T Lymphocytes + and CD8 +CD4 TCR/CD3 Complex in Immortalized Mature -Deficient Γ Signaling Through A

Signaling Through a CD3γ-Deficient TCR/CD3 Complex in Immortalized Mature CD4+ and CD8+ T Lymphocytes

This information is current as Alberto Pacheco-Castro, David Alvarez-Zapata, Pilar of September 25, 2021. Serrano-Torres and José R. Regueiro J Immunol 1998; 161:3152-3160; ; http://www.jimmunol.org/content/161/6/3152 Downloaded from

References This article cites 47 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/161/6/3152.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Signaling Through a CD3␥-Deﬁcient TCR/CD3 Complex in Immortalized Mature CD4؉ and CD8؉ T Lymphocytes1

Alberto Pacheco-Castro,2 David Alvarez-Zapata,2 Pilar Serrano-Torres, and Jose´R. Regueiro3

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4؉ and CD8؉ T lymphocytes from a human selective CD3␥ deficiency using Herpesvirus saimiri. The accessibility of the ,mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8؉ cells when compared with CD4؉ cells relative to their corresponding CD3␥-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-␣) and late (proliferation and secretion of TNF-␣), were normal in ␥-deficient cells, despite the fact that their TCR/CD3 complexes were

significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 Downloaded from triggering was severely impaired in ␥-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in ␥-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when ␥ is absent. They also provide evidence for the specific participation of the CD3␥ chain in the induction of certain cytokine genes in both CD4؉ and CD8؉ human mature T cells. These immortalized mutant cells may prove

to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex. The Journal of Immunology, 1998, http://www.jimmunol.org/ 161: 3152–3160.

cells detect the presence of Ags by way of a surface het- ing molecules (for a review, see Ref. 4). Isolated CD3⑀ or -␨ erodimer termed the TCR. TCR molecules are not ex- ITAMs cannot induce mature T cell proliferation (5) and ablation T pressed alone; they require association with a group of of CD3␦ blocks ␣␤, but not ␥␦ T cell development (6). monomorphic proteins, collectively called CD3. At least four types We have attempted to address this question for the CD3␥ chain of CD3 proteins, termed ␥, ␦, ⑀, and ␨, have been reported. CD3 by studying the functional behavior of human mature T cells de- proteins are believed to maintain TCR/CD3 expression and to par- rived from a natural selective CD3␥ deficiency (7). To circumvent ticipate in the delivery of signals that drive T cell maturation or the inherent difficulties of growing primary T cells and our inabil- by guest on September 25, 2021 apoptosis in the thymus and T cell activation or anergy in the ity to obtain CD8ϩ T cell lines (8), we have used Herpesvirus periphery (1). During early T cell development, some CD3 chains saimiri (HVS), a common lymphotropic virus of squirrel monkeys, may act alone or assist immature TCR ensembles, such as those known to immortalize both CD4ϩ and CD8ϩ human T lympho- containing pre-TCR␣ (2). However, their relative contribution to cytes (9, 10). Immortalized cells remain IL-2-dependent, but be- the signals that are propagated through the cytoplasm and that come Ag- and mitogen-independent for their continued growth result in distal activation events is a matter for discussion. CD3 (11). However, they do display normal downstream functional re- proteins may have partially overlapping functions, as all CD3 com- sponses (proliferation, cytokine synthesis, induction of activation 4 ponents display a shared amino acid motif called ITAM (immu- markers, cytotoxicity, etc.) when their TCR/CD3 activation path- noreceptor tyrosine-based activation motif) in their cytoplasmic way is triggered (12, 13). domains, which can by itself transduce several T cell differentiation and activation signals (for a review, see Ref. 3). Alternatively, they may have specialized functions, as ITAMs belonging to dif- Materials and Methods ferent CD3 chains show different affinities for downstream signal- Cell lines HVS-transformed T cell lines were derived from PBL of a healthy con- Inmunologıá, Facultad de Medicina, Universidad Complutense, Madrid, Spain genital CD3␥-deficient individual (DSF or III-2) (7, 14) and normal do- Received for publication December 4, 1997. Accepted for publication May 6, 1998. nors, as previously described (10). Briefly, PBLs were resuspended (2 ϫ 6 The costs of publication of this article were defrayed in part by the payment of page 10 cells/ml) in a mixture (1:1 proportions) of two culture media [RPMI charges. This article must therefore be hereby marked advertisement in accordance 1640 medium from Biochrom (Berlin, Germany) and cell growth (CG) with 18 U.S.C. Section 1734 solely to indicate this fact. medium from Vitromex (Vilshofen, Germany)] supplemented with 10% 1 This work was supported by PR112/96 (Ministerio de Educacioń y Cultura), 13/97 FCS (Flow Laboratories, Rockville, MD), 1% L-glutamine (BioWhitaker, (Comunidad Autońoma de Madrid) and SAF96/119 (Comisioń Interministerial de Berkshire, U.K.), 50 IU/ml human rIL-2 (Hoffmann-La Roche, Nutley, Ciencia y Tecnologıá) grants to Jose´R. Regueiro. Alberto Pacheco-Castro and David NJ), and 1 ␮g/ml phytohemagglutinin (PHA; Difco Laboratories, Detroit, Alvarez-Zapata were supported by the Ministerio de Educacioń y Cultura. MI). The same day of isolation for CD8ϩ cells, or at day 3 for CD4ϩ cells, 2 A.P.-C. and D.A.-Z. are joint first authors. they were resuspended at 2 ϫ 106 cells/ml in CG/RPMI medium contain- 3 Address correspondence and reprint requests to Dr. Jose´R. Regueiro, Inmunologıá, ing 50 IU/ml human rIL-2 and exposed once to 1 ml of HVS supernatant Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. E-mail ad- in 24-well plates (Costar, Cambridge, MA). Thereafter, medium was re- dress: [email protected] placed every 3 to 4 days (no PHA, only rIL-2). An immortalized phenotype 4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation was indicated by the death of control cultures (i.e., non-HVS-exposed) vs motif; HVS, Herpesvirus saimiri C-488; CG, cell growth medium; PE, phycoerythrin; the sustained growth, presence of HVS genomes, and T lymphoblast cell MFI, mean fluorescence intensity. morphology of test cultures, as described (9, 15). HVS-exposed T cells had

