCR signaling disruptions and lineage choice errors. Similarly, the appearance of MHC-II selected CD8+ T cells in CD4-silencer knockout mice was also originally explained as revealing a stochastic mechanism underlying CD4/CD8 lineage choice (Leung et al., 2001). However, deletion of the CD4 silencer element additionally deleted cryptic CD4 enhancer elements transcriptionally active in signaled thymocytes, as CD4 coreceptor expression on MHC-II signaled thymocytes in CD4 silencer deficient mice was reduced relative to wildtype mice (Leung et al., 2001). In fact, careful examination of published thymocyte profiles from these CD4-silencer deficient mice reveal that CD4 coreceptor expression dramatically declined on signaled DP thymocytes during their differentiation into CD4+8lo intermediate thymocytes, explaining why MHC-II specific lineage choice was error-prone in these animals.
Expression of endogenously encoded CD4 coreceptors is transcriptionally upregulated during MHC-II specific selection, but the molecular mechanism by which this occurs is not yet clear. Transcriptional regulation of the endogenouos Cd4 gene locus still remains incompletely understood (Ellmeier et al., 1999; Leung et al., 2001), but we suspect that Cd4 enhancer elements exist that increase Cd4 gene transcription in response to TCR mediated positive selection signals. Even though the existence of TCR-responsive enhancer elements in the Cd4 gene remain a matter of speculation, the transcription factor Tox appears to be necessary for increased Cd4 gene expression during positive selection, as CD4 expression on signaled thymocytes from Tox-deficient mice is downregulated, rather than upregulated, during MHC-II specific selection (Aliahmad and Kaye, 2008). And we would like to note that, unlike endogenous CD4 proteins whose expression increases during MHC-II specific positive selection, the expression of CD4 proteins encoded by the T cell-specific transgenes examined here declined during MHC-II specific positive selection. Thus we suspect that the transcriptional activity of many or most T cell specific transgenes may similarly decline during differentiation of signaled DP into CD4+8lo intermediate thymocytes with potential consequences for thymocyte development.
Finally, our conclusion that error-free CD4 lineage choice requires CD4 upregulation to stabilize MHC-II specific TCR/ligand engagements and to sustain MHC-II specific TCR signaling conflicts directly with the concept that, during positive selection, TCR signaled CD4hiCD8hi (DPhi) thymocytes downregulate expression of both CD4 and CD8 coreceptors to become CD4loCD8lo (DPlo) thymocytes which then differentiate into CD4+CD8lo intermediate cells that ultimately differentiate into either CD4+ or CD8+ T cells (Aliahmad and Kaye, 2008; He and Kappes, 2006; Lucas and Germain, 1996; Sant'Angelo et al., 1998). Importantly, it should be appreciated that precursor-progeny assessments have never actually documented that CD4+ T cells arise from DPlo thymocytes that had previously been DPhi. In fact we do not think that that is the phenotypic pathway by which mature CD4+ T cells arise. Rather, we think the phenotypic pathway by which MHC-II specific T cell differentiation occurs is far more straightforward (Fig. 7B): MHC-II specific TCR signals induce newly arising DPlo thymocytes (Fig. 7B, population 4) to upregulate CD4, to downregulate CD8, and to differentiate into CD4+8lo intermediate thymocytes (Fig. 7B, population 5); CD4+8lo intermediate thymocytes then differentiate into mature CD4+ T cells (Fig. 7B, population 6). In this developmental scheme, CD4 coreceptor expression is constantly increasing on MHC-II signaled thymocytes, progressively increasing the stability of MHC-II specific TCR/ligand interactions and promoting error-free CD4 lineage choice (Fig. 7B). Notably, in this developmental scheme, DPhi thymocytes (Fig. 7B, population 3) are thought to be cells that have failed MHC-II specific positive selection but might still be signaled by MHC-I ligands to differentiate into CD8+ T cells.
In conclusion, the kinetics of CD4 coreceptor expression during MHC-II specific positive selection importantly influence the integrity of CD4 lineage choice, especially in TCR transgenic mice whose thymocytes compete for limiting ligand. Building on current knowledge about the kinetics of CD8 coreceptor expression (Kioussis and Ellmeier, 2002; Singer et al., 2008), this study reveals that coreceptor kinetics during positive selection promote error-free CD8 lineage choice by disrupting MHC-I specific TCR signaling and promote error-free CD4 lineage choice by prolonging MHC-II specific TCR signaling.
