Activation of naı¨ve CD4 T cells by anti-CD3 reveals an important role for Fyn in Lck-mediated signaling

Katsuji Sugie*, Myung-Shin Jeon†, and Howard M. Grey*‡

Divisions of *Vaccine Discovery and †Cell Biology, La Jolla Institute for and Immunology, San Diego, CA 92121

Contributed by Howard M. Grey, August 23, 2004 Although there was no impairment in IL-2 secretion and prolifer- Fyn-deficient T cells by -pulsed APCs led to a robust ation of Fyn-deficient naı¨veCD4 cells after stimulation with anti- proliferative and IL-2 response, whereas anti-CD3-mediated gen and antigen-presenting cells, stimulation of these cells with stimulation revealed a profound signaling defect in Fyn-deficient anti-CD3 and anti-CD28 revealed profound defects. Crosslinking of T cells. This defect could be corrected by the cocrosslinking of purified wild-type naı¨veCD4 cells with anti-CD3 activated Lck and CD3 with CD4. Furthermore, the signaling defect of Fyn- initiated the signaling cascade downstream of Lck, including phos- deficient T cells stimulated with anti-CD3 was restricted to naı¨ve phorylation of ZAP-70, LAT, and PLC-␥1; calcium flux; and dephos- T cells and was not observed when Fyn-deficient effector T cells phorylation and nuclear translocation of the nuclear factor of were analyzed. Biochemical experiments on anti-CD3 stimulated activated T cells (NFAT)p. All of these signaling events were cells strongly suggest that the presence of Fyn was necessary for diminished severely in Fyn-deficient naı¨ve cells activated by CD3 an effective recruitment and͞or activation of Lck in the region crosslinking. Coaggregation of CD3 and CD4 reconstituted this of the TCR. Lck-dependent signaling pathway in Fyn؊/؊ T cells. These results suggest that when signaling of naı¨veT cells is restricted to the Materials and Methods antigen receptor, Fyn plays an essential role by positive regulation Animals. Pigeon cytochrome c (PCC)-specific AD10 TCR trans- of Lck activity. genic (Tg) mice were bred on a B10.A background (16, 17) in our animal facility. B10.A, B6.129 SF2J, and FynϪ/Ϫ B6.129 SF2J Ϫ/Ϫ ignaling through the T cell antigen receptor (TCR) is initi- mice were purchased from The Jackson Laboratory. Fyn Sated by the activity of the Src family tyrosine kinases, Fyn and mice were bred onto AD10 TCR Tg genetic background in our Lck (1). It has been shown in numerous studies using Lck- animal facility (15). deficient cell lines and mice whose peripheral T cells are and Reagents. The following antibodies were used in deficient in Lck that this enzyme plays a critical upstream role ␧ in the signaling cascade leading to T cell development, activation, this study: biotinylated anti-mouse CD3 (2C11), biotinylated and differentiation (2). In contrast to Lck, the role of Fyn kinase anti-mouse CD4 (RM4-5), anti-mouse CD28 (37.51), anti-mouse in T cell activation and development is less well defined. With the IL-2 (JES6-1A12), biotinylated anti-mouse IL-2 (JES6-5H4), exception of natural killer T (NKT) cells (3, 4), Fyn deficiency and recombinant mouse IL-2 (BD Biosciences and Pharmingen); has little or no effect on T cell development in the thymus, and anti-mouse nuclear factor of activated T cells p (NFATp) peripheral T cells from Fyn-deficient mice, to the extent they (4G6-G5), anti-Lck (3A5), and anti-Fyn (FYN3) (Santa Cruz Biotechnology); anti-phospho-Y783 PLC-␥1, anti-phospho-Y319 have been studied, show variable and incomplete defects in ␥ mounting immune responses (5–7). These findings are consistent ZAP-70, anti-phospho-Y171 LAT, anti-PLC- 1, and anti- with the concept that, for the most part, Fyn is a redundant Src ZAP-70 (Cell Signaling Technology, Beverly, MA); and anti- kinase without a unique function in the signaling of T cells. The LAT (Upstate Biotechnology, Lake Placid, NY). Alexa Fluor finding that some of the substrates for Lck [such as the immu- 633, Alexa Fluor 488, and Indo-1-AM were purchased from noreceptor tyrosine-based activation motifs (ITAMs) of TCR␨ Molecular Probes. Streptavidin and ionomycin were purchased as well as CD3␥␦␧] can also serve as substrates for Fyn (8) from Pierce and Calbiochem, respectively. Enolase purified from supports this view. Furthermore, some substrates of