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 Allergy 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 antigen-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 T cell 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 Antibodies 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 antibody- 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.