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Keywords:

  • Knockout mice;
  • Signal transduction;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Signals generated by ligation of the T-cell receptor (TCR) are critical in both thymocyte development and peripheral activation and differentiation. TCR signaling mechanisms have been explored using both in vitro 1 and in vivo 2 systems. These studies have led to a model in which crosslinking of the cell surface TCR leads to phosphorylation and activation of both Src and Syk family kinases resulting in phosphorylation of the lipid raft resident-transmembrane adaptor protein linker for activated T cells (LAT) 3. Phosphorylated LAT recruits cytosolic Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) Grb2-like adaptor downstream of Shc (GADS) and PLCγ1 4. The formation of a complex composed of LAT, SLP-76, GADS and PLCγ1 is critical for the propagation of signals to downstream second messengers. A deficiency in any one of these components results in defects in biochemical markers of T-cell activation in cell lines 5–8 and T-cell developmental defects in genetically modified mice 9–12.

The study of SLP-76 in mature T-cell signaling has only been partially addressed due to the critical role for SLP-76 in thymocyte development. Germline deletion of SLP-76 leads to a complete block at the CD4CD8CD25+CD44 (double negative 3 (DN3)) to CD4+CD8+ (double positive (DP)) transition 9, 11. Transgenic expression or knockin rescue of germline SLP-76 deficiency with mutant forms of SLP-76 result in varied degrees of developmental defects, but generate single positive thymocytes and mature T cells for study 13–15. Cre-loxP-mediated conditional deletion of SLP-76 with a tissue-specific CD4-Cre transgenic (SLP-76CD4Cre) revealed a requirement for SLP-76 in positive selection 16. These mice exhibit varying degrees of peripheral T lymphopenia with abnormal cell surface expression of TCR/CD3 and cell surface activation markers 14–16. In addition, we have recently shown that sustained expression of WT SLP-76 is required for normal memory T-cell generation 17–19. A systematic evaluation of SLP-76 requirements in antigen-inexperienced peripheral T cells has not been described.

To study TCR-mediated signaling in normally developed, non-antigen experienced T lymphocytes, we employ a system in which temporally controlled deletion of SLP-76 can be induced after normal T-cell development. Efficient deletion of SLP-76 is achieved in adult mice using a tamoxifen (tam)-responsive Cre recombinase (CreT2). In contrast to the effect observed with CD4Cre-mediated deletion, naïve peripheral T cells from mice with tam-induced SLP-76 deletion maintain cell surface expression of TCR but remain unable to propagate TCR-generated signals. Primary immune responses are dramatically reduced using an in vivo model of experimental autoimmune encephalomyelitis (EAE) induction. Lastly, timed activity of Cre induction can be used to “switch” expression of WT to a mutant form of SLP-76 in peripheral T cells, allowing in vivo structure-function studies confined to mature cell compartments. Our studies establish a model for regulated deletion of SLP-76 after thymic development and reveal a central role for SLP-76 in initiating immune responses by naïve T cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

To directly address whether peripheral T lymphocytes lacking SLP-76 are capable of transducing signals generated by TCR crosslinking, we used an inducible bi-transgenic system that could delete in a temporally controlled fashion (Fig. 1A). Conditional SLP-76 mice (SLP-76Flox) with the Rosa26-based reporter R26RYFP 20 were intercrossed with SLP-76+/− mice expressing CreT2 21; a modified form of the Cre recombinase that is inactive unless exposed to the estrogen receptor antagonist tam 22. Prior to tam administration, CreT2 is inactive; there is no evidence of yellow fluorescent protein (YFP) expression or SLP-76 germline deficiency. Specifically, thymic and peripheral T lymphocytes exhibit WT numbers, phenotypes and functions (data not shown). Mice lacked gross evidence of the abnormal lymphatic/vascular separation that has been described in SLP-76null mice 23. For clarity, SLP-76Flox/−R26RYFPCreT2+ mice that delete SLP-76 are designated SLP-76cKO, control littermates with a WT allele of SLP-76 (SLP-76Flox/+R26RYFPCreT2+) are designated SLP-76cHET.