ZAP-70, Saccharomyces cerevisiae and rabbit muscle were purchased from such as Vav (9) and SLP 76 (8), are also substrates of Fyn. Sigma. PCC 88-104 peptide was synthesized on a Symphony However, other studies have shown that Fyn has its own unique synthesizer (Rainin Instruments) and purified as described (18). substrates, some of which play significant roles in T cell activa- Cells. Naı¨ve CD4 cells were purified from lymph nodes and tion. These include ADAP (SLAP-130͞FYB) (10), Pyk2 (11), spleens of 8- to 12-week-old mice by using CD4 T cell isolation WASp (12), and CBP (13). Other indications that Fyn plays some kits and LS separation columns (Miltenyi Biotec, Auburn, CA). IMMUNOLOGY unique role in TCR-mediated signaling come from our previous Flow cytometry demonstrated that Ͼ95% of purified cells were studies that have shown that stimulation of T cells with low ϩ CD4 CD62Lhigh. For APCs, irradiated (3,000 rad; 1 rad ϭ 0.01 affinity ligands that function as TCR antagonists leads to Gy) B10.A spleen cells depleted of T cells by Thy 1.2 MicroBeads preferential activation of Fyn in the absence of any detectable and LS separation columns (Miltenyi Biotec) were used. CH27, changes in the activity of Lck or ZAP-70 (14, 15). Also, a mouse B lymphoma cell line that expresses I–Ek, was also used Fyn-deficient T cells are particularly inefficient at responding to as APCs in some experiments. weak TCR agonists (7). Because of these more recent findings that suggest a unique Cell Cultures. Cells were cultured in RPMI medium 1640 (In- role for Fyn in T cell activation, we have reinvestigated the vitrogen) supplemented with 2 mM L-glutamine, 50 ␮M 2-ME, capacity of Fyn-deficient T cells to respond to TCR-mediated nonessential amino acids (Invitrogen), 1 mM sodium pyruvate, signaling in this study. We compared naı¨ve CD4 T cells from Fyn-deficient and wild-type mice that contained a TCR trans- gene for their capacity to respond to either antigen presented by Abbreviations: PCC, pigeon cytochrome c; Tg, transgenic; TCR, T cell antigen receptor; APC, antigen-presenting cells (APCs) or to respond to - antigen-presenting cell; NFAT, nuclear factor of activated T cells; AP-1, activator protein 1; mediated CD3 crosslinking. These experiments demonstrated ITAM, immunoreceptor tyrosine-based activation motif. that the relative importance of Fyn depended highly on the mode ‡To whom correspondence should be addressed. E-mail: [email protected]. of stimulation and the activation status of T cells; stimulation of © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406168101 PNAS ͉ October 12, 2004 ͉ vol. 101 ͉ no. 41 ͉ 14859–14864 Downloaded by guest on September 26, 2021 10% FCS (Omega Scientific, Tarzana, CA), and antibiotics. To generate effector cells, 1 ϫ 106 naı¨ve CD4 cells from Fynϩ/ϩ and FynϪ/Ϫ AD10 TCR Tg mice were cultured with 2 ϫ 107 irradiated APCs from B10.A mice in the presence of 1 ␮g͞ml PCC 88-104 peptide for 8 days with the addition of recombinant mouse IL-2 (1 ng͞ml) on day 4.

IL-2 Secretion and Proliferation. Purified naı¨ve or effector CD4 cells were plated at 1 ϫ 105 cells per well in 0.2-ml 96-well flat-bottomed tissue culture-untreated plates (Falcon) coated with anti-CD3 with or without the addition of anti-CD4. Soluble Fig. 1. Fyn is required for naı¨ve CD4 cells to respond to anti-CD3 and ␮ ͞ anti-CD28 stimulation. (a) Naı¨veCD4 cells purified from AD10 TCR Tg mice on anti-CD28 (1 g ml) was also added to the culture. Alterna- ϩ/ϩ Ϫ/Ϫ tively, CD4 cells were plated at 1.0 ϫ 105 cells per well with 5.0 ϫ Fyn and Fyn backgrounds were stimulated either with plate-bound 5 ␮ ͞ anti-CD3 and soluble anti-CD28 or antigen (PCC 88-104) and irradiated APCs. 10 APCs and PCC 88-104 peptide (1 g ml) in 96-well flat- (b) Effector CD4 cells generated in vitro were stimulated either with plate- bottomed plates (Falcon). Supernatants were taken between 24 bound anti-CD3 and soluble anti-CD28 or antigen (PCC 88-104) and irradiated and 48 h of culture to measure IL-2 by ELISA (BD Biosciences APCs. and Pharmingen). For proliferation, the uptake of tritiated thymidine [1 ␮Ci (1 Ci ϭ 37 GBq); ICN] at 48–64 h of culture was determined. were purchased from Promega and end-labeled with ␥-[32P]ATP. After 