Multiple co-stimulatory signals are required for triggering proliferation of T cells from human secondary lymphoid tissue
Samir G. Agrawal1,4,6,
Jeanine Marquet1,4,
Joël Plumas5,
Hélène Rouard1,
Marie-Hélène Delfau-Larue1,
Philippe Gaulard2,
Laurence Boumsell4,
Félix Reyes3,
Armand Bensussan4 and
Jean-Pierre Farcet1,4
1 Departments of Immunology,
2 Histopathology and
3 Clinical Hematology, and
4 INSERM Research Unit U448, Henri Mondor Hospital, 94010 Créteil, France
5 Immunology Department, ETS Isère-Savoie, BGMT UA 2021, 38701 Grenoble, France
Correspondence to:
J.-P. Farcet, Laboratoire d'Immunologie Biologique, Hôpital Henri Mondor, 94010 Créteil, France
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Abstract
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Vaccine-based therapies are being developed for a variety of cancers and their efficacy will be determined by their ability to stimulate T cells in the secondary lymphoid tissue. We found that T cells isolated from human secondary lymphoid organs (LT-T), in contrast to peripheral blood T cells (PB-T) are hyporesponsive to cross-linked anti-CD3 mAb (CD3c) even in the presence of exogenous IL-2. Using mAb to trigger CD2 and CD28 co-stimulatory molecules, we found that such dual co-stimulation of LT-T induces profound and sustained responses including CD25 expression, IL-2 secretion and proliferation. Different levels of co-stimulation produced a hierarchical pattern of responses in LT-T, which correlated with the degree of CD3TCR down-regulation. Mature antigen-presenting cells (APC) restored the capacity of LT-T to proliferate to stimulation of the CD3TCR complex. Blocking studies demonstrated that optimal proliferation was critically dependent on co-stimulation via CD2 and CD28 engaged by their ligands on the APC. Therefore, LT-T have increased co-stimulatory requirements as compared to PB-T, i.e. multiple co-stimulatory signals coupled to CD3TCR triggering. Furthermore, LT-T were found to be dependent on APC for survival, in contrast to PB-T. Clearly, LT-T do not behave in a comparable way to PB-T and in vitro experiments assessing novel cancer vaccines should therefore use LT-T as the most appropriate population of responder T cells.
Keywords: anergy, antigen-presenting cell, anti-tumor vaccination, CD2, CD3TCR down-modulation, CD28, lymph node, non-Hodgkin's lymphoma
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Introduction
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The first reports on antitumoral vaccination trials using sensitized dendritic cells as antigen-presenting cells (APC) in cancer patients have been encouraging (13). It is assumed that the adaptive immune response is initiated in secondary lymphoid organs were T cells trafficking from the bloodstream encounter the vaccinal antigen presented by dendritic cells from the injection site. Ex vivo, proliferation and differentiation of specific T cells are dependent upon many parameters, with respect to both the APC and the responding T cells. The stability of the MHCantigenic peptide complex (4) and the level of expression of accessory/adhesion molecules (5) determine the immunogenicity of APC. For T cells, their functional status, i.e. naive, memory or anergized, conditions the outcome of the response. We and others have reported that, in contrast to peripheral blood T cells (PB-T), T cells isolated from biopsies of secondary lymphoid organs (LT-T) involved in non-Hodgkin's Lymphoma (NHL) are hyporesponsive to `two-signal' activation, via the CD3TCR and CD28 molecules (68). The proliferative response of LT-T to mitogenic stimuli, such as immobilized anti-CD3 mAb (CD3c), was poor even in the presence of exogenous IL-2. However, bypassing proximal signaling with phorbol myristate acetate (PMA) and ionomycin led to LT-T proliferation, which points to an alteration in the membrane steps of T cell activation. Furthermore, we have shown that LT-T from non-malignant lymph nodes, tonsils and traumatic spleen display a similar proliferative hyporesponsiveness and an increased susceptibility to spontaneous apoptosis during the first 24 h of in vitro culture (6). Such characteristics are reminiscent of those reported for human effector T cells, isolated from lamina propria (9), synovium (10) and intestine (11). Altogether, these results suggest that LT-T responsiveness is conditioned by T cell interaction with the tissue microenvironment and that full activation requires appropriate membrane stimuli which are different from those driving activation in PB-T. In order to determine the signals required to activate LT-T we used agonist mAb as probes for the CD3TCR and various co-stimulatory molecules. Next, using activated autologous B cells as mature APC and blocking antibodies, we further defined the role of co-stimulatory molecules in optimizing the CD3TCR-driven proliferative response of LT-T. Finally, whether APC could rescue LT-T from apoptosis was examined.