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Figure 1. SLP-76 deletion in peripheral T cells. (A) Schematic of temporal deletion approach: Mice with the SLP-76 gene floxed were crossed to mice expressing a tam-regulated Cre recombinase (CreT2). (Top) Tam was administered daily for five consecutive days by oral gavage and mice were analyzed from day 10 to 30 from the start of tam treatment. (Bottom) Prior to tam, a portion of the SLP-76 gene was floxed; a transcriptional stop cassette that blocks transcription of YFP from the Rosa26 locus was also floxed. In the presence of tam, CreT2 was active resulting in recombination between loxP sites, deletion of SLP-76 and of the stop cassette. This resulted in cells lacking SLP-76 but expressing YFP. (B) Following tam treatment, splenocytes from SLP-76cHET and SLP-76cKO mice were sorted based on YFP and CD4+ expression. Deletion was assessed at the genomic DNA level by PCR (top) and the RNA level by Tacman-based real-time PCR (bottom). Error bars represent standard deviation of triplicate reactions for each cDNA sample (ND, non-detectable). Representative of three individual experiments. (C) Contour plot of CD4 and CD8-gated splenocytes from a SLP-76cKO mouse. Relative percentages of cells within the gated regions are shown. Representative of greater than ten individual mice.

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SLP-76 can be efficiently deleted from mature T lymphocytes

To confirm the efficiency of SLP-76 excision and generation of SLP-76-deficient T cells, lymphocytes from spleen and lymph nodes were harvested from tam-treated mice. PCR analysis of genomic DNA confirms deletion at the floxed locus in CD4+YFP+ cells (Fig. 1B). YFP cells isolated from the same mice contain a mixture of deleted and non-deleted alleles complicating interpretation of this population. Steady-state RNA expression is also substantially reduced in cells isolated from the SLP-76cKO mice relative to those from the SLP-76cHET mice and non-detectable in YFP+SLP-76cKO cells (Fig. 1B, bottom).

To confirm that individual T cells lacked detectable SLP-76 protein, we utilized flow cytometry to assess protein levels. Approximately 15–20% of CD4+ gated cells and 3–5% of CD8+ gated cells from SLP-76cKO mice retain the SLP-76 protein after tam-induced deletion (Fig. 1C). Expression of YFP is highly correlative with loss of SLP-76 protein; YFP+ cells rarely stained positive for SLP-76. T lymphocytes from SLP-76cHET animals treated with tam express equivalent levels of SLP-76 in the YFP+ and YFP populations, which are slightly less than those found in SLP-76+/− cells (Supporting Information Fig. S1 and Fig. 2A). Importantly, SLP-76cKO mice have normal numbers of splenic T lymphocytes. Cell surface levels of TCR, CD44, CD62L and CD25 are similar to those from SLP-76cHET and tam-treated C57BL/6 (Supporting Information Fig. S2). Thus, effective inducible in vivo deletion of SLP-76 can be achieved resulting in phenotypically normal, naïve, SLP-76-deficient T cells for study.

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Figure 2. SLP-76cKO T cells are unable to transduce TCR-mediated signals. (A) FACS-sorted CD90.2+YFP+ T cells were left unstimulated or stimulated with anti-CD3 (500A2) for the time periods indicated followed by lysis and western blotting to detect SLP-76, pLAT and pPLCγ1. Total PLCγ1 is shown as a loading control. Representative of three individual experiments. (B) Cell suspensions from lymph node of indicated mice loaded with Indo1 were stimulated by co-crosslinking cell surface CD3 and CD4 (indicated by arrow). Intracellular calcium was measured as a ratio of FL4/FL5. Ionomycin was added to each sample during the last 30 s of acquisition. Representative of two experiments. (C) Splenocytes stimulated for 15 min with anti-CD3 (500A2) or PMA were assessed for phosphorylation of ERK. Dot plots are gated on CD4+ cells. Numbers within the gated areas are relative percentages of YFP+ and YFP gated cells that stain positive for pERK. Representative of cells from six SLP-76cHET and four SLP-76cKO mice.