20 min of incubation, complexes were sepa- Intracellular Free Calcium. CD4 cells were loaded with Indo-1-AM rated on 6% nondenaturing polyacrylamide gels. as described in ref. 19, incubated with biotinylated anti-CD3 with or without biotinylated anti-CD4 at room temperature for 30 Results min, centrifuged, and resuspended at 1 ϫ 106 cells per ml. Cells Effect of Fyn Deficiency on TCR-Mediated Activation of Naı¨ve CD4 were analyzed on a LSR flow cytometer (BD Biosciences). Data Cells. To determine the role of Fyn in the activation of naı¨ve CD4 analysis was performed by using FLOWJO software (Tree Star, ϩ cells, we used wild-type or Fyn-deficient mice that expressed a Ashland, OR). To measure intracellular free Ca2 concentration transgene for a TCR specific for PCC 88-104 peptide presented 2ϩ ([Ca ]i) in CD4 cells stimulated with antigen and APCs, CD4 by the class II MHC, I–EK. Purified CD4 T cells from these mice cells that formed conjugates with APCs were identified as were stimulated in two different ways, either (i) by the cognate ϩ/ϩ described (20). Briefly, naı¨ve CD4 cells purified from Fyn and PCC 88-104 pulsed onto APCs, or (ii) to confine Ϫ/Ϫ Fyn AD10 TCR Tg mice were labeled with Alexa Fluor 633 stimulation to a simpler system involving TCR-mediated stim- and loaded with Indo-1-AM. CH27 APCs were labeled with ␮ ͞ ulation, with plate-bound anti-CD3 antibodies and soluble anti- Alexa Fluor 488 and pulsed with 10 g ml PCC 88-104 peptide. CD28. T cell activation was measured by the production of IL-2. CD4 and CH27 cells were mixed, centrifuged briefly, incubated Strikingly, different results were obtained with respect to the at 37°C for 2 min, resuspended, and analyzed by flow cytometry. ability of FynϪ/Ϫ T cells to respond to these two modes of 2ϩ ͞ [Ca ]i analysis was performed on Alexa 633 Alexa 488 double- stimulation. As shown in Fig. 1a, Fynϩ/ϩ and FynϪ/Ϫ T cells positive T cell–APC conjugates. responded equivalently when stimulated by antigen-pulsed APCs. In contrast, the same Fyn-deficient T cells, when stimu- Immunoblotting. NFATp was detected in whole-cell lysates by lated by anti-CD3 and anti-CD28 antibodies, demonstrated a immunoblotting as described (21). To analyze tyrosine- severe defect in IL-2 production. Low to undetectable quantities phosphorylated ZAP-70, LAT, and PLC-␥1, 8 ϫ 106 naı¨ve CD4 of IL-2 were observed routinely in the cultures containing cells were incubated with biotinylated anti-CD3 with or without Ϫ Ϫ Fyn / T cells, whereas vigorous IL-2 responses were obtained biotinylated anti-CD4 (5 ␮g͞ml each) on ice for 30 min, centri- ϩ ϩ in the Fyn / T cell cultures. A similar, albeit less striking, defect fuged, and resuspended in ice-chilled PBS. After prewarming at Ϫ Ϫ in proliferation of anti-CD3 stimulated Fyn / T cells was also 37°C for 1 min, cells were crosslinked by addition of streptavidin (50 ␮g͞ml) and incubated at 37°C for 30 sec. Total-cell lysates observed (Fig. 7, which is published as supporting information were separated by 8% SDS͞PAGE under reducing conditions, on the PNAS web site). transferred onto Immobilon-P, and probed with anti- ؊/؊ 2؉ phosphopeptide antibodies and, subsequently, antibodies against Failure of Fyn T Cells to Increase Cytosolic Ca After TCR Crosslink- ZAP-70, LAT, and PLC-␥1 proteins, as indicated. ing. The striking contrast between the ability of naı¨ve Fyn- deficient T cells to produce IL-2 after stimulation with antigen- In Vitro Kinase Assays. Naive CD4 cells (2 ϫ 107 cells per sample) pulsed APCs and their inability to respond to anti-CD3-coated were preincubated with biotinylated anti-CD3 with or without plates in the presence of soluble anti-CD28 suggested that, in the biotinylated anti-CD4, crosslinked with streptavidin, and solu- absence of T cell–APC conjugates with the consequent trans- bilized in 1% Nonidet P-40͞150 mM NaCl͞20 mM Tris⅐HCl, pH location of various costimulatory, adhesion, and signaling mol- 7.5͞5 mM EDTA͞1% aprotinin͞10 ␮g/ml leupeptin͞1mM ecules in close proximity to the TCR, Fyn plays a critical ͞ nonredundant role in TCR-mediated stimulation. To assess the Na3VO4 1 mM PMSF. The anti-Fyn and anti-Lck immunopre- Ϫ Ϫ cipitates were subjected to in vitro kinase assays as described (15) signaling defect in Fyn / T cells, we examined the intracellular calcium after TCR crosslinking. The data shown in Fig. 2a by using enolase from rabbit muscle and S. cerevisiae (Sigma) for Ϫ Ϫ Lck and Fyn, respectively. substantiate the inability of Fyn / T cells to respond normally to TCR crosslinking. Whereas Fynϩ/ϩ T cells gave a vigorous Nuclear Extraction and Electrophoretic Mobility-Shift Assay. After calcium response after CD3 crosslinking, Fyn-deficient cells stimulation, T cells were lysed in 0.2% Nonidet P-40, and nuclei showed no detectable increase in intracellular calcium. As a ϩ were recovered and salt-extracted as described (22). Nuclear control, the calcium response to the Ca2 ionophore ionomycin extracts (1–5 ␮g) were incubated with 3 ϫ 104 cpm of double- was measured and shown to be normal (data not shown). The ϩ ϩ Ϫ Ϫ stranded oligonucleotide probes. The probes specific for activa- calcium response of Fyn / and Fyn / cells was examined also tor protein 1 (AP-1) (5Ј-CGCTTGATGAGTCAGCCGGAA- after their stimulation by antigen-pulsed APCs. Because of the 3Ј) and NF-␬B(5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј) necessity of centrifuging the T cells and APCs together to

14860 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406168101 Sugie et al. Downloaded by guest on September 26, 2021 Fig. 3. Cocrosslinking of CD3 and CD4 overcomes the signaling defect in FynϪ/Ϫ T cells. (a)Naı¨ve CD4 cells purified from Fynϩ/ϩ and FynϪ/Ϫ B6.129 SF2J mice were cultured in plates coated with anti-CD3 or anti-CD3 and anti-CD4 in the presence of soluble anti-CD28. (b)Naı¨ve CD4 cells from Fynϩ/ϩ (thick line) and FynϪ/Ϫ (thin line) mice were loaded with Indo-1-AM, preincubated with biotinylated anti-CD3 and biotinylated anti-CD4, and analyzed by flow cy- tometry. Cells were crosslinked with streptavidin at 100 sec of the analysis (arrow).

engagement of the TCR without costimulatory T cell molecules involved in the signaling (other than CD28), Fyn played a critical role in the initial phase of activation. Because Fyn has been shown to be constitutively associated with the TCR (24), we postulated that a critical difference between anti-CD3-mediated stimulation and antigen͞APC-mediated stimulation might be the absence of a Src kinase in close proximity of the TCR when Fig. 2. Fyn is required for calcium response after crosslinking of CD3 on naı¨ve FynϪ/Ϫ T cells were stimulated by anti-CD3. To test this idea, we CD4 cells. (a)Naı¨ve CD4 cells from Fynϩ/ϩ (thick line) and FynϪ/Ϫ (thin line) AD10 TCR Tg mice were loaded with Indo-1-AM, preincubated with biotin- examined the effect of CD3 and CD4 cocrosslinking on the ylated anti-CD3, and analyzed by flow cytometry. CD3 was crosslinked with calcium response and IL-2 production. Under these conditions, streptavidin at 100 sec of the analysis (arrow). (b)Naı¨ve CD4 cells from Fynϩ/ϩ CD4-associated Lck would be brought into proximity of the (bold line) and FynϪ/Ϫ (thin line) AD10 TCR Tg mice were labeled with Alexa TCR. It was found that cocrosslinking CD3 and CD4 almost Ϫ Ϫ 633 and loaded with Indo-1-AM. CH27 cells were labeled with Alexa 488 and completely corrected the defect in IL-2 production of Fyn / pulsed with 0 or 10 ␮g͞ml PCC 88-104 peptide. CD4 cells and CH27 cells were naı¨ve T cells (Fig. 3a Right) and the calcium response was mixed, centrifuged, incubated at 37°C for 2 min, and applied to a flow indistinguishable from that of wild-type CD4 T cells (Fig. 3b). cytometer. The calcium levels of CD4 cells that formed conjugates with CD27 cells, identified as Alexa 633͞Alexa 488 double-positive population, were ϩ/ϩ Ϫ/Ϫ Dephosphorylation and Nuclear Localization of NFATp (NFATc2, analyzed. (c) Fyn (thick line) and Fyn (thin line) effector CD4 cells were ؊ ؊ / ؉/؉ loaded with Indo-1-AM, preincubated with biotinylated anti-CD3, and NFAT1) in Fyn and Fyn T Cells. One of the most important crosslinked with streptavidin at 100 sec (arrow). consequences of the rise in intracellular calcium after TCR- mediated signaling is the activation of calcineurin and dephos- phorylation and nuclear transport of NFAT. To determine promote the formation of conjugates, it was not possible to study whether the failure of anti-CD3 