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Requirement for Tec Kinases Rlk and Itk in T Cell Receptor Signaling and Immunity

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Science  23 Apr 1999:
Vol. 284, Issue 5414, pp. 638-641
DOI: 10.1126/science.284.5414.638

Abstract

T cell receptor (TCR) signaling requires activation of Zap-70 and Src family tyrosine kinases, but requirements for other tyrosine kinases are less clear. Combined deletion in mice of two Tec kinases, Rlk and Itk, caused marked defects in TCR responses including proliferation, cytokine production, and apoptosis in vitro and adaptive immune responses to Toxoplasma gondii in vivo. Molecular events immediately downstream from the TCR were intact inrlk −/− itk −/− cells, but intermediate events including inositol trisphosphate production, calcium mobilization, and mitogen-activated protein kinase activation were impaired, establishing Tec kinases as critical regulators of TCR signaling required for phospholipase C–γ activation.

Stimulation of T lymphocytes through the TCR elicits broad responses required for proper immune function, including cell proliferation, cytokine production, and apoptosis. Key components of TCR signaling are nonreceptor tyrosine kinases, and cells lacking Lck or Zap-70 are essentially unresponsive to antigen (1, 2). The Tec kinases, exemplified by BTK, are a distinct family with members specifically expressed in lymphoid lineages (3, 4). BTKmutations cause severe immunodeficiencies, with defective B cell development and function (4). No disease is yet associated with the loss of Tec kinases in T lymphocytes, which express Itk, Rlk, and Tec (3). Itk-deficient mice have only mildly impaired responses to infection (5), suggesting functional redundancy between these kinases.

Rlk (also called Txk) is a Tec kinase expressed in developing and mature T lymphocytes (6). Rlk resembles other Tec kinases in that it lacks the COOH-terminal regulatory tyrosine and myristoylation sequences of Src kinases, has a proline-rich region that binds SH3 domains, and is activated by phosphorylation by Src kinases. However, Rlk lacks the phosphatidylinositol phosphate–binding pleckstrin homology domain common to other Tec kinases and is activated independently of phosphoinositide 3-kinase (PI3K) activity. Instead, Rlk has a palmitoylated cysteine-string motif, required for subcellular localization (7). To better understand the contribution of Tec kinases to T cell signaling, we analyzed mice mutated in Rlk, Itk, or both kinases.

Because alternate translation initiation generates at least two Rlk isoforms, a full-length protein and a shorter species lacking the cysteine string (7), we introduced a targeted mutation downstream of the second initiation codon of rlkinto mouse embryonic stem (ES) cells (Fig. 1, A and B). Heterozygous F1offspring of chimeric mice were interbred and generated normal numbers of homozygous rlk mutants with no evidence of kinase-active Rlk or truncated Rlk protein species in lymphoid cell extracts (Fig. 1C). Preliminary analyses of these mice suggested that all thymocyte and mature T cell populations were present with no major defects in T cell functional responses in vitro (see below).

Figure 1

Disruption of rlk gene. (A) Rlk gene disruption (24). Gray box, 3′ flanking probe. H III, Hind III. (B) Southern (DNA) blot analyses of ES clone DNA probed with 3′ flanking probe. (C) (Left) Rlk kinase assay from splenocytes (7). (Right) Immunoprecipitated Rlk immunoblotted with antisera to the NH2-terminus of Rlk. Samples were resolved by 10% SDS-PAGE for full-length Rlk (top panels) and by 4 to 20% SDS-PAGE for smaller protein species (predicted truncated products of 16 and 10 kD, bottom panels). IP, immunoprecipitation; IB, immunoblot; HC, heavy chain; LC, light chain. Numbers at left indicate the molecular mass (in kilodaltons). (D) CD4+ and CD8+splenic T cell subsets. Cellularity of spleens (×107) and percentage of CD4+ and CD8+ cells represent the average of 7 to 12 mice per genotype. Flow cytometric antibodies were obtained from PharMingen.

Northern blot analyses of RNA from lymphoid organs ofrlk −/− mice showed slightly increased amounts of itk mRNA (8). Thus, up-regulation of related Tec kinases may partially compensate for the lack of Rlk. To address this possibility, we generated rlk −/− itk −/− mice by interbreeding. Itk-deficient mice have reduced numbers of mature T cells, particularly CD4+ cells, causing a decreased CD4:CD8 ratio (9).rlk −/− itk −/− mutants, however, had normal total T cell numbers (Fig. 1D). Both CD4+ and CD8+ cell numbers are increased relative to itk −/− mice; therefore,rlk −/− itk −/− animals still have a decreased CD4:CD8 ratio (Fig. 1D), suggesting altered regulation of lymphoid development and homeostasis (10).