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Methods
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Samples and cell preparation
Material from a variety of human secondary lymphoid organs was studied: whole tonsils from children and adults undergoing tonsillectomy; lymph node biopsies showing benign follicular hyperplasia or malignant follicular NHL; and splenectomies performed for autoimmune disorders, trauma or NHL. All biopsies were received fresh in a sterile container without any medium and processed immediately. The preparation of cell suspensions has been described previously (6). Peripheral blood mononuclear cells were isolated by Ficoll-Paque centrifugation of peripheral blood from patients with NHL and healthy donors. All cells were cryopreserved in 10% DMSO with 20% FCS and stored in liquid nitrogen until testing.
Highly purified T cell populations were obtained by negative selection using a cocktail of hapten-modified antibodies (CD11b, CD16, CD19, CD36 and CD56) followed by anti-hapten magnetic microbeads (Pan-T cell isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). For the purification of some tissue samples, anti-CD19 magnetic microbeads were also added to take account of the greater proportion of B cells. All purified T cell populations were CD19, CD56 and >96% (and most were >98%) CD3+. Cell cultures were performed with complete medium: RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mM sodium pyruvate.
Reagents and mAb
The following directly conjugated mAb were purchased from Caltag (Burlingame, CA): CD25FITC (CD25-3G10), CD3TriColor (S4.1) and CD19TriColor (SJ25-C1). mAb produced locally were: anti-CD3 mAb (CD3ana3) which was used in cross-linking conditions, i.e. pre-immobilized onto wells at a 1/2500 dilution of ascites; a mitogenic pair of soluble anti-CD2 mAb (CD2XII and D66, both used at a 1/600 dilution of ascites); and soluble anti-TCR
ß mAb (IP26A, 1/10,000 dilution of ascites). Anti-CD58 mAb (BRIC5, used at a 1/500 dilution of ascites) was obtained through exchanges of the Vth International Workshop on the Differentiation Antigens (12) and anti-CD28 (CD28.2, used at 1 µg/ml) was kindly donated by D. Olive (Marseille, France). Human CTLA-4Ig (R & D Systems, Abingdon, UK) and control human Ig (purified in our laboratory from normal human serum) were both used at 5 µg/ml.
PMA was used at 10 ng/ml (Sigma, St Louis, MO). The following cytokines were used at doses specified in the text: IL-1ß (Pepro Tech, Rocky Hill, NJ), IL-2 (Eurocetus, Amsterdam, Holland), IL-4 (Sanofi, Labège, France), IL-7 (Sanofi), IL-10 (Diaclone, Besancion, France), IL-12 (R & D Systems), IFN-ß (Sigma), IFN-
(PBL Biomedical Laboratories, New Brunswick, NJ), tumor necrosis factor (TNF)-
(Diaclone) and transforming growth factor (TGF)-ß (R & D Systems). Human recombinant VCAM-1 and ICAM-1 (R & D Systems) and fibronectin (purified from human plasma and kindly provided by A. Créange, Créteil, France) were precoated onto culture wells (50 µl at 5 µg/ml and 1 µg/ml and 50 µg/ml in PBS respectively) by incubation overnight at 4°C or for 4 h at 37°C. The efficacy of the coating was verified by visual documentation of the adhesion of resting and PMA-activated T cells to wells.
Flow cytometry and quantification of CD3TCR complexes
Pre-titrated quantities of mAb were added for 15 min at 4°C, and cells were then washed in staining buffer (PBS, 5% FCS and 0.02% sodium azide) and fixed in 1% paraformaldehyde. All tests were analyzed on a Epics XL flow cytometer (Coulter, Hialeah, FL). Isotype control antibodies were used to establish the PMT settings. Quantification of the number of surface CD3TCR complexes was performed using the QIFIKIT (Dako, Glostrup, Denmark) according to the manufacturer's instructions with staining of CD3TCR complexes by CD3ana3 mAb and control of background staining with an irrelevant isotype control mAb.