crosslinking to induce a dis- ϩ the initial rise in intracellular Ca2 , but the subsequent plateau cernible calcium response in FynϪ/Ϫ T cells was associated with phase of the response was analyzed readily, and as shown in Fig. ϩ/ϩ Ϫ/Ϫ ϩ ϩ Ϫ Ϫ a defect in NFAT dephosphorylation, Fyn and Fyn T cells 2b, Fyn / , and Fyn / T cells responded similarly to this mode were stimulated by CD3 crosslinking, and the NFATp in whole- of stimulation. Thus, as was observed for IL-2 secretion, a cell lysates was analyzed by immunoblotting. The data shown in striking difference in the capacity of Fyn-deficient cells to Fig. 4 are consistent with the calcium data, and they indicate that respond to antibody-mediated TCR crosslinking and antigen NFATp dephosphorylation occurred in Fynϩ/ϩ cells after anti- plus APC stimulation was also evident when Ca2ϩ responses were studied. IMMUNOLOGY

Effector T Cells from Fyn-Deficient Mice Respond Normally to TCR Crosslinking. The response of previously primed effector or memory T cells differs in several respects from naı¨ve T cells, including an increased antigen sensitivity (16) and a decreased dependency on costimulation (23). Because of these differences, we wanted to determine whether the defect in IL-2 production and the lack of a calcium response that was observed after CD3 crosslinking of naı¨ve FynϪ/Ϫ T cells would be seen also with previously primed effector T cells. As shown in Fig. 1b, effector Fynϩ/ϩ and FynϪ/Ϫ T cells secreted similar amounts of IL-2 when stimulated with anti-CD3 and anti-CD28. Similarly, the calcium Ϫ/Ϫ Fig. 4. NFATp dephosphorylation induced by CD3 crosslinking is impaired in response to CD3 crosslinking of previously primed Fyn T cells FynϪ/Ϫ naı¨ve CD4 cells. Naı¨ve CD4 cells were purified from Fynϩ/ϩ and FynϪ/Ϫ was almost as vigorous as wild-type effector T cells (Fig. 2c). B6.129 SF2J mice. The cells were preincubated with biotinylated anti-CD3 alone or together with biotinylated anti-CD4 and crosslinked with streptavi- ؊ ؊ Naı¨ ve Fyn / T Cells Respond Normally When Stimulated by din at room temperature for 3 min. Total-cell lysates were subjected to Cocrosslinking of CD3 and CD4. The data presented above indicated immunoblotting with anti-NFATp mAb. The arrows correspond to phosphor- that when stimulation of naı¨ve CD4 T cells was restricted to ylated (Upper) and dephosphorylated (Lower) NFATp.

Sugie et al. PNAS ͉ October 12, 2004 ͉ vol. 101 ͉ no. 41 ͉ 14861 Downloaded by guest on September 26, 2021 CD3 crosslinking but not in FynϪ/Ϫ T cells (compare lanes 2 and 5). In contrast, when Fynϩ/ϩ and FynϪ/Ϫ T cells were incubated with biotinylated anti-CD3 and anti-CD4 and cocrosslinked with streptavidin, NFATp was dephosphorylated efficiently and equivalently in both Fynϩ/ϩ and FynϪ/Ϫ T cells (lanes 3 and 6). As a further analysis of the defect in T cell signaling after anti-CD3 crosslinking in FynϪ/Ϫ cells, transport of NFATp into the nucleus after stimulation of FynϪ/Ϫ and Fynϩ/ϩ cells was determined. Consistent with the data obtained on NFATp dephosphorylation, FynϪ/Ϫ T cells stimulated with anti-CD3 and CD28 failed to transport NFATp into the nucleus (see Fig. 8, which is published as supporting information on the PNAS web site). Other calcium-regulated transcription factors, such as NF-␬B and AP-1, are also involved in IL-2 gene transcription. Electro- phoretic mobility-shift assay was performed on nuclear extracts of anti-CD3͞CD28 or antigen͞APC-stimulated naı¨ve CD4 T cells from FynϪ/Ϫ and Fynϩ/ϩ TCR Tg T cells. In agreement with the data obtained on NFATp nuclear localization, both NF-␬B and AP-1 were found in greatly reduced quantities in the nuclei Ϫ/Ϫ ͞ of Fyn T cells stimulated with anti-CD3 CD28 compared Fig. 5. Fyn deficiency results in decreased tyrosine phosphorylation of ϩ/ϩ ϩ/ϩ Ϫ/Ϫ with Fyn cells. In contrast, Fyn and Fyn T cells had ZAP-70, LAT, and PLC-␥1 induced by crosslinking of CD3. Naı¨ve CD4 cells similar amounts of nuclear NF-␬B and AP-1 after stimulation purified from Fynϩ/ϩ and FynϪ/Ϫ B6.129 SF2J mice were preincubated with with antigen-pulsed APCs (see Fig. 9, which is published as biotinylated anti-CD3 alone (lanes 2 and 5) or together with biotinylated supporting information on the PNAS web