Table I. Surface markers of CD4ϩ and CD8ϩ CD3␥-deﬁcient (DSF4 and DSF8, respectively) and -sufﬁcient (CTC4 and CTO8, respectively) HVS T cells

Cell Type

CD4ϩ CD8ϩ

Percentagea Percentagea

Molecule mAb CTC4 DSF4 MFI ratiob CTO8 DSF8 MFI ratiob

CD3c Leu4 98 96 3.3 Ϯ 0.9 100 96 6.0 Ϯ 1.1 CD3 IOT3b 99 82 3.5 Ϯ 0.2 100 89 4.3 Ϯ 1.4 CD3 OKT3 97 74 3.7 Ϯ 0.7 99 73 4.7 Ϯ 0.2 CD3 X35 99 78 4.0 Ϯ 0.5 98 75 4.3 Ϯ 1.4 TCR␣␤c BMA031 98 27 5.6 Ϯ 0.7 99 9 12.1 Ϯ 1.1 TCR␣␤ OKT3a 98 17 5.7 Ϯ 0.3 98 18 7.7 Ϯ 0.3 CD4 Leu3a 99 99 0.9 Ϯ 0.2 0 0 — CD8 Leu2a 0 0 — 99 100 0.6 Ϯ 0.2 CD2 T11 100 100 0.8 Ϯ 0.1 100 100 1.4 Ϯ 0.4 CD18 IOT18 100 96 0.9 Ϯ 0.3 99 99 1.0 Ϯ 0.3 CD5 CD5 25 40 1.1 Ϯ 0.5 27 90 1.2 Ϯ 0.7

CD45RO Leu45RO 100 100 0.6 Ϯ 0.1 100 100 0.3 Ϯ 0.1 Downloaded from HLA-DR HLA-DR 100 100 0.7 Ϯ 0.3 100 100 1.2 Ϯ 0.5