APC and APC supernatant generation
Highly purified B cell populations were obtained from the biopsies by positive selection using CD19 magnetic microbeads (Miltenyi Biotec). B cells were activated for 57 days in 25 cm2 culture flasks with irradiated (75 Gy) murine L cells transfected with human CD40 ligand (L-CD40L; kindly donated by F. Brière, Dardilly, France) and 5 ng/ml IL-4. Activated B cells were dislodged from the L-CD40L cells by a sharp lateral jolt, harvested and washed. Activated autologous B cells, which were always >98% CD19+, CD80+ and CD86+, were used as APC. APC supernatant was produced by collecting activated B cells the day before use and transferring them to 96-well U-bottom plates in complete medium, but without L-CD40L cells or IL-4. The supernatant from these wells was harvested on the day of the experiment.
T cell survival and apoptosis
All experiments were performed on pure T cell populations isolated from rapidly thawed cryopreserved samples. Viability was assessed by Trypan blue exclusion and 105 Trypan blue cells/well were planted in 96-well U-bottom plates (Costar) in 0.2 ml of complete medium. The effect of various cytokines on cell survival was tested by addition at the start of cultures. For APC supernatant, 100 µl of supernatant was used per well, keeping the final volume constant at 0.2 ml. For experiments involving mixtures of T cells and autologous APC, 5x104 cells of each were added per well. Triplicate wells were used for all conditions.
At the start and at 24 h of each culture, we obtained, in duplicate, a direct absolute count of the number of viable T cells by combining Annexin V and propidium iodide (Annexin VFITC kit; Bender MedSystems, Vienna, Austria) staining with Flow-Count fluorospheres (Coulter). The population of `live' lymphocytes was gated on using the morphological criteria of forward and side scatter. We have previously shown that <1% of cells in the `live' gate are propidium iodide+ (6), so we were able to omit propidium iodide and combine Annexin V staining with anti-CD3 TriColor and Flow-Count fluorospheres to obtain an absolute count of the number of viable T cells by flow cytometry in T cell/APC mixtures. Results are presented as the percentage gain in survival in the test conditions as compared to medium alone: percentage gain in survival = (VT24 VM24)/(VM0 VM24)x100%, where VM0 and VM24 are the number of viable T cells in medium at the start and at 24 h of culture respectively, and VT24 is the number of viable T cells in the test condition at 24 h.
T cell proliferation assays
T cells (5x104/well) were planted in 96-well U-bottom plates in a final volume of 0.2 ml of complete medium. Cells were cultured in triplicate wells with medium alone or with the following stimuli: precoated anti-CD3 mAb (CD3ana3), 10 ng/ml PMA; IL-2 (50 U/ml), a mitogenic pair of anti-CD2 mAb (CD2XII and D66), anti-TCR
ß mAb (IP26A) and 1 µg/ml anti-CD28. Combinations of these stimuli were also used, as detailed in the text. Cultures were incubated in a humidified incubator with 5% CO2 in air at 37°C for 7 days. During the last 8 h of the culture, 1 µCi [3H]thymidine (Amersham, Little Chalfont, UK) was added per well. Cells were harvested on a Packard Harvester (Packard, Meridian, CT) and incorporation of radioactivity was determined using a microplate scintillation counter (Top Count; Packard). Experiments with autologous APC were performed as above with the addition of 5x104 irradiated (50 Gy) activated B cells per well. For inhibition experiments, all reagents were added at the start of cultures: CD2XII alone, anti-CD58 mAb (BRIC5), CTLA-4Ig, human Ig or combinations thereof.
IL-2 bioassay
T cells (105) were planted in 96-well U-bottom plates in a final volume of 0.2 ml of complete medium and activated as described in the text. Supernatant was harvested at 24 and 48 h, and stored at 80°C until analyzed. IL-2 was measured in a functional bioassay using the CTLL2 cell line as previously described (13).
Statistical analysis
All data were analyzed using the non-parametric Wilcoxon test.