site). anti-CD4 (lanes 3 and 6) and crosslinked with streptavidin at 37°C for 30 sec. Total-cell lysates were subjected to immunoblotting with anti-phosphopep- ␥ Early Tyrosine Phosphorylation Events in Fyn؉/؉ and Fyn؊/؊ T Cells tide antibodies specific for pY319 ZAP-70, pY171 LAT, and pY783 PLC- 1. After stripping, the membranes were probed with anti-protein antibodies specific After TCR Crosslinking. Upstream of the increase in intracellular for ZAP-70, LAT, and PLC-␥1. calcium, several important tyrosine phosphorylation events take place. The ITAMs of CD3 and TCR␨ chains are phosphorylated by Lck and Fyn, and ZAP-70 is phosphorylated by Lck; subse- contrast, FynϪ/Ϫ T cells stimulated by anti-CD3 crosslinking quently, ZAP-70 phosphorylates other substrates, including showed no increase in Lck activity, as measured by autophos- LAT, which then binds several SH2-containing molecules, in- phorylation or enolase phosphorylation (Fig. 6a, lane 5). The bar cluding PLC-␥1, the activity of which at the plasma membrane graph in the lower part of Fig. 6a shows the average fold increase is critical for a calcium response. To determine whether any of from three experiments. Enolase phosphorylation by Lck in- these upstream tyrosine phosphorylation events might be de- ϩ/ϩ Ϫ/Ϫ ϩ/ϩ Ϫ/Ϫ creased 57% in Fyn cells after CD3 crosslinking, and auto- fective in Fyn T cells, Fyn and Fyn T cells were Ϫ/Ϫ stimulated by anti-CD3 or anti-CD3 and anti-CD4 and the phosphorylation increased by 20%. In Fyn T cells, CD3 total-cell lysate was subsequently analyzed by immunoblotting crosslinking resulted in only a 6% increase in both Lck auto- using anti-phosphopeptide antibodies specific for the active phosphorylation and enolase phosphorylation. Cocrosslinking of forms of ZAP-70, LAT, and PLC-␥1. Fig. 5 shows the results. When Fynϩ/ϩ cells were stimulated by anti-CD3 crosslinking, active forms of ZAP70, LAT, and PLC-␥1 were clearly formed (Fig. 5, lane 2). After CD3 and CD4 cocrosslinking, the amount of phospho-ZAP-70 greatly increased over the level attained by anti-CD3 alone, whereas phospho-LAT and PLC-␥1 showed no (LAT) or only modest (PLC-␥1) further increases (Fig. 5, lane 3). In contrast, anti-CD3 stimulation of FynϪ/Ϫ T cells resulted in much less phosphorylation of ZAP-70, LAT, and PLC-␥1. Densitometry of the anti-phosphopeptide immunoblots indi- cated that anti-CD3 stimulated Fynϩ/ϩ cells (Fig. 5, lane 2) contained 6-fold more phospho-ZAP-70, phospho-LAT, and phospho-PLC-␥1 than FynϪ/Ϫ cells (Fig. 5, lane 5). However, cocrosslinking of CD3 with CD4 to a large extent corrected the signaling deficiency of FynϪ/Ϫ T cells (Fig. 5, lane 6). ⌻he data obtained with phospho-ZAP-70 indicated that Lck- mediated phosphorylation and activation of ZAP-70 was defec- tive in FynϪ/Ϫ cells. This observation could be due to a decrease in cellular Lck enzymatic activity and͞or inefficient localization Fig. 6. Regulation by Fyn of Lck activation induced by CD3 crosslinking. Naı¨ve of Lck to the region occupied by TCR and ZAP-70. To examine CD4 cells from Fynϩ/ϩ and FynϪ/Ϫ AD10 TCR Tg mice were preincubated with the functional capacity of Lck directly, Fynϩ/ϩ and FynϪ/Ϫ cells biotinylated anti-CD3 with or without the addition of biotinylated anti-CD4. were stimulated with anti-CD3 or anti-CD3 and CD4 and Lck or Crosslinking was accomplished by the addition of streptavidin at 37°C for 30 Fyn immunoprecipitates were subjected to in vitro kinase assays. sec. Anti-Lck (a) or Fyn (b) immunoprecipitates were subjected to in vitro kinase assays and analyzed by SDS͞PAGE for autophosphorylation and phos- Fig. 6 shows both the autophosphorylation and the phosphory- phorylation of the exogenous substrate enolase. The graphs under the auto- lation of an exogenous substrate, enolase. As expected, unstimu- radiographs summarize the results of three independent experiments. Eno- Ϫ/Ϫ lated wild-type and Fyn T cells showed considerable consti- lase phosphorylation (filled bars) and autophosphorylation (dotted bars) were tutive Lck activity, which was modestly increased after CD3 normalized to the unstimulated controls. , P ϭ 0.03, between Fynϩ/ϩ and ϩ ϩ * crosslinking in the case of Fyn / T cells (Fig. 6a, lane 2). In FynϪ/Ϫ cells.