a Expressed as representative percentages of gated lymphoid cells with fluorescence intensities above the upper limit of the negative control for the indicated molecules using the corresponding Abs. All cells were negative for CD28, CD45RA, CD16, CD20, and TCR␥␦ molecules. Four other unrelated CD3-␥-sufficient HVS T cell lines (two CD4ϩ, two CD8ϩ) showed comparable phenotypes to CTC4 and CTO8, respectively. b Expressed as the range (mean Ϯ SD) of mean fluorescent intensity of ␥ϩ relative to ␥Ϫ cells in at least three experiments. c See representative plots in Figure 1A. http://www.jimmunol.org/ been maintained in long-term culture for more than 16 mo when the experi- number of sites per bead against the corresponding corrected MFI value of ments reported here were performed. V␤ usage analyses were performed as each bead type, and a linear regression analysis was performed (Inplot; described elsewhere (16). GraphPad Software, San Diego, CA). The correlation coefficient was always Ͼ0.98. The number of bound mAb molecules per cell as a function Phenotypical analyses of the corrected MFI (Leu4 MFI Ϫ negative control MFI) was determined by extrapolating from the standard curve. The result of such calculation is The following mAbs were used for cytofluorometric analyses: Leu4 (anti- ␣ the mean number of cell-bound mAb molecules per cell in the considered CD3), Leu2a (anti-CD8 ), Leu3a (anti-CD4), Leu19 (anti-CD56), Leu23 population (or subpopulation). (anti-CD69), Leu45R0 (anti-CD45R0), Leu11c (anti-CD16), Leu16 (anti- Data were collected on 2 to 5 ϫ 104 viable cells as determined by by guest on September 25, 2021 CD20), and HLA-DR from Becton Dickinson (Mountain View, CA); electronic gating on forward scatter and side scatter light parameters. MsIgG (used as negative control) and phycoerythrin (PE)-conjugated goat anti-mouse IgG (HϩL) (used as secondary Ab) from Caltag (South San Francisco, CA); IOT18 (anti-CD18), IOT3b (anti-CD3), and BMA031 (anti- TCR␣␤) from Immunotech (Marseille, France); 2H4 (anti-CD45RA) and TCR/CD3 internalization assays T11 (anti-CD2) from Coulter Clone (Hialeah, FL); anti-CD5 from Serotec The assays were done as described by Dietrich et al. (17). Briefly, cells (Sussex, U.K.); KOLT-2 (anti-CD28) from CLB (Amsterdam, The Neth- were washed twice in PBS, resuspended (5 ϫ 105 cells/ml) in CG/RPMI erlands); OKT3 (anti-CD3), and OKT3a (anti-TCR␣␤) from Ortho Diag- rIL-2-free medium and incubated at 37°C for 30 min in the presence or nostic (Raritan, NJ); TCR␦1 (anti-TCR␥␦) from T Cell Science (Cam- absence of 20 ng/ml PMA. After stimulation, cells were washed in PBS bridge, MA); anti-IL-2 and anti-TNF-␣ from R&D Systems (Abingdon, containing 1% FCS and stained with Leu4 mAb (anti-CD3⑀) for 30 min at U.K.); anti-CD154 (CD40L) from PharMingen (San Diego, CA). TG5 (an 4°C. Then, cells were washed in PBS/1% FCS buffer and analyzed by flow anti-CD3␥ rabbit antiserum raised against the CD3␥ C-terminal peptide cytometry as described above. Results were expressed as the percentage of GLQGNQLRRN) and X35 (anti-CD3) were kindly provided by D. Alex- MFI of control cells incubated without PMA. ander (Babraham Institute, Cambridge, U.K.) and D. Bourel (Centre Re- gional de Transfusion Sanguine, Rennes, France), respectively. ␤F1 (anti- TCR␤) and OKT6 (anti-CD1) mAb ascites were kindly provided by M. L. Functional assays Toribio (Centro de Biologıá Molecular, Madrid, Spain). For single- and two-color immunofluorescence, 1 ϫ 106 cells were in- Intracellular calcium release was induced in cells loaded with the fluores- cubated for 30 min at 4°C with appropriate FITC- or PE-conjugated mAb cent dye Fluo-3AM (Sigma, St. Louis, MO) according to a standard pro- in PBS/EDTA buffer containing 1% FCS. After two washes with PBS, cells cedure (14). Briefly, 2 ϫ 106 cells were washed twice and resuspended in were analyzed in a Epics Elite Analyzer cytofluorometer (Coulter). Iso- Ca2ϩ-free medium (Sigma) at a final concentration of 1 ϫ 106 cells/ml. type-matched irrelevant Abs were used to define background fluorescence. Then, cells were incubated in a stirring bath at 37°C for 30 min with 4 ␮M For intracellular stainings using TG5, ␤F1, or OKT6, cells were first per- Fluo-3AM, washed once more with Ca2ϩ-free medium, and resuspended at meabilized and fixed as explained below (cytokine synthesis assays). a final concentration of 0.5 to 1 ϫ 106 cells/ml for flow cytometry analysis. The precise quantification of CD3 molecules per T cell was conducted Changes in relative fluorescence intensity were recorded as a function of by indirect immunofluorescence in parallel with calibrated beads, follow- time before and after the sequential addition of the following reagents: 1) ing the manufacturer’s protocol (Qifikit, Biocytex, Marseille, France). anti-CD3 mAb (IOT3b from Immunotech, 100 ␮l at 12.5 ␮g/ml); and 2) Briefly, 1 ϫ 106 PBL were first stained with PE-conjugated CD4 (Leu3a) cross-linking reagent, human-adsorbed goat anti-mouse IgG (HϩL) from or CD8 (Leu2a) mAb for 30 min at 4°C. After two washes with PBS, PBL, Caltag, 40 ␮l at 1.25 mg/ml. or HVS, cells were incubated with or without (negative control) an anti- Proliferation was measured by standard [3H]thymidine uptake assays CD3⑀ mAb (Leu4) for 30 min at 4°C. Cells and different calibrated beads (14). Immortalized cells were starved for 7 days in the absence of IL-2, (with 0 (negative control), 5,000, 13,000, 26,000, 59,000, 110,000, adjusted to a final concentration of 5 ϫ 105 cell/ml in fresh mixed medium, 220,000, and 600,000 Fc-binding sites per bead) were washed twice with and incubated for 48 h in round-bottom 96-well plates (180 ␮l of cells/ PBS and incubated with FITC-conjugated anti-mouse IgG for 45 min at well) previously coated with different concentrations of anti-CD3 (IOT3b; 4°C. After two washes with PBS, cells and calibrated beads were analyzed Immunotech). Then cells were pulsed with [3H]thymidine (1 ␮Ci/well, by flow cytometry. For each positive bead, its mean fluorescence intensity Amersham, Buckinghamshire, U.K.) for another 16 to 18 h and harvested (MFI) value was recorded and corrected by subtracting the MFI of the onto glass fiber filters. Thymidine incorporation into cellular DNA was negative control. A standard curve was then calculated by plotting the evaluated as cpm in a scintillation ␤ counter (Packard, Meriden, CT). Other 3154 SIGNALING THROUGH A CD3␥-DEFICIENT TCR/CD3 COMPLEX stimuli (10 ng/ml PMA (Sigma); 7 million SRBC/well (BioMe´rieux, Char- bonnier les Bains, France); 750 ng/ml ionomycin (Sigma)) were also assayed. All experiments were done in triplicate wells and expressed as median cpm values. To measure CD3-mediated cytotoxicity, HVS-effector cells (E) were starved (no rIL-2) overnight in CG/RPMI medium containing 5% FCS, washed, and subsequently resuspended in CG/RPMI medium containing 10% FCS without rIL-2 at a final concentration of 2.5 ϫ 106 cells/ml. Target (T) cells (P815 mouse mastocytoma), previously loaded with 51Cr, were resuspended in the same medium at a concentration of 1 ϫ 105 cells/ ml. A total of 5 ϫ 103 P815 cells were used in all E:T ratios (25:1, 10:1,