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T cells deficient in SLP-76 fail to transduce TCR-mediated signals

To assess the effect of SLP-76 deficiency on TCR signaling in mature T lymphocytes, CD90+YFP+ FACS-sorted T lymphocytes were stimulated with soluble anti-CD3 antibody and assayed at various time points. Crosslinking of the TCR with anti-CD3 antibody results in phosphorylation of LAT suggesting that proximal signaling is grossly intact (Fig. 2A). In contrast, despite normal cell surface expression of TCR and LAT phosphorylation in SLP-76cKO peripheral T cells, crosslinking with anti-CD3 antibody did not induce appreciable phosphorylation of PLCγ1 or increases in intracellular calcium (Fig. 2A and B). Similarly, single-cell phosphoflow analysis shows minimal (<2-fold increase over unstimulated) ERK phosphorylation in YFP+CD4+ and YFP+CD8+ cells following TCR crosslinking (Fig. 2C and Supporting Information Fig. S3A, respectively). Conversely, the number of cells with detectable phospho-ERK increased approximately tenfold in YFP+ cells from SLP-76cHET mice. Stimulation with phorbol ester bypassed the proximal block resulting in phosphorylation of ERK in both SLP-76cKO and SLP-76cHET cells. These results demonstrate that mature peripheral T cells lacking SLP-76 cannot transduce appropriate intracellular signals following TCR crosslinking.

We next assessed downstream indicators of TCR-mediated signaling in SLP-76cKO cells, including upregulation of activation markers and proliferative expansion. TCR crosslinking results in the upregulation of both CD69 and CD25 on YFP+ gated cells from SLP-76cHET but not SLP-76cKO T cells (Fig. 3A and B). Consistent with incomplete deletion, a small percentage of YFPneg T cells from SLP-76cKO upregulate the activation markers (Supporting Information Fig. S4A). Comparison of SLP-76cHET with C57BL/6 controls had no defect in activation marker upregulation suggesting that acute conversion from two to one allele of SLP-76 has no effect on these functions (Supporting Information Fig. S4B). To assess proliferative capacity, splenocytes from SLP-76cKO and SLP-76cHET mice lacking the YFP reporter were labeled with CFSE and stimulated with anti-CD3 in vitro. CD4+ and CD8+ SLP-76cKO T cells fail to proliferate (Fig. 3C), regardless of the concentration of antibody used, addition of anti-CD28 co-stimulation or IL-2 supplementation (Supporting Information Fig. S4A and data not shown). In contrast, bypassing proximal signaling with PMA and ionomycin stimulation is sufficient to upregulate activation markers and rescue proliferation (data not shown). Overall, mature T cells lacking SLP-76 are incapable of effective activation or proliferation in response to TCR crosslinking.

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Figure 3. SLP-76 is required in mature T cells for upregulation of activation markers and for proliferation. (A and B) Splenocytes isolated from SLP-76cHET and SLP-76cKO mice were left unstimulated (shaded histograms) or were stimulated overnight with soluble anti-CD3 (open histograms). Cells were surface stained with CD4, CD8, CD69 and CD25. Expression of (A) CD69 and (B) CD25 on CD4+YFP+ and CD8+YFP+ are shown. Representative of greater than five experiments. (C) Splenocytes were labeled with CFSE and stimulated in vitro for 72 h then stained for CD4 and CD8. Proliferation measured by dilution of CFSE in CD4 gated (left panel) and CD8 gated (right panel) is shown. SLP-76cKO cells are shown with a dotted line and SLP-76cHET cells with a solid line. Representative of two experiments. (D) CFSE-labeled Thy1.2+ T cells from SLP-76cHET and SLP-76cKO mice were adoptively transferred into RAG2−/−CD45SJL mice. Suspensions taken from lymph nodes seven days after transfer gated on CD45B6 donor, B220 then CD4 (left) or CD8 (right) are shown. Representative of two experiments. (E) Splenocytes from SLP-76cHet (dashed line) and SLP-76cKO (solid line) were adoptively transferred into CD45SJL and RAG2−/−CD45SJL recipients. BRDU was administered from day 5 through day 7. Lymph node suspensions gated on CD45B6 donor, YFP+ then CD4 (left) or CD8 (right) are shown. Shaded histograms represent BRDU incorporation into cells transferred into non-lymphopenic CD45SJL mice, where no LIP is expected. Representative of two experiments with a total of nine recipient mice in each experiment.