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Results
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CD2 and CD28 co-engagement triggered optimal LT-T proliferation
CD3TCR stimulation alone is sufficient to induce proliferation in PB-T, which is maximal at day 4 (6) and then decreases down to baseline at day 7, unless exogenous IL-2 is provided (Fig. 1
). We have previously shown that LT-T are hyporesponsive to CD3TCR stimulation in day 4 assays, even with exogenous IL-2 or a second signal via CD28 (6), and this was re-confirmed in this study for day 7 proliferation assays (Fig. 1
). Therefore, we investigated the role of other co-stimulatory molecules for LT-T proliferation. The alternative CD2 pathway has been shown to be operative in lamina propria T cells (14,15), which, like LT-T, are hyporesponsive to CD3TCR activation. Unlike PB-T, LT-T show constitutive CD2R epitope expression, although the levels are variable (data not shown). However, LT-T stimulation via CD2 alone did not lead to proliferation, whereas co-engagement of CD2 and CD28 with mAb led to very strong responses which were maximal at day 7 (Fig. 1
). In this system, the combination of CD2 and CD28 stimulation was pivotal, as the addition of exogenous IL-2 or CD3TCR triggering were unable to augment the response. Combinations other than dual CD2/CD28 stimulation were ineffective (see Fig. 1
). We also investigated the role of LFA-1 and VLA-4, two integrins which co-stimulate the proliferation of human PB-T (16,17), as we hypothesized that adhesive interactions might be even more important for LT-T. The purified ligands VCAM-1 and fibronectin (for VLA-4) and ICAM-1 (for LFA-1) were unable to induce proliferation of LT-T in combination with either TCR (data not shown) or CD2 triggering (Fig. 2
). However, if LT-T were first driven to proliferate by CD2 activation and exogenous IL-2, VCAM-1 and fibronectin, but not ICAM-1, enhanced the proliferative response (Fig. 2
).

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Fig. 1. LT-T are hyporesponsive to TCRCD3 stimulation but proliferate strongly to CD2 and CD28 co-engagement. T cells (50,000) were planted in U-bottom 96-well plates and stimulated with the indicated conditions for 7 days at 37°C in humidified atmosphere of 5% CO2. CD2 = a mitogenic pair of anti-CD2 mAb (CD2X11 and D66, both at 1/600 dilution of ascites); CD3c = immobilized CD3ana3 (anti-CD3 ascites produced in our laboratory); CD28 = anti-CD28 mAb (clone CD28.2, 1 µg/ml); IL-2 (50 units/ml); PMA (10 ng/ml). [3H]Thymidine was added for the last 8 h of culture. The results are expressed as the mean of six different PB-T (white bars) and 12 different LT-T (shaded bars). An asterisk above a column indicates a statistically significant difference between the two groups: CD3c (P = 0.0018); CD2 (P = 0.012); CD3c/CD2 (P = 0.039); IL-2 + CD3c (P = 0.002); CD28 + CD3c (P = 0.029).
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Fig. 2. Adhesion molecules can provide co-stimulation for LT-T but cannot replace CD28 signaling. LT-T cells (50,000) were planted in U-bottom 96-well plates and stimulated with the indicated conditions for 7 days at 37°C in humidified atmosphere of 5% CO2. CD2 = a mitogenic pair of anti-CD2 mAb (CD2X11 and D66, both at 1/600 dilution of ascites); IL-2 (50 U/ml); VCAM-1, fibronectin and ICAM-1 were immobilized onto wells as described in Methods. [3H]Thymidine was added for the last 8 h of culture. This experiment is representative of four independent experiments, with similar results using T cells isolated from lymph nodes involved by NHL (n = 2) and from a traumatic spleen (n = 1).
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Thus, LT-T proliferated maximally with dual CD2 and CD28 activation using mAb, and the essential role of CD28 signaling could only partially be replaced by IL-2 and not by LFA-1 or VLA-4 co-ligation.