5:1, and 1:1). Effector and target cells were incubated at 37°C/5% CO2 for 4 h in the presence of 0.2 ␮g/ml anti-CD3 (IOT3b) (positive test), in the absence of anti-CD3 Ab (spontaneous lysis), or in the presence of an isotype-matched irrelevant Ab (0.2 ␮g/ml w6/32, anti-HLA class I). Lysis was measured as 51Cr release in a ␥ counter (Packard). Values are given as [(E/T cpm Ϫ basal cpm)/(max cpm Ϫ basal cpm)] ϫ 100, where E/T cpm is the median cpm value for each E:T ratio assayed, max cpm indicates 51Cr-maximum release (median value, induced by addition of 100 ␮l/well, 2% SDS), and basal cpm denotes the amount of 51Cr released spontane- ously from P815 cells in the absence of E cells. Experiments were done in 96-well connical bottom-plates and each combination was assayed in triplicate. Downloaded from To measure CD69 and CD154 (CD40L) induction after stimulation, cells were starved in CG/RPMI medium without human rIL-2 for 7 days and resuspended at 5 ϫ 105 cells/ml in 96-well plates in the absence or presence of 1 ␮g/ml plastic-bound anti-CD3 mAb (IOT3b) for6hat37°C. Then, cells were washed twice in PBS, stained with anti-CD69 or anti- CD154 mAb for 30 min at 4°C, washed twice in PBS, and analyzed by ﬂow

cytometry as described above. http://www.jimmunol.org/

Cytokine synthesis assays To analyze intracellular cytokine induction, rIL-2-starved cells were resuspended at 5 ϫ 105/ml in 96-well plates and stimulated for 6 h with or without 1 ␮g/ml immobilized anti-CD3 mAb (IOT3b). For the last 2 h, 10 ␮g/ml Brefeldin A (Sigma) was added to the cultures to block secretion. Cells were harvested, washed twice in PBS buffer, and fixed with 500 ␮l of 4% formaldehyde in PBS for 20 min at room temperature. Then, the cells were stained intracellularly for cytokine content using a modified method based on that described by Assenmacher et al. (18). Briefly, cells by guest on September 25, 2021 were washed twice in PBS containing 0.1% saponin (Sigma), incubated with anti-IL-2 or anti-TNF-␣ FITC-conjugated mAb in 100 ␮lofPBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer. The cytometric analyses were performed in an Epics Elite Analyzer as described above. To determine cytokine secretion after stimulation, rIL-2-starved cells were resuspended at 5 ϫ 105 cells/ml in CG/RPMI medium without IL-2 in a 96-well plate (105 cells/well) with or without 1 ␮g/ml plastic-bound FIGURE 1. Comparative TCR/CD3 accessibility in HVS-immortalized anti-CD3 mAb (IOT3b) or 10 ng/ml PMA plus 750 ng/ml ionomycin. To CD3␥-deficient (␥Ϫ) and -sufficient (␥ϩ) T cells. A, Comparative accessi- block autocrine use of IL-2, 10 ng/ml anti-CD25 mAb (Coulter) was added bility of surface CD3⑀ (Leu4) and TCR␣␤ (BMA031) epitopes in ␥Ϫ to the relevant cultures. After 48 h, duplicate culture supernatants were (white histograms) vs ␥ϩ (black histograms) T cells, either CD4ϩ (upper) ␣ ␥ collected and assayed for their cytokine content (IL-2, TNF- , IFN- , IL-5, or CD8ϩ (lower). The bold vertical lines indicate the upper limit of back- or IL-6) by commercial ELISA assays (Bender MedSystems, Vienna, Aus- ground fluorescence using isotype-matched irrelevant mAbs. The results tria). The limits of detection were 15 pg/ml, 16 pg/ml, 5 pg/ml, 4 pg/ml, and 4 pg/ml, respectively. are representative of five independent experiments. B, Intracellular accessibility (white histograms) to CD3␥ (TG5 rabbit antiserum) and TCR␤ ϩ Ϫ ϩ Statistical analysis (␤F1 mAb ascites) in ␥ (upper) and ␥ (lower) CD8 T cells as compared with background fluorescence (black histograms) using nonimmune Student’s t test was used for all comparisons. Only p values below 0.05 rabbit serum or CD1 ascites, respectively. The results are representative of Ϯ were considered significant. Data are presented as mean SD, except three independent experiments. Similar results were obtained with CD4ϩ T where indicated. cells. Results Phenotypical characterization of HVS-immortalized T All cell lines showed a characteristic mature activated T cell lymphocytes profile (Table I and Ref. 19). However, as shown in Figure 1A After infection of PBLs from the CD3␥-deficient individual and and Table I, DSF4 and DSF8 T cells showed a selective im- two unrelated normal donors with HVS supernatant, four different paired accessibility of TCR/CD3-associated epitopes, ranging HVS-immortalized T cell lines were obtained: two ␥-deficient from virtually no accessibility of TCR␣␤ framework epitopes HVS T cell lines termed DSF4 (CD4ϩ) and DSF8 (CD8ϩ), and (around 6- and 12-fold less than controls using BMA031, re- two ␥-sufficient HVS T cell lines termed CTC4 and CTO8, with spectively) to around 3-fold and 6-fold, respectively, less ac- the equivalent phenotypes. All cell lines rapidly reached a stable cessibility of CD3⑀ epitopes (Leu4) as compared with controls. growth rate (doubling times ranged 3.2 to 4.3 days). As expected, Quantitative analysis of the cytofluorometric data obtained with all cell lines contained HVS genomes, as shown by PCR analysis Leu4 essentially confirmed these findings, and revealed an ab- using HVS-specific primers (not shown). solute number of 45,000 and 30,000 accessible sites per cell in The Journal of Immunology 3155

Table II. Absolute number of accessible Leu4 (anti-CD3) sites (ϫ 10Ϫ3) on PBL and HSV T cells a

Cell Type

CD4ϩ CD8ϩ

␥ϩ ␥Ϫ Ratio ␥ϩ ␥Ϫ Ratio

HVS 150 45 3.3 125 30 4.1 PBL 56 9 6.2 47 3 15.6 Ratio 2.7 5.0 2.7 10

a Data represent mean values of at least three different experiments using calibrated beads as a standard. SD was below 10% of the indicated values.