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SLP-76-deficient T cells fail to undergo lymphopenia-induced proliferation (LIP)

T cells can undergo LIP that requires both cytokine and TCR signals 24, 25. LIP was assessed using CFSE dye dilution and alternatively with bromodeoxyuridine (BrdU) incorporation in conjuction with YFP expression. T cells isolated from SLP-76cHET and SLP-76cKO mice lacking the YFP reporter were labeled with CFSE and adoptively transferred into RAG2-deficient mice expressing the CD45SJL congenic marker. One week later, lymph node CD45B6 donor cells gated on CD4 or CD8 were assessed for LIP (Fig. 3D). A majority of both CD4+ and CD8+ T cells from SLP-76cHET donors underwent one or more rounds of division. In contrast, a majority of CD4 and CD8 cells derived from SLP-76cKO donors fail to undergo cell division as measured by CFSE dilution. A small percentage of SLP-76cKO-derived CD4+ T cells underwent one round of division and CD8+ cells underwent more than one division. This may be due to a small number of SLP-76-deficient cells proliferating in response to lymphopenia or the non-deleted cells. To differentiate these possiblilites, additional adoptive transfers were done using cells containing the YFP reporter RAG2−/− recipients and BRDU was administered during the final two days of potential LIP. YFP+ T cells originating from SLP-76cKO mice failed to incorporate BRDU (Fig. 3E) and represented a small percentage of cells found in the secondary lympoid organs of recipient mice (data not shown). Recipients of SLP-76cHET T cells had greater numbers of donor-derived cells that had efficiently incorporated BRDU (Fig. 3E). Taken together, these data strongly argue that SLP-76 expression is a cell-intrinsic requirement for LIP.

SLP-76 is required for induction of EAE

To determine the extent to which SLP-76 contributes to a functional immune response in vivo, we examined the effect of SLP-76 absence during active EAE. All mice, both SLP-76cKO and control (which included SLP-76cHET, C57BL/6 and (C57BL/6×129)F1), received tam. Following tam treatment, mice were immunized with myelin oligodendrocyte glycoprotein (MOG) 35–55 emulsified in complete Freund's adjuvant (CFA) according to the standard protocol 26 and observed for evidence of clinical disease. None of the SLP-76cKO mice immunized with MOG peptide showed evidence of disease, while 83% of the control mice developed hind-limb weakness (Fig. 4A and Table 1). Onset of clinical disease in control mice was characteristic of MOG35–55-induced EAE in C57BL/6 and B6.129 mice 27 (Fig. 4A). Histological examination of brain and spinal cords from WT mice revealed inflammatory infiltration and demyelination, while minimal inflammation of CNS tissue was observed in SLP-76cKO mice (data not shown). CD4+ T cells isolated from the CNS tissue of control mice expressed high levels of CD44, consistent with previous activation (Fig. 4B). Conversely, the few CD4+ cells found within the CNS of SLP-76cKO mice had lower levels of CD44, indicative of limited prior activation. Thus, mice with conditional deletion of SLP-76 lack both lymphocytic CNS infiltrates as well as clinical signs of EAE, suggesting a central role for SLP-76 in the initiation of autoimmune pathology and disease.

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Figure 4. SLP-76cKO mice are resistant to active EAE induction. (A) Mice were treated with tam for five consecutive days prior to immunization with MOG35–55 and assessed for clinical signs of EAE. Control mice (SLP-76WT, C57Bl/6 or C57Bl/6x129 mice; squares; n=4). SLP-76cKO mice (triangles; n=3). Mean with standard deviation of clinical scores from one of three individual experiments is shown. (B) Mononuclear cells were isolated from the CNS of mice at day 28 post immunization with MOG35–55. Cells were stained and analyzed by flow cytometry. Left panels are gated on live, mononuclear cells and a representative contour plot is shown. Right panels are gated on CD4+ as indicated and histograms indicate levels of cell surface CD44. Representative of data from two separate experiments with a total of six mice.

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Table 1. EAE in SLP-76cKO and SLP-76control (a) mice
 IncidenceOnset (mean±SEM)Maximal scoreMean scoreb)
  • a

    Combined data from a total of three individual experiments.

  • c)

    a) The SLP-76control group includes CreT2- and non-transgenic mice in addition to SLP-76cHET.

  • b)

    b) Assessed at day 21±SEM; two-tailed Student' t-test was performed for comparison between SLP-76 control and SLP-76c) cKO mice assessed at day 21 (p<0.0001) Disease severity was scored on a scale of 0–5: 0, no illness; 1, limp tail; 2, hindlimb paresis; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, moribund or dead.