C57BL/6 (B6), β2mo, and CD4o mice were purchased from The Jackson Laboratory (Bar Harbor, ME). β2moCD4o double deficient were generated and bred to mice expressing the line 30 CD4 reporter transgene (CD4r) from Dr. Dan Littman (NYU) (Sawada et al., 1994). All CD4 transgenes in the present study were expressed in β2moCD4oCD4r+ unless otherwise indicated (Table 1). The 8DP4 transgene has previously been characterized (Sarafova et al., 2005) and expresses CD4 under the control of E8IIICD8α enhancer and CD8α promoter elements (Ellmeier et al., 1998). CD4T3 transgenic mice express CD4 under the control of hCD3δ promoter/enhancer elements and were constructed in our laboratory from a transgenic vector kindly provided by Dr. Dan Littman (NYU) (Davis et al., 1993). The CD4hCD2 transgene expresses CD4 under the control of hCD2 promoter and enhancer elements, as previously described (Van Laethem et al., 2007). A brief description of the CD4 transgenic mice used in this study is provided (Table 1). Where indicated, mice also expressed the AND TCR transgene (Kaye et al., 1989). Mixed radiation bone marrow chimeras were constructed by reconstituting 950R irradiated host mice with a total of 107 T-depleted bone marrow cells from CD45.1 and CD45.2 donor mice. All mice were cared for in accordance with NIH guidelines.
Re-engineered CD4 coreceptor proteins expressing different cytosolic tails were generated from cDNAs encoding CD4, CD8α and CD8β by conventional cloning procedures. Amino acid sequences at the modified junctions were as follows: 4aa ext/tm junction - VNQT/DIYIWAPLAGIC; 4bb ext/tm junction - VNQT/DITTLSLL; 44t tm/cyto junction - GLCILCCV/RCRHQQRQ. The resulting chimeric cDNAs were inserted into the hCD2-based cassette and were used to generate transgenic mice, as previously described (Van Laethem et al., 2007). Transgenic offspring were mated to β2moCD4o mice or to AND TCR transgenic β2moCD4o mice. Transgenic offspring were identified by the presence of CD4+CD8+ cells in the blood. The absence of endogenous CD4 and β2m were confirmed by PCR. All mice in this study were heterozygous for the transgene(s) they expressed.
Monoclonal antibodies with the following specificities were used in this study: CD3 (145-2C11 Pharmingen); CD4 (GK1.5, Pharmingen); CD8α (CT-CD8 α, Caltag); CD24 (M1-69, Pharmingen); CD69 (H1.2F3, Pharmingen); hCD2 (CD0215-4, Caltag); H-2Kb (AF6-88.5, Pharmingen); TCRβ (H57-597, Pharmingen); CD45.1 (A20, Pharmingen); CD45.2 (104, Pharmingen); TCR-Vβ3 (KJ25, Pharmingen); TCR-Vα11 (RR8-1, Pharmingen).
CD4 fluorescence on TCR-signaled DP thymocytes (identified as TCRβhiCD4r+CD8hi cells), intermediate thymocytes (identified as TCRβhiCD4r+CD8int cells) and CD4SP thymocytes (identified as TCRβhiCD4r+CD8− cells) was either expressed as mean fluorescence intensity (MFI) or quantitated into linear Total Fluorescence Units (TFU) using an empirically derived calibration curve constructed for each logarithmic amplifier. Relative CD4 expression on each cell population was calculated relative to signaled DP thymocytes. To avoid fluorescence compensation errors that might have affected our flow cytometric data, we verified that CD4 vs CD8 profiles from samples stained with only two colors were always identical to CD4 vs CD8 profiles from multi-color stained samples. In addition, three different fluorochrome combinations were used for TCR vs CD4 vs CD8 staining so that results were reproducible on different machines using different lasers and different fluorochromes.
Thymocytes (2×107 cells/group) were solubilized in 1% Brij96 and CD4 molecules were immunoprecipitated with purified anti-CD4 mAb (GK1.5, Pharmingen) and protein G-sepharose beads. Whole cell lysates and immunoprecipitates were resolved by SDS-PAGE on 10% acrylamide (Invitrogen) under reducing conditions and transferred to nitrocellulose membranes (Amersham). Blots were incubated with anti-Lck (3A5, Pharmingen) and anti-actin antibodies followed by horseradish peroxidase-conjugated protein A. CD4 protein was detected using RM4.5 rat mAb (Pharmingen) followed by horseradish peroxidase-conjugated goat anti-rat antibodies. Reactivity was detected by enhanced chemiluminescence.
Pre-selection DP (CD69−TCRβloCD4+CD8+) and intermediate (CD69+TCRβint/hiCD4+CD8lo) thymocytes were obtained by electronic sorting of thymocyte suspensions and total RNA was immediately isolated using the RNEasy kit (Qiagen). RNA was reverse transcribed into cDNA by oligo(dT) priming with the SuperScript™ III First Strand Synthesis System (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed with an ABI PRISM 7900HT Sequence Detection System using the SYBR green detection system (Qiagen) with the following primers: Gata3 (F: 5’-GTCCTCATCTCTTCACCTTCC-3’; R: 5’-GAGTCCGCAGGCATTGCAAAG-3’); Tox (F: 5’-CAGGACCCCTACTATTGCAAC-3’; R: 5’-GCAGGCCATTGTGATTCATGG-3’); ThPOK (F: 5’-ACATGAGGACCCACACTGGTG-3’; 5’-CTTCCTCTTCCTCCTCCTCAG-3’); Runx3 (F: 5’-GCGACATGGCTTCCAACAGC-3’; R: 5’-CTTAGCGCGCCGCTGTTCTCGC-3’); Actb (F: 5’-GAGAGGGAAATCGTGCGTGA-3’; R: 5’-ACATCTGCTGGAAGGTGGAC-3’). Gene expression values were normalized to those of Actb (β-actin gene) in the same sample.