DSF4 and DSF8 cells, respectively, compared with 150,000 and 125,000 accessible sites per cell in CTC4 and CTC8, respectively (Table II). CD3␥-sufficient HVS T cells, whether CD4ϩ or CD8ϩ, increased their accessibility to Leu4 around 3-fold as compared with fresh peripheral blood T lymphocytes (Table II). Downloaded from In contrast, ␥-deficient HVS T cells increased their accessibility to Leu4 by a factor of 5 in the case of CD4ϩ cells and, inter- FIGURE 2. PMA-induced TCR/CD3 down-regulation in CD3␥-defi- estingly, by a factor of 10 in CD8ϩ cells. cient (␥Ϫ, white bars) and -sufficient (␥ϩ, black bars) HVS T cell lines, ϩ ϩ To confirm the absence of CD3␥ protein in ␥-deficient T cells, either CD4 (left)orCD8 (right). Cells were incubated in the presence of permeabilized cells were stained intracellularly with an anti-CD3␥ 20 ng/ml of PMA for 30 min at 37°C, washed and stained with PE-conjugated anti-CD3⑀ (Leu4) mAb, and analyzed by flow cytometry as de- antiserum (TG5) and, as a positive control, with an anti-TCR␤ http://www.jimmunol.org/ ␤ scribed in Materials and Methods. Results are given relative to cells in- mAb ( F1). The results confirmed the lack of TG5-reactive ma- Ϯ ␥ cubated without PMA (control). Each bar represents the mean SD of five terial in -deficient T cell lines (Fig. 1B) Similar results were ob- independent experiments. *, Denotes p Ͻ 0.05 as compared with the cor- tained by immunoprecipitation (data not shown). responding ␥ϩ values. To test whether the HVS T cells obtained were representative of ␣␤ϩ a few TCR clones or, alternatively, they represented a poly- First, calcium flux, an immediate activation event after ␤ clonal population, V 1–20 regions were analyzed by RT-PCR TCR/CD3 engagement was tested and found to be undistinguish- after more than 6 mo of culture. Results revealed that more than able in ␥-sufficient vs ␥-deficient HVS cell lines (2% vs 3% as 85% of the V␤ genes tested (1–4, 5.1, 5.2/3, 6.1/3, 7–12, 13.1, basal calcium content levels, 2% vs 4% in the presence of soluble 13.2, 14–20) were expressed in CD4ϩ HVS T cells (not shown). by guest on September 25, 2021 anti-CD3 mAb, and 75% vs 80% after CD3 cross-linking, respec- These patterns are compatible with the variable predominance of tively (Fig. 3). V␤ gene usage in HVS-immortalized peripheral T cells previously Second, proliferation, a comparatively late event, was tested and described (20). found also to be similar in all T cell lines, both CD4 and CD8 PMA-induced internalization of the TCR/CD3 complex was selectively impaired in CD3␥-deficient T cells Previous reports have shown that CD3␥ is crucial for protein kinase C-dependent TCR/CD3 down-modulation (17, 21, 22). It was thus relevant to test PMA-induced TCR/CD3 internalization in the immortalized ␥-deficient T cell lines, to further sub- stantiate their lack of CD3␥. As described, both CD4ϩ and CD8ϩ CD3␥-sufficient HVS T cells lost around half of their TCR/CD3 complexes from the membrane after stimulation (Fig. 2). CD3␥-deficient cells, in contrast, were essentially un- perturbed in their relative TCR/CD3 surface expression by the same stimulus, and the difference with CD3␥-sufficient cells was statistically significant. These data cannot be explained by a complete inability of CD3␥-deficient cells to modulate their surface TCR/CD3 complexes or by the fact that they already expressed fewer surface complexes, because Ab-mediated internalization, which is believed to be protein kinase C-indepen- FIGURE 3. CD3-mediated calcium mobilization in CD4ϩ CD3␥-defi- dent (22), was completely normal (19). cient (␥Ϫ) and -sufficient (␥ϩ) HVS T cell lines. Calcium mobilization in response to anti-CD3 cross-linking was analyzed in CD4ϩ CD3␥-deficient Several early and late TCR/CD3-induced activation events were and -sufficient HVS T cells and is shown in a representative experiment normal in the absence of CD3␥ (n ϭ 3) as time (x-axis) vs relative fluorescence intensity (y-axis) vs number of cells [(z-axis, ranging from white (no cells) to black (most cells)]. To assess qualitatively the signals propagated by the mutant Calcium levels were measured consecutively, first basally, second after TCR/CD3 complex, we next assayed a number of functional pa- addition of IOT3b mAb (M), and third after cross-linking the mAb (X). rameters on CD3␥-deficient and -sufficient HVS T cells using Each manipulation is marked by a vertical white lane lasting approximately TCR/CD3-dependent (anti-CD3 mAb), and -independent (PMA 1 min. Similar results were obtained with CD8ϩ CD3␥-deficient and -suf- plus ionomycin) stimuli. ficient HVS T cells. 3156 SIGNALING THROUGH A CD3␥-DEFICIENT TCR/CD3 COMPLEX Downloaded from

FIGURE 4. Proliferative responses of CD3␥-deficient and -sufficient HVS T cell lines, either CD4ϩ or CD8ϩ, to different stimuli. A, Proliferation of CD3␥- deficient (white bars) and -sufficient (black bars) cells in the absence of stimulus (medium) or stimulated with 1 ␮g/ml of plastic-bound anti-CD3 mAb, with PMA http://www.jimmunol.org/ plus ionomycin (PMAϩION) or with sheep red blood cells (SRBC) as described in Materials and Methods. Results are given as mean cpm values Ϯ SD of six independent experiments. B) Proliferation of CD3␥-deficient (Ⅺ) and -sufficient (f) HVS T cells in response to increasing amounts of anti-CD3 (IOT3b) mAb. Data are given as mean cpm values Ϯ SD of five independent experiments. by guest on September 25, 2021