SLP-76control c)10/1215.5±0.741.8±0.3
SLP-76cKO0/13N/A00

Tam/CreT2-induced deletion can be used for structure–function analysis in mature T lymphocytes

We next applied the conditional deletion model of SLP-76 to the study of structure–function in normally developed primary cells. TCR crosslinking of Jurkat cells, expressing only a mutant form of SLP-76 in which three tyrosines have been mutated to phenylalanine (Y3F), results in impaired activation of PLCγ1 and calcium influx 28, 29. Similarly, in SLP-76-deficient mice expressing a T-cell-specific SLP-76Y3F transgene, thymocyte cellularity is significantly decreased and TCR-mediated signaling in thymocytes is markedly abnormal 15. To address whether the presence of the Y3F mutation in normally developed, primary T cells leads to abnormal function of T-cell signaling, we bred mice expressing the SLP-76Y3F transgene onto the conditional knockout background. Adult mice were fed tam to induce deletion of the WT allele, leaving the mutated SLP-76Y3F form of the protein as the only remaining SLP-76 molecule expressed (SLP-76Flox[RIGHTWARDS ARROW]Y3F). Examination of splenocytes from SLP-76Flox[RIGHTWARDS ARROW]Y3F mice revealed normal proportions of CD4+ and CD8+ T cells and normal levels of cell surface TCR (Fig. 5A). Biochemical analysis of SLP-76Flox[RIGHTWARDS ARROW]Y3F cells stimulated with anti-CD3 revealed WT levels of phosphorylation of ZAP-70 in response to TCR crosslinking, but virtually absent PLCγ1 phosphorylation. Additionally, ERK phosphorylation was detectable but reduced (Fig. 5B). In response to TCR crosslinking, CD69 is moderately upregulated in CD4+ cells but only minimally in CD8+ T cells from SLP-76Flox[RIGHTWARDS ARROW]Y3F mice (Fig. 5C). This subtle difference between CD4+ and CD8+ cells suggests that the mutant form of the protein may be differentially required. Lastly, proliferation of SLP-76Flox[RIGHTWARDS ARROW]Y3F T cells was substantially less than WT levels, but increased when compared with SLP-76cKO cells (Fig. 5D). These results indicate that T cells restricted to signaling through the SLP-76Y3F mutant form of the adaptor have moderate signaling defects and suggest a decrease in TCR signal strength in SLP-76Flox[RIGHTWARDS ARROW]Y3F T cells.

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Figure 5. Peripheral T cells expressing a mutant form of SLP-76 fail to transduce TCR-mediated signals despite normal development and normal TCR levels. WT and SLP-76flox[RIGHTWARDS ARROW]Y3F mice were treated with tam for five days. (A) Expression of CD4 and CD8 (top panels) and TCR-β (bottom panels) on splenocytes was analyzed by FACS. Representative of four experiments (B) Cell lysates prepared from splenocytes stimulated with anti-CD3 for the indicated times were used for western blotting to detect phospho-ZAP-70 (p-ZAP-70), phospho-LAT (p-LAT), phospho-PLCγ1 (p-PLCγ1), total PLCγ1, phospho-ERK (p-ERK) and total ERK. Representative of two experiments. (C) Splenocytes from SLP-76WT (dashed line), SLP-76flox[RIGHTWARDS ARROW]Y3F (dotted line), SLP-76cKO (solid line with anti-CD3 overnight and CD69 levels were assessed by flow cytometry on gated CD4+ (top panel) and CD8+ (bottom panel) T cells. Shaded areas represent staining of unstimulated cells. Representative of two experiments. (D) Thymidine incorporation was measured after overnight anti-CD3 stimulation of splenocytes from SLP-76WT (squares), SLP-76cKO (circles) and SLP-76flox[RIGHTWARDS ARROW]Y3F (triangles) mice. Representative of two experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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 WT control lymphocytes both before and after deletion. SLP-76-deficient T cells expressing WT 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, 16, 30, 31. Similar to conditional loss of Lck and Fyn 32, 33, 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 34–36. Increased TCR signaling resulting from deletion of negative regulators hematopoietic progenitor kinase 1 or Sts1/Sts2 worsens EAE 37, 38. 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 murine T cells 40. In addition, preliminary studies have shown no differences in trafficking of SLP-76cKO and SLP-76cHET 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, APC 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 is 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 question 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 hematopoietic 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 double negative 3 (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 hematopoietic 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, is complicated by potential defects in SLP-76-deficient APCs.