14862 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406168101 Sugie et al. Downloaded by guest on September 26, 2021 CD3 and CD4 led to a substantial increase in Lck activity in both PLC-␥1, the enzyme responsible for cleavage of phosphatidyl- Fynϩ/ϩ and FynϪ/Ϫ T cells (Fig. 6a, lanes 3 and 6), with an inositol and the generation of IP3. Because this pathway is average increase in activity of 2.5- to 3-fold, as determined by strongly Lck-dependent, we investigated the possible role of Fyn autophosphorylation and enolase phosphorylation. Fyn activity in the function of Lck in naı¨ve T cells stimulated by CD3 after CD3 or CD3 plus CD4 crosslinking of wild-type T cells was crosslinking. Surprisingly, we found a profound Fyn dependency analyzed also (Fig. 6b). After CD3 crosslinking, enolase phos- on this Lck-dependent signaling pathway. First, activation of phorylation increased by 70% and autophosphorylation in- ZAP-70, LAT, and PLC-␥ were all Ϸ6-fold lower in FynϪ/Ϫ T creased by 56%. After CD3͞CD4 cocrosslinking, Fyn activity cells than in Fynϩ/ϩ T cells; second, in vitro kinase assays of Lck was further increased, with enolase phosphorylation being in- after anti-CD3 stimulation of naı¨ve T cells revealed no increase creased by 2.5-fold and autophosphorylation being increased by in Lck activity in FynϪ/Ϫ T cells, whereas a significant, albeit 3.2-fold. modest, increase was seen in Fynϩ/ϩ T cells. A role of Fyn in Lck function is consistent with two other Discussion published reports. First, Bothwell and coworkers (25) showed This study was carried out with the goal of determining whether that decreasing Fyn kinase expression in a T cell line by Fyn tyrosine kinase had a unique role in the signaling of CD4 T transfection with antisense RNA resulted paradoxically in a cells through TCR͞CD3. Our results demonstrate that the decrease in Lck kinase activity as well. Second, Tang et al. (26), capacity for Fyn-deficient T cells to be activated depends criti- by using a similar system to that which we have used, demon- cally on the mode of stimulation and the prior experience of the strated that FynϪ/Ϫ T cells stimulated by anti-CD3 crosslinking T cells. Naı¨ve FynϪ/Ϫ CD4 T cells stimulated by CD3 crosslinking had a reduction in LAT phosphorylation compared with anti- had profound defects in the early signaling events of tyrosine CD3 stimulated wild-type T cells. Conversely, Julius and col- phosphorylation and calcium response, which resulted in a leagues (27) showed by using Lck-deficient T cells that Fyn markedly reduced ability to produce IL-2 and proliferate. In activity could be positively regulated by Lck. Although our data contrast, previously activated FynϪ/Ϫ T cells showed no such suggest the regulation of Lck by Fyn (Fig. 6a), they are also activation defects. Furthermore, when naı¨ve FynϪ/Ϫ T cells were consistent with the regulation of Fyn by Lck because cocrosslink- stimulated by antigen-pulsed APCs or cocrosslinking of CD3 and ing of CD3 and CD4 approximately doubled Fyn kinase activity CD4, no defects in naı¨ve FynϪ/Ϫ T cell responses were observed. as compared with crosslinking of CD3 alone (Fig. 6b). Our Previous characterizations have led to somewhat varied con- observations, together with the previous reports, suggest that in clusions with regard to the capacity of FynϪ/Ϫ peripheral T cells naı¨ve CD4 T cells, Lck and Fyn have a synergistic effect on one to respond to stimulation. Appleby et al. (6) characterized the another that leads to increased activity of both enzymes after proliferative responses of FynϪ/Ϫ T cells to anti-CD3 plus PMA, stimulation through the TCR. allogeneic spleen cells, and . In all cases, FynϪ/Ϫ T We do not know the mechanism by which Fyn influences Lck cells responded somewhat less vigorously (15–70%) than wild- function in naı¨ve T cells. It is possible that Fyn can phosphor- type T cells. Stein et al. (5) found a modest decrease in ylate Lck directly and modify its activity, or, by adding to the proliferation and a more profound decrease in IL-2 production phosphorylation of the TCR associated ITAMs, increase the in response to anti-CD3 and phorbol 12-myristate 13-acetate concentration of the Lck substrate ZAP-70. However, we favor (PMA) stimulation, whereas allogeneic mixed reac- the possibility that Fyn, by some unknown mechanism, induces tion (allo-MLR) and superantigen proliferative responses were the redistribution of Lck to the region where TCR, CD3, and similar to those of Fynϩ/ϩ T cells. Utting et al. (7) found no defect ZAP-70 are localized, thereby facilitating the phosphorylation in the