(Fig. 4A). It is noteworthy that all lines showed a normal dose-de- shown in Figure 6, unstimulated cells, both ␥-deficient and -suf- pendent proliferation in response to increasing amounts of plastic- ficient, expressed comparable levels of CD154. After CD3 activa- bound anti-CD3 (Fig. 4B), although CD3␥-deficient T cells expressed tion, all cells significantly up-regulated CD154 expression by a 3- to 5-fold less accessible CD3 epitopes than CD3␥-sufficient ones. factor of 6 (CD4ϩ cells) or 9 (CD8ϩ cells). In contrast to CD154, Third, TCR/CD3-redirected cytotoxicity was tested in HVS T CD69 expression was undetectable in these particular HVS T cells cells. As shown in Figure 5, both ␥-deficient and ␥-sufficient (Fig. 6B), although this is not always the case (Ref. 23, and our CD8ϩ cells showed comparable cytotoxicity responses at different unpublished results). After CD3 engaging, however, comparable E:T cell ratios. levels of CD69 induction were recorded in all T cell lines. Fourth, we tested other early activation events after CD3-trig- Taken together, these results suggest that the CD3␥ chain gering, namely the induction of CD69 and CD154 (CD40L). As of the TCR/CD3 complex was dispensable for several The Journal of Immunology 3157

production after CD3 stimulation was impaired both in CD4ϩ and in CD8ϩ ␥-deficient HVS T cells ( p Ͻ 0.05 vs ␥-sufficient cells). To rule out the existence of an intrinsic (that is, TCR/CD3-independent) defect of IL-2 production in ␥-deficient HVS T cells, they were stimulated with PMA plus ionomycin, which by-passes membrane signals. This strong stimulant mixture discriminates between potentially inducible genes (responsive) and irreversibly blocked genes (unresponsive), as shown in Th1 vs Th2 cytokine profiles (24). In these conditions, all T cell lines, both ␥-deficient and -sufficient, produced similar amounts of IL-2 (19- to 32-fold more than their corresponding basal values, Fig. 7). The observed defect in CD3-induced cytokine induction was selective for IL-2, because

ϩ TNF-␣ synthesis after anti-CD3 stimulation was comparable be- FIGURE 5. CD3-mediated cytotoxicity by CD8 CD3␥-deficient (open tween ␥-deficient and -sufficient T cell lines within each T cell symbols) and -sufficient (solid symbols) HVS T cells. HVS T cells were ϩ ϩ assayed at the indicated E:T cell ratios on P815 (Fc␥Rϩ murine mastocy- phenotype (3- to 4-fold in CD4 cells, 10- to 23-fold in CD8 toma) for 51Cr release in the presence (triangles) or absence (spontaneous cells), although high basal TNF-␣ production was observed in both ϩ lysis; squares) of 0.2 ␮g/ml anti-CD3 (IOT3b), as described in Materials CD4 cell lines (Fig. 7). High basal TNF-␣ levels in HVS T cell and Methods. A similar spontaneous lysis was observed in the presence of has also been reported previously (9, 13). IFN-␥ production was an isotype-matched irrelevant Ab (w6/32, anti-HLA class I). Data are given also analyzed and shown to be similar in all unstimulated cell lines, Downloaded from as mean % lysis Ϯ SD of three independent experiments. but it did not increase after stimulation with anti-CD3 or PMA plus ionomycin, precluding additional functional studies. To further rule out the possibility that CD3␥-deficient T cells were derived TCR/CD3-induced activation events: immediate (calcium flux), from Th2 cells, we analyzed IL-5 and IL-6 synthesis. As shown in early (cytotoxicity, induction of CD69 or CD154), and late Figure 7, none of the tested HVS T cells produced significant (proliferation). levels of IL-5 (or IL-6, data not shown) under any culture http://www.jimmunol.org/ TCR/CD3-induced IL-2, but not TNF-␣, secretion was impaired conditions. in the absence of CD3␥ These results were confirmed with an independent assay for short-term lymphokine induction, namely intracellular cytokine The secretion of different lymphokines after stimulation was next staining. As shown in Figure 8 (upper), essentially no intracellular ␥ ϩ ϩ assayed by ELISA. As shown in Figure 7, both -deficient and IL-2 (iIL-2 ) cells were induced in ␥-deficient HVS T cells after -sufficient unstimulated T cells produced basal levels of IL-2 but, CD3 triggering (Ͻ5%). In contrast, around 35% (32 to 37%) of when stimulated with anti-CD3, only the latter significantly in- ␥-sufficient cells became iIL-2ϩ under similar conditions. Again, creased IL-2 production by a factor of 6 to 8. By contrast, IL-2 the defect was selective for IL-2 induction, because similar numbers of TNF-␣ϩ cells (70 to 85%) were observed in both ␥-defi- by guest on September 25, 2021 cient and ␥-sufficient cells in comparison to basal levels of 6 to 10%, irrespectively of their CD3␥ protein content (Fig. 8, lower). We have tested IL-2 induction in more than 30 different HVS T cell lines derived from normal donors as well as from several patients with defined immunodeficiencies (Ataxia-Telangiectasia, common variable immunodeficiency) and none behaved in the same way as ␥-deficient cells. Taken together, these data suggest that the CD3␥ chain of the TCR/CD3 complex was dispensable for the induction of certain cytokines (like TNF-␣), but not for others (like IL-2), in human CD4ϩ and CD8ϩ HVS-immortalized polyclonal T lymphocytes. Therefore, the propagation of signals through a ␥-deficient TCR/ CD3 complex can result in different functional consequences when compared with a ␥-sufficient TCR/CD3 complex.