Our result of deficient immune responses following conditional deletion of a TCR proximal adaptor is 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 knockin 30, 31. 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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice and induction of in vivo SLP-76 deletion

The generation of SLP-76Flox mice, in which exon 3 is flanked by loxP sites, has been previously described 16, 17, 19, 43. SLP-76Δ mice lack exon 3 of SLP-76 in the germline and were generated by breeding SLP-76Flox mice with protamine-1-Cre transgenic mice 44. SLP-76null mice, containing a germline deletion of SLP-76 generated by insertion of Neomycin at exon 1 9, and SLP-76Y3F mice, expressing the Y3F mutant form of SLP-76 under the transcriptional control of the CD2 promoter/enhancer 15, were obtained from Gary Koretzky (University of Pennsylvania). R26RYFP 20 were obtained from F. Costantini (Columbia University). SLP-76Flox/Flox mice were inter-crossed with CreT2 transgenic mice 21 to generate mice with tam-inducible deletion of SLP-76. All conditional SLP-76 mice were crossed with C57BL/6 mice for at least two generations. All mice studied received tam treatment. RAG2−/−CD45SJL mice were initially purchased from Taconic (model ♯000461-M/F). Mice were housed and bred in the University of Pennsylvania mouse facility. All animal experiments were performed in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee guidelines.

Tam administration

Tam (Sigma: T-5648, St. Louis, MO) was resuspended in ethanol at a concentration of 1 mg/μL then diluted into corn oil with frequent vortexing for a final stock concentration of 20 mg/mL. Using a weight-based regimen, 200 μg tam/g mouse body weight was administered by oral gavage each day for five consecutive days.

Flow cytometry

Fluorochrome conjugated antibodies to CD4, CD8, CD44, CD25, CD69, CD62L and TCR-β were purchased from Becton-Dickenson or eBioscience. FITC-labeled anti-SLP-76 was purchased from Becton-Dickenson, P-Erk from cell signaling and PE-anti-rabbit from Jackson Immunologicals. PE-labeled sheep anti-SLP-76 antisera were used as described 9. Surface and intracellular staining was performed using standard methods. Data were acquired using either FACS-Calibur or LSR-II instruments (Becton-Dickinson). FACS analysis was performed using FlowJo (Tree Star). Splenocyte YFP+ and YFP populations were obtained using flow cytometric sorting on a BD FACSAria.

Real-time PCR

Cells were sorted by high-speed FACS directly into Trizol Reagent (Invitrogen). RNA was isolated according to the manufacture's instructions. cDNA was made using a SuperScriptII First Strand kit (Invitrogen). Real-time PCR reactions were performed using murine SLP-76 and Actin primer/probe mix from Applied Biosystems. Reactions were performed in 10 μL total volume using a 7500 Fast Real-time PCR System with FastTaq Master Mix (Applied Biosystems). For analysis, samples were normalized to actin levels and then set relative to normalized YFP+ SLP-76cHET levels. Each sample was performed in triplicate.

Calcium flux

Calcium flux assays were performed as described 16. Briefly, lymphocytes were loaded with 2 μg/mL of Indo-1 (Molecular Probes) at 30°C for 30 min and concurrently stained with biotinylated anti-CD3 and anti-CD4 (clone RMA4-4), PE-labeled anti-CD4 (clone RMA4-5) and APC-labeled-anti-CD8 antibodies. Baseline levels were measured for 30 s before addition of streptavidin (Molecular Probes) as a crosslinker. Cytoplasmic calcium was measured by a change in Indo-1 fluorescence. Ionomycin was added to all samples during the last 30 s of data collection. A BD-LSR (Becton Dickinson) flow cytometer was used for data acquisition and FlowJo software (Tree Star) was used for analysis.

Western blotting

For experiments with sorted cells, splenocytes+/− lymph node cells were B220-depleted using B220 MACs purification (Miltenyi Biotech) or BioMag Goat Anti-Rat IgG magnetic beads (Qiagen) bound to purified anti-B220 (eBioscience). Cells then underwent flow cytometric sorting for expression of YFP and/or CD90.2 using a FACs Aria (BD) or MoFlo (DAKO-Cytomation) sorter. Alternatively, four million bulk splenocytes were left unstimulated or were stimulated by the addition of 5 μg/mL final anti-CD3 (500A2, Pharmingen) for the indicated times. Cells were then lysed in a buffer containing 1% Nonidet-P40, 150 mM NaCl, 50 mM Tris, pH 7.4 with 1 mM Na3VO4, 5 mM NaF, 1 mM PMSF, 5 mM Na pyrophosphate and Protease Inhibitor Cocktail (Sigma). Proteins were resolved by SDS-PAGE and transferred to a Trans-Blot Nitrocellulose membrane (Bio-Rad Laboratories). Antibodies used for blotting included phospho-PLCγ-1 (Tyr783), phospho-pp44/42 MAPK (Thr202/Tyr202), phospho-AKT (Ser-473), phospho-LAT (Tyr191) and PLCγ-1 from Cell Signaling Technology and ERK1/2 (Santa Cruz) and SLP-76 (eBioscience).