LT-T responsiveness to CD3TCR stimulation was restored by APC
The strong in vitro responses induced in LT-T by mAb stimulation of CD2 and CD28 are obviously not physiological. Nevertheless, these results suggested that LT-T activation by CD3TCR engagement must absolutely require a professional APC which expresses the appropriate co-stimulatory molecules. To verify this, as mature APC, we used autologous B cells activated for 7 days by IL-4 and murine L cells transfected with human CD40 ligand. LT-T did not proliferate to APC, CD3c or soluble anti-TCR
ß mAb alone (Fig. 3A
). Strong proliferation of LT-T to CD3TCR stimulation was induced in the presence of APC and, remarkably, the proliferation to CD3c/APC was equivalent to that induced by CD2/CD28 co-stimulation with mAb. Similar results were obtained using the soluble anti-TCR
ß mAb in the presence of APC, although the kinetics of the response were shortened (Fig. 3A
). The essential role of the CD2CD58 and CD28B7 pathways in the APC-mediated co-stimulation of LT-T was demonstrated by the 70% inhibition of proliferation obtained by simultaneous blockade of both pathways (Fig. 3B
).

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Fig. 3. APC restore the responsiveness of LT-T to TCRCD3 stimulationrole of the CD28/B7 and CD2/CD58 pathways. T cells (50,000) (from a traumatic spleen) were planted in U-bottom 96-well plates and stimulated with the indicated conditions at 37°C in humidified atmosphere of 5% CO2. CD2 = a mitogenic pair of anti-CD2 mAb (CD2X11 and D66, both at 1/600 dilution of ascites); CD3c = immobilized CD3ana3 (anti-CD3 ascites produced in our laboratory); CD28 = anti-CD28 mAb (clone CD28.2, 1 µg/ml); TCRs = anti-TCR ß mAb used in soluble form (clone IP26A, 1 in 10,000 dilution of ascites); APC = 50,000 irradiated (50 Gy) autologous B cells activated for 7 days with IL-4 and L-CD40L. [3H]Thymidine was added for the last 8 h of culture. The c.p.m. of APC cultured alone have been subtracted (day 1: 6952; day 2: 515; day 3: 282; day 6: 339). (A) Kinetics of T cell proliferation induced by TCR triggering in the presence of APC compared to CD2/CD28 co-engagement by mAb. (B) Inhibition of day 6 proliferation in the same experiment as (A). The top bar represents the baseline proliferation of LT-T cells by CD3c and APC. All reagents were added at the start of cultures: CTLA-4Ig (5 µg/ml); CD2X11, an anti-CD2 mAb directed against the CD58 binding epitope (1:600 dilution of ascites); CD58, anti-CD58 mAb (clone BRIC5, 1:500 dilution of ascites); and control human Ig (5 µg/ml). Similar results were obtained in two other independent experiments using LT-T.
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CD2 and CD28 co-engagement induced optimal TCR down-modulation in LT-T
First, the constitutive level of CD3TCR expression on LT-T was examined because T cell activation has been shown to be compromised when the level of surface TCR expression is reduced to <5000 TCR molecules per cell in T cell clones (18). LT-T were found to express less surface TCR than PB-T and a representative flow cytometric histogram is shown in Fig. 4
(A). It is important to note that the LT-T curve, like that for PB-T, is unimodal, indicating that the phenomenon of TCR down-modulation affects the entire population of LT-T. These findings were confirmed in a larger series of samples, regardless of the nature of the LT-T: while the MFI was 400 ± 50 for PB-T (n = 6), it was 260 ± 40 for reactive LT-T (n = 10) and 265 ± 40 for NHL LT-T (n = 9). Using a commercial calibration kit, we calculated that PB-T displayed an average of 40,000 CD3TCR complexes and LT-T 30,000. This down-modulation was specific for the TCR, as the MFI of CD45RO was greater in LT-T than PB-T (72.4 ± 10.7 versus 51.2 ± 7.3), while that of CD45RA was the same (51.2 ± 20.4 versus 52.8 ± 20.7).

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Fig. 4. LT-T display less surface TCRCD3 than PB-T: down-regulation by CD2/CD28 co-engagement. (A) A representative example of staining with anti-CD3 mAb of T cells isolated from a lymph node involved by NHL (NHL-TIL) (solid line, MFI = 235) and autologous PB-T (broken line, MFI = 401). The filled curve represents the isotype control. The abscissa shows CD3 fluorescence expressed on an arbitrary logarithmic scale and the y-axis represents the number of events. (B) Tonsillar T cells (50,000) were planted in U-bottom 96-well plates in complete medium and stimulated with: immobilized anti-CD3 mAb (CD3c); a mitogenic pair of anti-CD2 mAb, CD2XII and D66 (CD2); or CD3c or CD2 + anti-CD28 (1 µg/ml). Cells were harvested at the time points shown and stained with anti-CD3TriColor. The results are expressed as a percentage of the surface expression of TCRCD3 at day 0 immediately after purification.