Discussion Our data indicate that CD3␥ is not absolutely required for the surface expression of the TCR/CD3 complex in human T lymphocytes. This finding is in contrast with several mutant T cells (17, 21, 25–28), but supports the data of others in normal T or non-T cells (29, 30), in CD3␦-deficient mice (6) and in other CD3␥- deficient humans (Ref. 31; and M. J. D. van Tol, personal com- FIGURE 6. CD3-mediated induction of CD154 (CD40L) and CD69 ex- munication). This discrepancy could be due to the existence of pression in CD3␥-deficient (␥Ϫ) and -sufficient (␥ϩ) HVS T cells. Either ϩ ϩ residual CD3␥ chains, or fragments thereof, in the CD3␥-deficient CD4 (left)orCD8 (right) HVS T cells were incubated with medium (black histograms) or with immobilized anti-CD3 mAb (white histograms), cells, although our available biochemical evidence does not sup- washed, and stained with anti-CD154 (upper) or anti-CD69 (lower) mAbs, port this possibility (Ref. 14, and our unpublished results). How- as described in Materials and Methods. The bold vertical lines indicate the ever, it must be born in mind that all mutant T cells were clones, upper limit of background fluorescence using isotype-matched irrelevant frequently derived from tumors (Jurkat), and artificially selected in mAbs. The results are representative of three independent experiments. vitro on the basis of the absence of surface TCR/CD3 expression 3158 SIGNALING THROUGH A CD3␥-DEFICIENT TCR/CD3 COMPLEX Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 7. Cytokine secretion by CD3␥-deficient (␥Ϫ, white bars) and -sufficient (␥ϩ, black bars) HVS T cell lines, either CD4ϩ or CD8ϩ. T cells were cultured in the presence of medium alone (M), immobilized anti-CD3 mAb (CD3), or PMA plus ionomycin (PϩI). After 48 h, culture supernatants were collected and analyzed for their IL-2, TNF-␣, IFN-␥, and IL-5 content, as described in Materials and Methods. Results are given in pg/ml as mean Ϯ SEM (SD/͌n) of four independent experiments. For IL-2, the numbers above each bar indicate the cytokine levels normalized to that of cells incubated in medium. *Denotes p Ͻ 0.05 as compared with the corresponding ␥-sufficient normalized values. and, for the reconstitution of TCR expression by transfection with levels that have been determined in vivo so that the available T CD3␥ or -␦ plasmids, again selected specifically for expression at cells (particularly CD8ϩ cells) adapt to the lack of CD3␥. Al- endogenous levels. Natural mutant T cells, in contrast, have been ternatively, it may be a HVS-associated effect, because HVS probably positively selected in vivo on the basis of their adequate specifically targets TCR-linked signal transduction pathways and functional TCR/CD3 expression (see Note added in proof). (Tip/Lck, Stp, and C/Ras; Refs. 32 and 33). When cultured with Mature CD4ϩ or CD8ϩ T lymphocytes lacking CD3␥ have been CD3␥-deficient cells, HVS may serve to immortalize only those previously shown to suffer a dissimilar TCR/CD3 accessibility de- cells that maintain TCR levels above a certain threshold. The fect (8). However, it was difficult to draw firm conclusions from observed increase in the level of expression of CD45RO or CD8 those data due to the scant peripheral CD8ϩ T cells of the donor. (Table I) in ␥-deficient cells may be a further compensatory HVS immortalization has confirmed that the lack of CD3␥ affects mechanism to ensure immortalization. TCR/CD3 accessibility in CD8ϩ T cells consistently more than in Asymmetric effects on TCR expression or function of CD4ϩ vs CD4ϩ T cells, as revealed with several mAbs (Table I). This find- CD8ϩ T cells have been reported previously (34–36) and may be ing may be the reflection of a hitherto unrecognized biochemical due to the preferential association of p56lck with CD4 (37–39). difference between TCR/CD3 complexes in CD4ϩ vs CD8ϩ T This association, in turn, may regulate the targeting of TCR/CD3 cells, revealed only when CD3␥ is absent. Alternatively, as sug- to the endosomal compartment thereby regulating TCR/CD3 sur- gested previously (8), peripheral CD8ϩ T cells from this CD3␥ face levels. deficiency, and thus their immortalized progeny, may belong to a We previously showed that, although primary CD4ϩ cells from minor population (in normals) that is relatively expanded in the this patient proliferated in response to phytohemagglutinin and al- absence of CD3␥. logeneic feeders, we were not able to induce the growth of primary It is noteworthy that whereas ␥-sufficient T cells up-regulated CD8ϩ cells (8). This was one of the reasons for trying HVS im- their Leu4 binding sites two- to threefold upon immortalization, mortalization. We believe that the proliferation of CD8ϩ cells fol- ␥-deficient cells up-regulated them 5-fold (CD4ϩ cells) or 10- lowing HVS-transformation may be due to one (or several) of the fold (CD8ϩ cells). This result may suggest that TCR/CD3 sur- following reasons: 1) Conventional T cell lines require frequent face levels or accessibility are deliberately maintained at the TCR-mediated restimulation, and it has been shown that T cells The Journal of Immunology 3159

