We describe a CD45 allele, lightning, characterized by a mutation in the protein’s extracellular domain. Expression was reduced due to impaired protein stability and increased turnover, but importantly, regulated isoform splicing is unperturbed and the polymorphism is structurally insulated from the cytoplasmic phosphatase domain.
CD45 isoform expression is tightly regulated and may affect protein function through differential dimerization or differential association with the CD4 co-receptor (Dornan et al., 2002
; Xu and Weiss, 2002
). In contrast to previously published series of CD45 transgenic lines in which single isoforms of CD45 are expressed at a range of doses, the lightning
allelic series titrates protein expression while preserving isoform splicing. By including both Ptprc+/−
animals and the high-expressing CD45 ‘HE’ line in our studies, we conclude that the phenotypes we identify were not due to an idiosyncratic effect of the lightning
We demonstrated distinct requirements for CD45 in basal and inducible TCR signaling during thymic development that are obscured in the context of complete CD45 deficiency. Whereas Ptprc−/−
mice have previously revealed an absolute requirement for CD45 in positive selection, and a partial requirement during beta-selection (Byth et al., 1996
; Kishihara et al., 1993
; Mee et al., 1999
), the lightning
allelic series uncouples these processes, revealing that low CD45 doses are sufficient to rescue the former, but high doses are required to correct the latter. Additional indicators of impaired basal signaling in Ptprc−/−
thymocytes (CD5 expression, constitutive ζ-chain phosphorylation, and TCR surface expression) are rescued only with high CD45 expression. In contrast, biochemical indicators of inducible TCR signal intensity in DP thymocytes showed rescue of CD45-dependent defects even at very low CD45 expression.
Inducible signaling via MHC presentation of antigen recruits coreceptor-associated Lck to the TCR. In contrast, neither beta-selection, nor basal or weak non-selecting MHC signals effectively recruit or require coreceptor. Indeed, while constitutive ζ-chain phosphorylation in DP thymocytes has been demonstrated to depend upon non-selecting MHC and Lck, it is coreceptor independent (Becker et al., 2007
; van Oers et al., 1996a
; van Oers et al., 1993
). We propose therefore that independent pools of CD4-associated and ‘free’ Lck may be differentially regulated by CD45 and independently mediate basal and inducible signaling (Falahati and Leitenberg, 2007
; Leitenberg et al., 1996
The distinct requirements for CD45 expression during basal and inducible signaling might also relate to the dynamic localization of Csk (Brdicka et al., 2000
; Kawabuchi et al., 2000
). High amounts of Csk bound to PAG and localized to Lck in lipid rafts in the basal state might require high expression of CD45 to counteract Lck Y505 phosphorylation, whereas loss of Csk from lipid rafts upon TCR stimulation might explain why even low doses of CD45 are sufficient to mediate inducible TCR signaling (Figure S7F
). Indeed, consistent with this model we have demonstrated that reduced Csk dosage rescues basal but not inducible signaling phenotypes in L/− and L/L DP thymocytes.
CD45 is expressed at extremely high amounts on the surface of hematopoietic cells, by some estimates up to 25 µM (data not shown). Yet, the reason for this apparent overabundance remains uncertain, especially when no definitive physiologically relevant ligand has been identified in 20 years of searching. We propose that one reason why so much CD45 is required on the surface of cells may be to counteract membrane-associated Csk in the basal state.
Although very low doses of CD45 are sufficient to rescue inducible TCR signaling and positive selection, high amounts of CD45 dampen these processes. The allelic series unmasks a negative regulatory role for CD45 that is obscured in Ptprc−/− animals by its positive regulatory role.
CD45 has been postulated to negatively regulate TCR signaling by dephosphorylating the activating Lck tyrosine 394 (McNeill et al., 2007
). Dual hyperphosphorylation of Lck at both inhibitory and activating tyrosines has been reported in many CD45 deficient cell lines and mice (Ashwell and D'Oro, 1999
). Here we also identify dual hyperphosphorylation of Lck at tyrosines 394 and 505 with decreasing doses of CD45. Importantly, we observe progressive dephosphorylation at these sites with supraphysiologic expression of CD45, suggesting that any dimerization that occurs at high surface density is insufficient to completely inhibit phosphatase activity. Maximal Y394 phosphorylation at low CD45 expression levels correlates with maximal inducible TCR signaling in DP thymocytes. We suggest that in the presence of even low amounts of CD45, Y394 rather than Y505 phosphorylation “dominantly” controls the sensitivity of inducible TCR signaling.