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Thereafter, using mAb, the modulation of CD3TCR expression on LT-T was studied as this is known to correlate with the hierarchy of T cell responses (19,20). We found that LT-T surface expression of TCR was only modestly reduced (by 20%) following stimulation by CD3c (Fig. 4B
). Furthermore, co-stimulation via CD28 (CD3c/28) did not augment this response. In contrast, triggering of CD2 produced marked TCR down-modulation in LT-T, while dual CD2/CD28 triggering led to prolonged and profound (70%) reduction in surface TCR expression (Fig. 4B
).
CD25 expression, IL-2 secretion and proliferation: the hierarchical response of LT-T
We investigated the mechanisms underlying the LT-T response by simultaneously looking at CD25 expression, cytokine (IL-2) production and proliferation. Table 1
and Fig. 5
(C) show that LT-T only produced significant amounts of IL-2 with dual co-stimulation via CD2 and CD28 using mAb and that this was equivalent to the PB-T response. Predictably, in view of the above results on surface TCR expression, the same conditions (CD2/28) led to maximal up-regulation of CD25, both in terms of the percentage of positive cells (maximum 90% at day 3, Fig. 5A
) and the level of expression per cell (Fig. 5B
) and was the only condition to produce strong proliferation (Fig. 5D
). The hierarchical nature of LT-T responses was clearly shown by the various combinations of stimuli: CD3c alone induced modest up-regulation of CD25, which was increased by additional co-stimulation (CD3c/28; Fig. 5A and B
), but IL-2 production and proliferation was insignificant (Fig. 5C and D
). Interestingly, CD2 triggering alone not only produced strong up-regulation of CD25 but also a small amount of IL-2, yet did still not lead to LT-T proliferation. Even the addition of 50 U/ml of exogenous IL-2 to CD2 triggering only produced half the proliferation of LT-T as that induced by dual CD2/CD28 activation (Fig. 1
). Thus, the strong proliferation of LT-T to CD2/CD28 activation cannot simply be explained by the up-regulation of CD25 and IL-2 secretion.
APC delivered survival signals to LT-T
We have previously shown that LT-T, compared to PB-T, have an increased susceptibility to spontaneous apoptosis in vitro (6). In preliminary experiments, we found that unstimulated autologous B cells from the same biopsies as LT-T were unable to improve LT-T survival in vitro (data not shown). However, mature APC obtained as above (see Results Section 2) in contact with LT-T were able to save >50% of LT-T that died when cultured in medium alone for 24 h (Table 2
). The use of anti-CD3, CD2, CD28 mAb and/or VCAM-1, fibronectin or ICAM-1 in various combinations did not improve LT-T survival (data not shown). These results suggest that APC-mediated survival involves a complex network of signals provided by both soluble and membrane-bound factors. Indeed, APC supernatant improved the survival of LT-T, although the effect was smaller than with APC themselves (Table 2
). Many cytokines have been reported to play a role in the survival of human lymphocytes (2126), therefore, we tested a large panel of cytokines, performing doseresponse experiments for each of them. None of the IL-2R
chain cytokines tested (IL-2, IL-4 and IL-7) nor IL-1ß, IL-12, IFN-
, TNF-
or TGF-ß improved survival of LT-T (data not shown and 6). Only IL-10 and IFN-ß consistently improved the survival of LT-T (Table 2
).