confirmed in primary (our unpublished results) or immortalized T cells (present results). Taken together, the data suggest that CD3␥ may play a specialized role in coupling the remaining TCR/CD3 chains with downstream intracellular signaling circuitries. However, as we are look- ing at mature T lymphocytes that presumably have been rescued in the thymus, it is possible that they have been selected to be IL-2- uninducible through their ␥-deficient TCR/CD3 complex. Defini- tive proof will require reconstitution of IL-2 secretion by CD3␥ gene transfer into ␥-deficient cells. The observed selective cytokine induction defect may affect several coregulated Th1 genes and may explain the reported IgG2 and polysaccharide Ab response deficiency in ␥-deficient individuals (42) or their reduced peripheral T cell pool (8). Indeed, IgG2 switch has been proposed to be dependent on Th1 cytokines (43) some of which, including IL-2, may be uninducible by the ␥-deficient TCR/CD3 complex. HVS-immortalized mutant T cells from human CD3␥ deficiency were shown to faithfully preserve the phenotypical and functional features of the original T cells (Ref. 19, and present results). This Downloaded from finding has been also shown for several other human immunodeficiencies, including X-linked severe combined immunodeficiency (44), CD95 deficiency (23), Wiskott-Aldrich syndrome (45), MHC FIGURE 8. CD3-mediated intracellular cytokine induction in CD3␥- class II deficiency (46), and Ataxia-Telangiectasia (47). Thus, the deficient (␥Ϫ) and -sufficient (␥ϩ) HVS T cell lines. Either CD4ϩ (left)or immortalization method is clearly a valid experimental approach to ϩ CD8 (right) cells were cultured with medium (black histograms) or with generate clean model systems of natural mutant T cells. However, http://www.jimmunol.org/ immobilized anti-CD3 mAb (white histograms), blocked with Brefeldin A, there are certain intrinsic limitations: irregular phenotypes (CD25, fixed, permeabilized, and stained for intracellular IL-2 (iIL-2, upper)or CD56, CD69, CD154; Refs. 9, 10, 13, and 23), uninducible cyto- TNF-␣ (iTNF-␣, lower), as described in Materials and Methods. The bold kines in certain conditions (like IFN-␥ in this report), and low vertical lines indicate the upper limit of background fluorescence using isotype-matched irrelevant mAbs. The results are representative of four relative responses to specific Ags due to high background prolif- independent experiments. eration (13). Similar models and further studies on this and other T cell deficiencies (in CD3⑀, Zap70, CD40L, ADA, PNP, lymphokine induction, Ca2ϩ influx, etc.; Ref. 48) would be useful to respond only when a threshold of ϳ8000 engaged TCRs have been define specific pathways linking the biochemical signals arising reached (40). As ␥-deficient CD8ϩ PBL (but not CD4ϩ) have less from surface receptors to their associated transcriptional by guest on September 25, 2021 than 3000 Leu4 sites (Table II), it is possible that they are at a machinery. disadvantage in this particular system. 2) In contrast, autocrine Note added in proof. After submission of this paper, CD3␥-defi- proliferation following HVS immortalization takes place through cient mice have been reported (Haks M., P. Krimpenfort, J. Borst CD2/CD58 homotypic interactions (11), which are intact in ␥-de- and A. Kruisbeek. 1998. The CD3␥ chain is essential for devel- ficient CD8ϩ cells (Ref. 41, Table I, Fig. 4A, and our unpublished opment of both the TCR␣␤ and TCR␥␦ lineages. EMBO J. results). 3) Exposure to HVS has been shown to be very effective 7:1871), which are essentially similar to available natural human in rescuing even minute numbers of mature T lymphocytes in in cases (Ref. 7, and van Tol, M. J. D., O¨ . Sanal, R. Langlois van den vitro differentiation assays (15). Bergh, Y. van den Wal, M. T. L. Roos, A. I. Berkel, J. M. Vossen, Our data and published functional results (14, 19) indicate that and F. Koning. 1997. CD3␥ chain deficiency leads to a cellular CD3␥ in HVS-immortalized T cells is dispensable for the follow- immunodeficiency with mild clinical presentation. Immunologist ing TCR/CD3-induced functions: calcium flux, TCR/CD3 down- S1:41.) and which confirm that TCR/CD3 accessibility is more regulation, cytotoxicity, CD69 or CD40L up-regulation, TNF-␣ impaired in peripheral CD8ϩ T lymphocytes than in CD4ϩ cells synthesis, and proliferation. This result may help us understand the when ␥ is absent. survival of the ␥-deficient donor (presently healthy and in his teens) and of other ␥-deficient individuals (31), despite a suscep- tibility to bacterial and particularly viral infections (like viral men- Acknowledgments ingitis). In contrast, TCR/CD3-induced synthesis of IL-2, as well We thank A. Arnaiz-Villena and A. Corell for sharing DSF4 cells, as PMA-induced TCR/CD3 down-regulation, was severely im- M. L. Toribio and B. Alarcoń for mAb samples, and the Centro de Tećnicas paired in ␥-deficient cells. Therefore, the cytosolic signals initiated Inmunolo´gicas (Universidad Complutense de Madrid) for technical sup- by the TCR itself are propagated differentially when CD3␥ is ab- port. Hoffmann-LaRoche is gratefully acknowledged for continuous supply sent and can result in distinct functional outcomes. of rIL-2. These data confirm previous reports indicating that the cytoplasmic tail of CD3␥ is dispensable for CD3-induced TCR/CD3 down- References modulation, cytolysis and TNF-␣ or IFN-␥ synthesis (21), but not 1. Weiss, A., and D. Littman. 1994. Signal transduction by lymphocyte antigen for PMA-induced TCR/CD3 down-modulation. IL-2 synthesis was receptors. Cell 76:263. not tested in those experiments, but a deficiency in IL-2 induction 2. Levelt, C. N., and K. Eichmann. 1995. Receptors and signals in early thymic was reported in an unimmortalized CD4ϩ T cell line from the same selection. Immunity 3:667. ␥-deficient donor (14) and in PBL (41). Calcium flux, however, 3. Kappes, D. J., B. 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