Supplementary Figure Legends
Figure S1. Expression of the CD4 reporter transgene (CD4r). The CD4r transgene consists of CD4 transcriptional control elements to drive expression of hCD2 cDNA so that hCD2 protein expression functions as a reporter of CD4 transcription. In mice expressing the CD4r transgene, CD4-lineage T cells are hCD2+, whereas CD8-lineage T cells are hCD2− (Sawada et al., 1994). As shown, the CD4r transgene does successfully distinguish CD4-lineage from CD8-lineage T cells in thymus and spleen.
Figure S2. Evaluation of thymocytes from experimental mice.
A. Total thymocyte numbers (mean ± SE) from the mouse strains used in this study.
B. Comparison of CD4 surface expression levels on whole thymocytes. CD4 staining and CD4 MFI values from the indicated mouse strains are displayed.
Figure S3. Phenotypic analysis of thymocytes and LNT cells from experimental mice.
A. Thymocytes from the indicated mice were analyzed and mature CD8+ thymocytes identified as HSA−CD8hi cells (middle panels) and as TCRβhiCD8hi cells (lower panels) and their frequencies are shown. Total thymocyte numbers from each strain are also indicated. Note that among CD4hCD2 transgenic thymocytes (top middle panel), CD4−CD8+ thymocytes are TCRβ−/lo cells, indicating that they are immature pre-selection thymocytes that have not yet expressed the CD4 transgene.
B. Total thymocytes (left panel) and LN cells (right panel) from the indicated mice were assessed for TCRβ expression (top row), and gated TCRβhi cells further analyzed for CD4 versus CD8 expression (middle row) and CD4r (i.e. hCD2) versus CD8 expression (bottom row). As can be seen in profiles from CD4T3 mice (columns 3 and 6), the frequency of MHC-II selection errors revealed by the frequency of TCRβhiCD4r-CD8+ cells in the thymus and LN were identically ~5%.
Figure S4. Characterization of hCD2-driven transgenes encoding re-engineered CD4 coreceptor proteins.
A. hCD2-driven transgenic constructs encoding re-engineered CD4 proteins. With the exception of wildtype CD4 transgenic proteins, re-engineered CD4 constructs were named according to the origin of their extracellular, transmembrane, and intracellular domains. Accordingly, the re-engineered CD4 constructs were named 4aa (contains the cytosolic tail of CD8α), 4bb (contains the cytosolic tail of CD8β), and 44t (which lacks a cytosolic tail altogether). One color histograms display surface CD4 expression on thymocytes and lymph node cells from β2moCD4o mice expressing each CD4 transgene (histograms).
B. Association of re-engineered CD4 proteins with intracellular Lck. Detergent solubilized lysates of thymocytes from the indicated mice were immunoprecipitated (IP) to completion with anti-CD4 mAb, resolved by SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes which were then immunoblotted (IB) with the indicated antibodies. The top panel reveals CD4-associated Lck, which established a quantitative hierarchy: CD4hCD2>4aahCD2>4bbhCD2=44ThCD2. Re-engineered CD4 proteins present in the anti-CD4 immunoprecipitates were visualized in the second panel. As a visual aid, white dashes were used to mark the center of each CD4 band, whereas the unmarked bands represent the Ig heavy chain of the immunoprecipitating anti-CD4 mAb. The different CD4 molecules migrated at different speeds in the gel, reflective of their different molecular weights (second panel). In the lower panels, aliquots of whole cell lysates were immunoblotted to assess total Lck content as well as actin content, as loading controls.
Figure S5. Schematic illustration of why ligand competition exacerbates lineage choice errors in CD4 transgenic but not CD4 wildtype mice. Endogenous CD4 (CD4endo) expression is upregulated on signaled thymocytes so that signaled CD4+8lo AND thymocytes express higher CD4 levels than unsignaled AND DP thymocytes, with the result that unsignaled AND DP thymocytes cannot disrupt continued TCR signaling and CD4 lineage choice by CD4+8lo AND thymocytes. In contrast, transgenic CD4 (CD4hCD2) expression is downregulated on signaled thymocytes so that signaled CD4+8lo AND thymocytes express lower CD4 levels than unsignaled AND DP thymocytes, with the result that unsignaled AND DP thymocytes disrupt continued TCR signaling by CD4+8lo AND thymocytes which leads to erroneous CD8 lineage choice.
We thank Drs. Naomi Taylor, Remy Bosselut, Hyun Park, and Richard Hodes for helpful discussions and critical readings of the manuscript; Remy Bosselut for the original construction of re-engineered CD4hCD2 transgenes; Lawrence Granger and Anthony Adams for expert flow cytometry; Don Plugge for data analysis support; and Dr. Krasimira Tsaneva-Atanasova for help with statistical analyses. This research was supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research. The authors have no conflicting financial interests.
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