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Discussion
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In contrast to PB-T, LT-T cannot be triggered to proliferate using CD3c mAb alone. In LT-T, the CD2 and CD28 co-stimulatory pathways are functional when engaged by specific mAb, and synergize to drive LT-T proliferation which is not inhibited by simultaneous CD3c triggering (Fig. 1
). Accordingly, CD2 and CD28 co-activation induces profound and sustained CD3TCR down-modulation, and high amounts of IL-2 secretion. Also, in the presence of mature APC, LT-T activation by either cross-linked or soluble mAb as CD3TCR probes results in intense and sustained proliferation. Among co-stimulatory ligands provided by APC, the combination of CD58 and B7 (CD80 and CD86) molecules, which are the counter-receptors for CD2 and CD28 on LT-T, is critical in inducing maximum LT-T proliferation. However, as shown in Fig. 3
, simultaneous blockade of both pathways still leads to significant proliferation, suggesting that APC provide multiple co-stimulatory ligands other than CD58 and B7 molecules. These observations show that the lack of proliferation of LT-T using mAb to the CD3TCR complex, with or without CD28 co-stimulation, is not due to an irreversible blockade of membrane or downstream signal transduction pathways via the CD3TCR in LT-T, but rather due to the differential co-stimulatory triggering requirements for full CD3TCR driven activation of LT-T as compared to PB-T.
In man, using PB-T and T cell clones, co-stimulation has been shown to be mediated not only by CD28 (27,28), but also the integrins LFA-1 and VLA-4 (16,17,27), CD5 (27), and CD2 (29,30), while even more co-stimulatory molecules are emerging (31). They contribute to regulating T cell activation in different ways, (i) allowing and strengthening the formation of the immunological synapse within APCT cell conjugates (32,33), (ii) sustaining the aggregation of the CD3TCR complex and the detergent-insoluble glycolipidic membrane fraction (DIG) (34) which is enriched for the src family protein tyrosine kinases, and (iii) complementing CD3TCR downstream signaling which results in IL-2 production and both IL-2-dependent and -independent G1/S cell cycle transition (35,36). Our data shows that LT-T have an absolute requirement for multiple co-stimulatory signals, the most important being CD2 and CD28 triggering, which can be optimally provided by mature APC. CD2, a resident in the membrane DIG fraction, and CD28, outside the DIG fraction, are the two best studied co-stimulatory molecules thought to participate at every step in T cell triggering and intracellular signaling cascades. Their capacity to coordinate regulation of T cell activation has recently been evidenced using double knockout mice, especially in the setting of suboptimal triggering of the CD3TCR pathway (32).
Here, the hierarchical nature of LT-T responses has been demonstrated using mAb: engagement of CD3TCR alone modestly up-regulates CD25 expression on day 1 with no further increase on day 2, no IL-2 secretion and no proliferation; while dual CD2/CD28 co-stimulation induces high levels of CD25 expression, IL-2 secretion and intense proliferation, which is sustained for a whole week. CD2 triggering alone induces intermediate CD25 and IL-2 responses, but no proliferation. These observations in LT-T are consistent with the recent finding of hierarchical co-stimulator thresholds for distinct immune responses in PB-T (37). Also, it is shown that the previously reported strict correlation between the ability of a TCR ligand to induce TCR down-modulation and its ability to elicit a response, with each functional outcome requiring a specific level of TCR down-modulation (19,20), can be extended to co-stimulation. While CD3TCR triggering alone induces only minor and temporary CD3TCR down-modulation in LT-T, dual CD2/CD28 co-stimulation induces profound and sustained CD3TCR down-modulation. Although predictable in view of the known inter-relationship between CD2 and the CD3TCR complex (3842), this is the first report which correlates graded co-stimulation, graded CD3TCR down-modulation and hierarchical T cell responses.
