We describe an inducible, conditional system for in vivo deletion of SLP-76 expression in mature, antigen-inexperienced T lymphocytes, enabling novel analysis of the role of this critical signal transducer in a primary response in vivo. In order to circumvent the developmental abnormalities associated with SLP-76 absence or mutation, we use a peripherally expressed, drug inducible form of the Cre recombinase. Using this system, we delete SLP-76 in normally developed, peripheral, mature T cells that are phenotypically similar to wild-type control lymphocytes both before and after deletion. SLP-76 deficient T cells expressing wildtype levels of cell surface TCR exhibit defective TCR-mediated proximal signal transduction and immune response.
This report highlights the advantage of a temporal deletion strategy to assess signal transduction in antigen-inexperienced cells. While tissue-specific conditional deletion approaches allow for cell lineage specificity, they still suffer from potential developmental alterations. For example, low cell surface TCR levels are found in SLP-76CD4Cre
mice and other models in which TCR signaling has been disrupted during development [15
]. Similar to conditional loss of Lck and Fyn [32
], deletion of SLP-76 from mature T cells does not alter cell surface TCR levels. Normal cell surface levels of TCR are maintained at least as long as one month post tam-induced deletion and up to a year following deletion of SLP-76 in virally-induced memory T cell populations [19
]. Normal cell surface TCR levels suggest an Lck/Fyn/SLP-76 independent maintenance of cell surface TCR levels and add support to a model in which so called “tonic” TCR signals may be qualitatively different than antigen-induced TCR mediated signals.
The use of a temporal deletion system also allowed us to evaluate the role of SLP-76 in vivo
using EAE as a model system. EAE is a CD4 T cell-dependent inflammatory demyelinating disease of the CNS involving a complex inflammatory cascade of events [34
]. Increased TCR signaling resulting from deletion of negative regulators hematopoietic progenitor kinase 1 or Sts1/Sts2 worsens EAE [37
]. The lack of EAE induction seen here suggests that SLP-76 dependent TCR signaling is required for development of CNS autoimmune disease, but does not rule out alternative SLP-76 dependent mechanisms.
The requirements for SLP-76 expression for chemokine mediated trafficking have only begun to be addressed experimentally. Initial studies in SLP-76 deficient Jurkat T cells suggested that SDF1 mediated signaling required expression of SLP-76 [39
], but a recent study was unable to extend this finding into primary T murine T cells [40
]. In addition, preliminary studies have shown no differences in trafficking of SLP-76cKO
cells in a non-inflammatory setting (E.C. and J.S.M., unpublished). Alternatively, because Cre-mediated SLP-76 deletion is effective in all cell types, antigen presenting cell function may also be affected following tam treatment. Alterations in dendritic cell function have been reported in SLP-76-deficient mice [41
], including impaired adhesion downstream of integrin activation. A combination of events attributable to the pleiotropic effects of SLP-76 deletion are probably responsible for the abrogation in clinical and pathologic EAE. Memory T cell differentiation and homeostasis is altered by timing deletion of SLP-76 after acute LCMV infection[19
] suggesting that temporal targeting of SLP-76 after induction of EAE may alter the clinical course. Studies to address this questions using temporal deletion at various time points during an immune response are currently ongoing in the laboratory.
Our use of a ubiquitously expressed CreT2 transgenic has several advantages and disadvantages. Following tam administration, Cre activity is seen in all hematopoetic cell types that we have evaluated (data not shown). For example, following tam administration, we find decreased thymic cellularity with a majority of thymocytes expressing markers consistent with a DN3 developmental block. This results in little to no ongoing T cell development and makes thymectomy unnecessary for the studies presented here. In addition, ubiquitous expression of CreT2 allows for the evaluation of SLP-76 deficient cells in other hematopoetic lineages. On the other hand, widespread deletion does have several disadvantages. Specifically, determining that defects in signaling and function are T cell intrinsic cannot be formally assessed without adoptive transfer or mixed chimera experiments. Interpretation of in vivo studies, such as evaluation of EAE, are complicated by potential defects in SLP-76 deficient antigen presenting cells.
Our result of deficient immune responses following conditional deletion of a TCR proximal adaptor are in contrast to a recent report in which conditional deletion of LAT in peripheral T cells [42
] resulted in Th2 mediated autoimmune pathology similar to that seen with the LATY136F
]. We believe that the divergent functional consequences of SLP-76 deletion reported here compared with LAT deletion in mature T cells are most likely due to differences in the method of Cre delivery and previous activation state of the cells evaluated. Specifically, our studies delete SLP-76 in resting lymphocytes and assess the immune response in a lymphoreplete animal, the LAT studies utilized ex vivo
activation followed by exposure to the elevated cytokine levels in lymphodepleted mice. Further experiments will be needed to determine if this technical difference is the sole cause of the disparate results or if the roles of these two critical adaptors in peripheral T cells are truly so distinct.
The functional defects resulting from SLP-76 deletion in mature T cells are as profound as those resulting from deletion in developing thymocytes. Loss of SLP-76 at any stage of T cell development or differentiation leads to a block in signal transduction and loss of effector function. These data suggest that SLP-76cKO mice generated through a temporal deletion strategy behave functionally as inducible TCR-knockouts and that this system will be useful to address fundamental questions regarding the requirements for TCR signaling requirements in the function and homeostasis of multiple T cell lineages.