Althoughrlk −/− itk −/− mice have mature T cells, these cells failed to respond to TCR activation as measured by proliferation of splenocytes stimulated with antibody to CD3ɛ (anti-CD3ɛ) (Fig. 2A) or concanavalin A (Con A) (8). This defect was confirmed with purified T cells in the presence of irradiated wild-type antigen-presenting cells and in mixed lymphocyte reactions (8). In contrast to itk −/−cells, which have only variable defects in CD4+ cell proliferation (9),rlk −/− itk −/− cells had proliferative defects in both CD4+ and CD8+T cell populations (8). Treatment with phorbol myristate acid (PMA) and the calcium ionophore ionomycin induced proliferation equivalent to that of wild-type cells, suggesting that defects in these cells lie proximal to Ca2+ mobilization and protein kinase C (PKC) activation.

Figure 2

Disruption of Rlk and Itk impairs T cell functional responses. (A) Proliferative defects. Total splenocytes stimulated with 2C11 (anti-CD3ɛ) with or without exogenous recombinant human IL-2 (100 U/ml; Parke-Davis) or PMA (10 ng/ml) and ionomycin (1 μg/ml) (P/I) for 40 to 48 hours were pulsed with [3H]thymidine and harvested after 6 to 12 hours. (B) IL-2 and IFN-γ levels after stimulation of splenocytes with anti-CD3 (3 μg/ml) for 24 hours measured by enzyme-linked immunosorbent assay (ELISA). (C) CD25 expression after stimulation of splenocytes with 2C11. Baseline CD25 expression is in gray. WT, wild type. (D) TCR-mediated apoptosis (25).

Splenocytes from rlk −/− animals stimulated in vitro with either anti-CD3ɛ or anti–Con A secreted half the interleukin 2 (IL-2) produced by wild-type animals, whereasitk −/− cells had more severe reductions in IL-2 production, as previously reported (9, 11). However, deficiency of both kinases further reduced IL-2 and interferon-γ (IFN-γ) production to undetectable levels (Fig. 2B). Furthermore, proliferation ofrlk −/− itk −/− cells was only partially restored by exogenous IL-2 (Fig. 2A), probably as a result of defective up-regulation of IL-2 receptor-α chain (CD25) (Fig. 2C), even in the presence of exogenous IL-2 (8). We also observed decreased TCR-driven apoptosis, an important component of T cell homeostasis (Fig. 2D) (12), suggesting one mechanism for increased T cell numbers inrlk −/− itk −/− mice relative to itk −/− animals.

To investigate biochemical defects inrlk −/− itk −/− T cells, we examined signaling events downstream of the TCR. Patterns of CD3-mediated tyrosine phosphorylation were not grossly altered, and the most proximal consequences of T cell activation, including phosphorylation of TCR zeta chains and Zap-70 (1), were intact in all genotypes (Fig. 3A).

Figure 3

Ca2+ and MAP kinase pathways inrlk −/− itk −/−mice. (A) Tyrosine phosphorylation of TCR zeta chain and Zap-70 in response to TCR stimulation (23). (B) Calcium flux in response to TCR stimulation (26). First arrow designates addition of biotinylated 2C11 and second arrow denotes cross-linking with streptavidin. (C) PLC-γ phosphorylation. Lysates from 2C11-stimulated purified T cells (5 × 106 ) were immunoprecipitated with anti–PLC-γ (Upstate), immunoblotted with anti-phosphotyrosine, and then reprobed with anti–PLC-γ. (D) IP3 production (27). (E) MAP kinase (MAPK) activation. Total cell lysates were resolved by SDS-PAGE (4 to 20% gradient) and probed sequentially with anti–phospho-MAPK that detects dually phosphorylated (active) ERK1 and ERK2 and anti-ERK1 (Santa Cruz) that recognizes ERK1 and ERK2. (F) T cell proliferation in response to anti-TCR and PMA. Proliferation was assayed as in Fig. 2A.

In contrast, Ca2+ mobilization upon T cell activation (Fig. 3B) was reduced in itk −/− cells (11) and substantially worsened by deficiency of both Itk and Rlk. Ca2+ mobilization in T cells is initiated by membrane association and activation of phospholipase C–γ (PLC-γ), which catalyzes the breakdown of phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3), a stimulant for Ca2+ mobilization, and diacylglycerol (DG), an activator of PKC (13). Within 2 min of TCR stimulation, tyrosine phosphorylation of PLC-γ was observed in wild-type cells, but not inrlk −/− itk −/− oritk −/− T cells (Fig. 3C). However, 5 min after stimulation, PLC-γ was phosphorylated in both wild-type and mutant cells, with only a fewfold reduction inrlk −/− itk −/− cells. Wild-type cells also showed maximal IP3 levels at 2 to 3 min after stimulation, consistent with rapid phosphorylation of PLC-γ and peak Ca2+mobilization. In contrast, double knockout cells produced virtually no IP3, even at time points when PLC-γ phosphorylation was observed (Fig. 3D). Our results suggest that only early PLC-γ tyrosine phosphorylation correlates with IP3 production and that this defect may be the major cause of altered Ca2+ mobilization inrlk −/− itk −/− cells.