The nature of the activation response varies depending on the history and developmental state of the T cell. In this and previous work (6), we have investigated the differential triggering requirements for LT-T and PB-T proliferation, and found that the CD3TCR pathway triggered by cross-linked anti-CD3 mAb required multiple co-stimulatory signals for LT-T proliferation, in contrast to PB-T. PB-T leave the bloodstream through high endothelial venules and enter the lymph node, where scanning of the APC surface is maximized in the 1025 µm diameter corridors in the cortex (43). Consequently, T cells traversing a lymph node are continuously engaging and disengaging their TCR with MHCself-peptide complexes (44). Our demonstration of the selective down-modulation of the TCR in LT-T as compared to PB-T, affecting the whole LT-T population, can be attributed to the partial agonist quality of self-peptides (4548) and the degeneracy of the TCR for these peptides (49,50), which would lead to constant, subthreshold stimulation of the TCR and induce a state of partial activation in LT-T. Both anergic T cells and partially activated T cells have been characterized in human and murine models by: an inability to proliferate to full two-signal (TCR and CD28) stimulation (5153); arrest in G0/G1 of the cell cycle (54,55), an activated phenotype and resistance to Fas-mediated apoptosis (56,57), and small increases in Bcl-xL and Bcl-2 as compared to fully activated T cells (56). These are all features of LT-T (6 and this study). Although we did not perform biochemical studies because of the small number of T cells available from lymph node biopsies, partial activation is likely to result in alteration of the signaling cascade in LT-T as compared to PB-T. Constitutive phosphorylation and activity of p56lck were previously reported in LT-T from NHL (58) and murine lymph node T cells show constitutive p56lck activity leading to TCR
phosphorylation, recruitment of non-phosphorylated ZAP-70 and absence of TCR
pp23 phosphoprotein (59,60). Altogether, the phenotypic and functional findings in LT-T, i.e. the hyporesponsiveness to CD3TCR stimulation, which is not reversed by exogenous IL-2, has been interpreted as anergy, and in the context of cancer as a mechanism for tumor escape (58). We do not believe that LT-T are anergic for the following reasons: (i) anergy is clonal, i.e. antigen specific, whereas LT-T are polyclonal (6), (ii) activation of the CD3TCR pathway is not blocked and (iii) IL-2 synthesis is not inhibited (61), given that the appropriate multiple co-stimulatory signals are provided.
In contrast to PB-T, LT-T show poor spontaneous survival in vitro (6). In this study, we have shown that APC can deliver survival signals to LT-T in vitro, and that this is mediated by both cell-surface and soluble factors (table 2
). The engagement of LT-T cell surface molecules by anti-CD3, -CD2 and -CD28 mAb, or combinations thereof, and/or the adhesion molecules, VCAM-1, ICAM-1 and fibronectin, did not improve LT-T survival, although these conditions led to proliferation of the LT-T viable subset when cultured for 7 days. These results highlight both the heterogeneity within a population of LT-T with respect to survival and the unique ability of APC to mediate both survival and graded responses of LT-T including proliferation. In a variety of murine models, it has been established that continuous engagement of the TCR by MHCpeptide complexes is essential for peripheral T cell survival (62,63). It seems probable that mature human T cells, like their murine counterparts (6466), are dependent on continuous subthreshold interactions with MHC molecules for survival in the periphery and that these signals are provided primarily by MHC-rich APC in the secondary lymphoid organs. The human LT-T investigated in this study are also unique, as compared to all other T cell populations reported in the literature (23,25,6769), in that their in vitro cytokine-mediated survival was restricted to IL-10 and IFN-ß, with no effect of the tested IL-2R
chain-signaling cytokines.
Consequently, human LT-T as a whole population differ markedly from PB-T in their activation and survival requirements. LT-T, which constitutively display a partially activated phenotype, depend exquisitely on multiple co-stimulatory signals, the most important being dual CD2/CD28 engagement, to respond to CD3TCR triggering. This co-stimulation is optimally provided by mature APC which also ensure LT-T survival. The APC dependence of these LT-T functions appears to be acquired when PB-T leave the bloodstream to enter the lymphoid tissue. LT-T, and not PB-T, being the responding T cells in any vaccination protocol, LT-T are the most appropriate responder population in the cellular assays used to characterize the function of dendritic cells, in as much as dendritic cells are the APC of choice in antitumor vaccination (70).
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Acknowledgments
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This work was supported by grants from ARC (no. 9523), the Ligue Nationale Contre le Cancer (Comité du Val de Marne) and the University Paris XII.
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Abbreviations
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APC antigen-presenting cells |
CD3c cross-linked anti-CD3 mAb |
LT-T lymphoid tissue T cells |
NHL non-Hodgkin's lymphoma |
PB-T peripheral blood T cells |
PMA phorbol myristate acetate |
TGF transforming growth factor |
TNF tumor necrosis factor |
 |
Notes
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6 Present address: Department of Haematology, Southampton General Hospital, Southampton SO16 6YD, UK 
Transmitting editor: J. F. Bach
Received 16 August 2000,
accepted 15 December 2000.
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