Phospho-flow analysis

In total, 2×106 splenocytes were incubated with α-CD3 (500A2, BD Pharmingen) at 2.5 μg/mL or PMA (Sigma) at 1 μg/mL for 15 min in reaction volumes of 200 μL. Reactions were stopped with 3 mL of 1×BD Phosflow lyse/fix buffer prewarmed to 37°C. Cells were washed and surface stained in FACS buffer (PBS with 3% FCS and 0.01% Sodium azide), and then equilibrated and probed for pERK in BD Perm/wash buffer. Primary α-pERK (Cell Signaling) was used at 1:200, and secondary α-rabbit-Ax488 (Molecular Probes) was used at 1:100. Flow cytometry was performed with an LSR II cytometer (BD) and analyzed using FlowJo software (Tree Star).

Proliferation and activation marker upregulation

Splenocytes (0.5×106) were cultured in a flat-bottom 96-well plate with titrated doses of anti-CD3 (2C11) for 4 days. Cells were pulsed with 3H-thymidine for the last 20 h of the culture and harvested the following day. To measure the upregulation of CD69, splenocytes were cultured as above. Eighteen hours later, cells were stained with anti-CD4, anti-CD8, anti-CD69 and anti-CD25 and evaluated by flow cytometry.

LIP

Cells derived from mice lacking the R26RYFP reporter were Thy1.2+ purified using MACS magnetic beads (Miltenyi Biotech), labeled with CFSE and 2×106 cells were intravenously injected into RAG2−/−CD45SJL mice. Seven days later, single cell suspensions from lymph node and spleen were analyzed by flow cytometry. Alternatively, 2×106 SLP-76cHet and SLP-76cKO splenocytes were injected intravenously into RAG2−/−CD45SJL or CD45SJL non-lymphopenic control mice. On days five through seven, recipient mice were administered BRDU. On day seven, single cell suspensions from lymph node and spleen were assessed for BRDU incorporation using a BRDU Flow Cytometry Assay Kit (BD Biosciences) according to the manufacturer's protocol.

Induction and evaluation of EAE

Seven- to nine-wk-old mice were immunized with MOG peptide 35–55 (CS Bio; Menlo Park, CA) according to the standard protocol 26. Briefly, subcutaneous injections of 200 μg MOG35–55 emulsified in 500 μg CFA (Sigma) were divided over the four flanks of each mouse. On the day of immunization and 48 h later, mice were injected intraperitoneally with 200 ng pertussis toxin (List Biological laboratories; Campbell, CA) diluted in 200 μL of PBS. Clinical evaluation was undertaken on subsequent days based on the following scores: 0, no weakness; 1, limp tail; 2, mild hind limb paresis; 3, severe hind limb paresis; 4, hind limb paralysis; 5, quadraplegia or moribund. Scoring was undertaken in a blinded fashion by one examiner (G. F. W.).

Mononuclear cell isolation from the CNS

CNS mononuclear cells were isolated as previously described 45. Briefly, mice were perfused with ice-cold PBS. Spinal cord and brain tissue were isolated and homogenized in RPMI 1640 medium with 10% FCS, followed by filtration through nytex (Sefar America; Depew, NY). Percoll (GE Healthcare Biosciences; Uppsala, Sweden) was added to a final concentration of 30% and the suspension was centrifuged at 1300×g for 30 min at 4°C. The Percoll and lipid layers were aspirated and the cell pellet was washed in 5 mL RPMI 1640 medium with 10% FCS.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank F. Costantini and G. Koretzky for providing mouse lines. We thank L. Kovoor for technical assistance. We thank D. Farber for critical review of this manuscript. This work was supported in part by grants from the NIH/NIAID (K08 AI055806 and R01 AI085160 to J. S. M. and K01AR52802 to M. S. J.) and the John Merrill Award from the American Society of Nephrology/American Society of Transplantation (to J. S. M.). G. F. W. and T. M. L. receive support from the National Multiple Sclerosis Society.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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