DG, the other product of PLC-γ, activates mitogen-activated protein (MAP) kinases ERK1 and ERK2 through PKC stimulation of the Ras/Raf pathway (14). Decreases in phosphorylated (active) ERK1 and ERK2 were also observed after anti-CD3 stimulation of T cells from bothitk −/− andrlk −/− itk −/− mice (Fig. 3E) (15). To specifically examine DG-mediated MAP kinase activation, we stimulated cells with anti-CD3 and low concentrations of PMA, a phorbol ester that directly activates PKC. Anti-CD3 plus PMA rescued proliferation inrlk −/− itk −/− cells (Fig. 3F) and normalized activation of ERK1 and ERK2 (8). Hence, the defective MAP kinase activation inrlk −/− itk −/− cells probably results from defects in DG-PKC–mediated pathways (16). Similarly, ionomycin also partially bypassesrlk −/− itk −/− defects in CD3-driven proliferation (8). Thus, Tec family kinases are critical in TCR signaling for full activation of PLC-γ and both arms of its downstream effector pathways.

To assess whether these in vitro defects translate into immunodeficiency in vivo, we challenged the Tec kinase–deficient mice with the intracellular pathogen Toxoplasma gondii. Mice of all mutant genotypes survived early infection, which requires natural killer (NK) cell activity, suggesting normal innate immunity (Fig. 4A) (17). However, 30 days after infection,rlk −/− itk −/− mice appeared visibly ill and had a mean survival time (MST) of 41 days. Itk-deficient animals died with an MST of 69 days, whereas only half ofrlk −/− animals had succumbed by 102 days. In these experiments, 80% of wild-type animals in the same genetic background survived over 18 weeks (126 days; Fig. 4A). These results suggest a graded resistance to T. gondii that correlates with expression of Tec family kinases.

Figure 4

Impaired resistance to T. gondi. Mice were infected intraperitoneally with 20 cysts of T. gondii (ME49 strain). (A) Survival curves. Pooled data from two independent infections (wild type, n = 11; rlk −/− andrlk −/− itk −/−,n = 9; itk −/−,n = 8). Numbers indicate MST; d, days. (B) (Left) Serum IFN-γ levels 6 days after acute T. gondii infection determined by ELISA. (Right) Con A (3 μg/ml) and STAg (1 μg/ml) induced IFN-γ production at 48 hours from splenocytes from chronically infected (day 30) mice. IFN-γ levels from STAg-stimulated uninfected wild-type splenocytes are less than 1 ng/ml. (C) Brain cyst count 30 days after T. gondii infection (28). CNS, central nervous system.

Correspondingly, serum IFN-γ concentrations 5 days after infection were normal in mice of all genotypes, suggesting unimpaired NK cell function (Fig. 4B). In contrast, splenocytes fromrlk −/− itk −/− mice 30 days after infection cultured with Con A or low-dose soluble tachyzoite antigen (STAg) produced decreased IFN-γ (Fig. 4B), indicating a deficit in adaptive antigen-driven responses, consistent with decreased cytokine production in naı̈verlk −/− itk −/− T cells (see Fig. 2B). Itk-deficient splenocytes had near normal IFN-γ production in response to these stimuli, thereby demonstrating increased responsiveness of itk −/− cells after in vivo challenge (see Fig. 2B). Finally, 30 days after infection,rlk −/− itk −/− mice had increased brain cyst numbers, indicating decreased ability to limit infection at this T cell–dependent stage (Fig. 4C). Thus, the enhanced susceptibility ofrlk −/− itk −/− mice toT. gondii demonstrates the physiologic importance in vivo of the marked defects observed in vitro.

Together, the above biological and biochemical evidence demonstrates that Tec family kinases are essential components of TCR signaling required to mount a successful immune response. Although defects in either T cell–specific Tec kinase, Itk or Rlk, have only mild to moderate effects on T cell function, elimination of both kinases resulted in a profound loss of mature T cell function in vitro and in vivo. Our data show that nonreceptor tyrosine kinases are important not only for initiation of antigen-driven responses but also for downstream propagation of these signals. The biochemical defects reported here resemble those observed in Slp-76–deficient cells (18), suggesting that Tec kinases and Slp-76 are part of a complex that activates PLC-γ. The requirement for multiple Tec kinases in TCR signaling is also intriguing, particularly given differential sensitivities of Rlk and Itk activation to PI3K inhibition (7). Products of PI3K are required for Btk activation of sustained Ca2+ mobilization in response to secretory immunoglobulin M (19). Thus, Rlk, a distinct Tec kinase that functions independently of PI3K, may provide a second level of regulation to antigen receptor signaling pathways in T cells.

Current models suggest that immune responses in lymphocytes are exquisitely controlled, requiring multiple finely tuned levels of activation (20). Thus, the Tec kinases, unlike the more proximally acting Lck and Zap-70, may not serve as primary triggers of the immune response but instead may further refine the strength of lymphocyte signaling by acting as critical modulators of PLC-γ and downstream effectors.

  • * These authors contributed equally to this work.

  • Present address: Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.

  • To whom correspondence should be addressed. E-mail: pams{at}nhgri.nih.gov

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