Reducing Csk dosage rescues basal signaling in allelic series thymocytes and implies that CD45 is required to counteract Csk phosphorylation of Lck Y505 in the basal state. Lck Y505 phosphorylation dominantly controls Lck activity under these conditions. By contrast, reduced Csk dosage does not affect inducible signaling in DP thymocytes, implying that phosphorylation of Lck Y505 does not mediate inducible signaling phenotypes of allelic series mice. Since high CD45 expression dampens inducible TCR signaling, we conclude that CD45 negatively regulates Lck activity through an alternate substrate, most likely the activation loop tyrosine Y394. Our data support a model whereby Lck Y394 is directly dephosphorylated by CD45, independently of Csk and its unique substrate Lck Y505.
Although dual activating and inhibitory tyrosine phosphorylation of Lck has been observed in CD45 deficient T cells and T cell lines, other CD45-deficient hematopoietic cells, including B cells and macrophages, do not exhibit hyper-phosphorylation of SFK activation loop tyrosines (Zhu et al., 2008
). This is more consistent with the regulation of Fyn activating tyrosine phosphorylation in our allelic series thymocytes, and suggests that Lck regulation by CD45 is unique among the SFKs. This may be the result of differential localization of the SFKs since Lck is uniquely associated with CD4 coreceptor and, consequently, with CD45 (Veillette et al., 1988
). The activating tyrosine of Lck may only function as an important CD45 substrate at high CD45 doses and in close proximity such that Lck is uniquely regulated by CD45 with respect to other SFKs.
We have identified differential regulation of inducible signaling in DP and SP subsets by CD45 and have demonstrated that Fyn rather than Lck accounts for these differences. Importantly, Fyn does not play a non-redundant role in either basal signaling or negative regulation of inducible signaling by CD45. Unpublished data from our lab suggests that the CD45 requirement for signaling by peripheral T cells is similar to that in SP4 rather than DP thymocytes, implying that DP thymocytes are unique among T cells. Indeed, prior studies had suggested that very low levels of CD45 expression were sufficient to reconstitute thymic development, while much higher levels were required to rescue TCR signaling in peripheral T cells (Kozieradzki et al., 1997
; McNeill et al., 2007
; Ogilvy et al., 2003
). We propose that this discrepancy is actually due to the distinct CD45 requirements of DP thymocytes and mature T cells which are in turn the result of differential roles of Fyn and Lck in these subsets.
This differential cell-type specific TCR ‘wiring’ has interesting implications for DP thymocyte signaling. Perhaps low Fyn expression forces DP thymocytes to be more dependent upon co-receptor-associated Lck. This would further enforce MHC-dependence during thymic positive selection (Van Laethem et al., 2007
). As well, such co-receptor associated Lck is itself more tightly regulated by CD45 than “free” Lck (Falahati and Leitenberg, 2007
). By dialing down activating Y394 phosphorylation of Lck at physiologic doses of CD45, the dynamic range of antigen recognition by DP thymocytes is enhanced at a crucial developmental checkpoint.
allelic series demonstrates that hypomorphic alleles of known genes can unmask previously obscured functions. Indeed, the physiologic relevance of CD45 dose is highlighted by recent studies demonstrating that mice expressing intermediate CD45 are resistant to fatal Ebola and Anthrax infections to which both wild type and CD45−/−
mice succumb (Panchal et al., 2009a
; Panchal et al., 2009b
We conclude by suggesting that high physiologic expression of CD45 on the surface of T cells serves two functions: First, to counteract Csk activity during basal signaling. Second, to negatively regulate TCR signaling specifically in DP thymocytes, thereby expanding the dynamic range of ligand-receptor interactions at a critical developmental checkpoint. This provides a unique solution to the central problem of the adaptive immune system: how, in the face of a universe of unknown antigens, to generate working receptors that respond sensitively, but not too sensitively.