proliferative response of FynϪ/Ϫ CD8 T cells stimulated of CD3 and ZAP-70 by Lck. It is possible that this redistri- with anti-CD3. However, these authors added IL-2 to the bution of Lck may involve its localization to lipid rafts. cultures, making the results difficult to interpret. Our data are Although considerable Lck is detected in lipid rafts in naı¨ve T most consistent with those of Stein et al. (5) because their results cells (28), this Lck moiety has been shown to have very little with APC-mediated responses (MLR and superantigen) showed kinase activity (13, 27), and it may be necessary to have no defect in FynϪ/Ϫ T cells, whereas anti-CD3 stimulation enzymatically active Lck transported into lipid rafts to allow showed a modest defect in proliferation and a profound defect effective signaling to occur after CD3 crosslinking. In this in IL-2 production. The initial reports of the immune function of regard, it is also of interest that effector cells express markedly FynϪ/Ϫ mice included a study of the calcium response of spleen enhanced levels of lipid rafts and Lck associated with plasma cells to anti-CD3 stimulation. Appleby et al. (6) showed a membrane as compared with naı¨ve T cells (28). This finding 60–70% reduction only when suboptimal concentrations of would be consistent with our observation that effector cell anti-CD3 was used to stimulate splenocytes but little or no activation by CD3 crosslinking is Fyn-independent. The ob-

reduction when optimal amounts of anti-CD3 were used. Stein servation that cocrosslinking of CD3 and CD4 corrects the IMMUNOLOGY et al. (5) also reported a partial reduction in the calcium signaling deficiency of naı¨ve FynϪ/Ϫ T cells is also in keeping response. The less dramatic defect in the calcium response found with the hypothesis that one role of Fyn is to facilitate the by these previous studies, compared with our findings, is most translocation of Lck into proximity of the TCR and that, by likely explained by our use of purified CD4 cells and the forcing the apposition of CD4-associated Lck to the TCR by combination of biotinylated anti-CD3 and streptavidin to stim- cocrosslinking CD3 and CD4, the signaling defect in FynϪ/Ϫ ulate them. In the two initial reports on FynϪ/Ϫ mice, whole cells is overcome. spleen cell populations were used and CD3 crosslinking was What might be the physiologic significance of Fyn- accomplished by Fc receptor-bearing cells in the preparations dependent stimulation of naı¨ve T cells through the TCR? that contained accessory molecules (such as MHC, ICAM, and There is compelling evidence that TCR-peptide plus MHC B7) that could help to stimulate a calcium response. interaction occurs between naı¨ve T cells and APCs in the The major pathway for stimulating a calcium response in T absence of cognate antigen stimulation and that this interac- cells after TCR crosslinking involves the tyrosine phosphoryla- tion is critical for the survival and homeostatic proliferation of tion of the ITAMs on CD3␨ chain by Lck and Fyn, the binding T cells (29, 30). In vitro studies have shown that antigen- of ZAP-70 to the ITAMs, the phosphorylation and activation of independent interactions between naı¨ve T cells and dendritic ZAP-70 by Lck, and the phosphorylation by ZAP-70 of the cells result in sufficient T cell signals being generated for a low adaptor protein LAT. Phosphorylated LAT serves as a scaffold level Ca2ϩ response to be detected (31). This response depends to bring several molecules to the plasma membrane, including on TCR aggregation at the –T cell interface.

Sugie et al. PNAS ͉ October 12, 2004 ͉ vol. 101 ͉ no. 41 ͉ 14863 Downloaded by guest on September 26, 2021 These data, together with our previous studies that indicate We thank Drs. Toshi and Yuko Kawakami, Jianyong Huang, and Fyn activation preferentially occurs after stimulation with Keith Bahjat (La Jolla Institute for Allergy and Immunology) for helpful low-affinity ligands (14, 15), suggest the possibility that TCR advice; Jessica Goss, Edmund Ie, Yasuko Kawabara, and Samuel Connell for technical assistance; and Nancy Martorana for interactions with low-affinity self ligands on dendritic cells assistance in preparation of the manuscript. This work was supported in provide a sufficient stimulus for signaling a Fyn-dependent part by a grant from the National Institutes of Health (to H.M.G.). This pathway that is involved in the survival and homeostatic is publication no. 652 from the La Jolla Institute for Allergy and proliferation of naı¨ve T cells. Immunology.

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