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(Journal of Leukocyte Biology. 2001;70:252-260.)
© 2001 by Society for Leukocyte Biology

Feedback activation of T-cell antigen-presenting cells during interactions with T-cell responders

Mark D. Mannie and Mindi R. Walker

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC

Correspondence: Dr. Mark D. Mannie, Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858-4354. E-mail: manniem{at}mail.ecu.edu


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ABSTRACT
 
Like many T cells in the myelin basic protein (MBP)-specific T-cell repertoire, CD4- GP2.3H3.16 (3H3) T cells recognize guinea pig MBP as an agonist but recognize autologous rat (R)MBP as a mixed agonist/antagonist. 3H3 T cells do not exhibit proliferative responses to RMBP but nonetheless respond to RMBP by accumulation of T-cell surface I-A/peptide complexes and generation of T-cell antigen-presenting cell (T-APC) activity. This study showed that presentation of RMBP by 3H3 T-APC is long-lived but is lost during interactions with cognate responders or on overt activation of T-APCs. Presentation of RMBP to encephalitogenic T cells resulted in the reciprocal activation of 3H3 T-APCs as evidenced by blastogenesis, proliferation, and induction of interleukin-2R and OX40 markers on 3H3 T-APC. These data indicate that T-APCs, like B-cell APCs, undergo clonal expansion after presentation of a cognate antigen to T-cell responders.

Key Words: EAE/MS • antigen presentation • MHC • T-cell receptors


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INTRODUCTION
 
T cells from mice [1 2 3 4 5 6 ], rats [7 8 9 10 11 12 13 14 ], and humans [15 16 17 18 19 20 21 22 23 24 25 ] have the capacity to express class II major histocompatibility complex (MHC) glycoproteins and present antigen to other T cells. Activated T cells may directly synthesize class II MHC glycoproteins and assemble MHC-peptide complexes for surface expression. Another major pathway for T-cell acquisition of class II MHC involves the intercellular transfer of MHC-peptide complexes from professional antigen-presenting cells (APCs) to T-cell responders [26 27 28 29 ]. Although T-cell APCs (T-APCs) elicit proliferation of responders, many studies have shown that MHC class II-restricted interaction among T-helper cells ultimately results in anergy or apoptosis of responders as a manifestation of tolerance induction [18 , 23 , 24 , 30 31 32 33 34 35 ].

This study was based on the use of the CD4- GP2.3H3.16 (3H3) clone of myelin basic protein (MBP)-specific T cells [36 37 38 ]. These T cells recognize guinea pig (GP)MBP as an agonist but recognize rat myelin basic protein (RMBP) as a T-cell receptor (TCR) antagonist in proliferation and interleukin (IL)-2 production assays. RMBP nonetheless has sufficient agonistic activity to elicit T-cell surface expression of class II MHC glycoproteins [38 ]. Our study revealed that 3H3 T cells proliferate when these T-APCs present RMBP peptide/class II MHC complexes to pathogenic responders that recognize the same MHC ligand as an agonist. This mechanism of feedback activation might thereby cause an accompanying clonal expansion of T cells that recognize a TCR antagonist concomitantly with T cells that recognize the same antigen as an agonist. Because recognition of antagonistic ligands might generate tolerogenic T-APC activity [34 , 36 , 37 ], this mechanism might regulate the balance between regulatory T cells and pathogenic effector T cells during the course of an immune response.


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MATERIALS AND METHODS
 
Animals and reagents
Lewis rats (Harlan-Sprague Dawley, Indianapolis, IN) were maintained at East Carolina University School of Medicine, Greenville, NC. MBP was purified from rat or guinea pig spinal cords (Rockland Immunochemicals, Gilbertsville, PA). Anti-I-A OX6 immunoglobulin IgG1 [39 ], anti-I-E OX17 IgG1 [39 ], anti-interleukin (IL)-2R OX39 IgG1 [40 ], and anti-OX40 IgG2b [41 ] were produced as culture supernatants, were concentrated by ultrafiltration through Amicon (Beverly, MA) spiral-wound membranes (100-kDa exclusion), and were added to cells as a 1:20–1:50 dilution. Purified anti-B7.1 (3H5) monoclonal antibodies (mAbs) [42 ] (PharMingen, La Jolla, CA) were used at a concentration of 2.5 µg/mL. Concanavalin A (conA) (Sigma, St. Louis, MO) was used at a final concentration of 2.5 µg/mL.

Lewis rat T-cell lines and cell culture conditions
T-cell lines used in this study are described in Table 1 . T cells were propagated in complete RPMI 1640 medium supplemented with IL-2 (CM). Complete RPMI medium contained 10% heat-inactivated fetal bovine serum (Summit, Boulder, CO), 2 mM glutamine, 100 µg/mL of streptomycin, 100 U/mL of penicillin (Whittaker Bioproducts, Walkersville, MD), and 50 µM 2-mercaptoethanol (Sigma) (cRPMI). The CM contained 0.4 % (v/v) supernatant of an Sf9 insect cell culture infected with a recombinant rat IL-2-containing baculovirus.


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  		Table 1. Lewis Rat T Cell Lines Used in This Study

Preparation of T-APCs
3H3 T cells (5x105/mL) and irradiated splenocytes (SPLs) (5x106/mL) were cultured for 2–3 days with RMBP or conalbumin. Fumed silica (Sigma) was added as designated to deplete phagocytic APCs. 3H3 T cells were then purified from residual professional APCs on a discontinuous gradient of Percoll. 3H3 T cells were or were not propagated (105/mL) in CM for designated periods and then were irradiated and used to stimulate responder T cells. The only source of antigen in these assays was that added during the initial culture with irradiated SPLs.

Measurement of in vitro proliferation
Responder T cells (2.5x104/well) were cultured with irradiated (3,000 rads of {gamma}-radiation) T-APCs unless designated otherwise. Cultures were pulsed with 1 µCi of [3H]thymidine (6.7 Ci/mmol) (NEN Research Products, Boston, MA) during the last day of a 48-h culture and were harvested to measure [3H]thymidine incorporation by scintillation counting unless designated otherwise.

Isolation of peritoneal exudate M{phi}
Lewis rats were injected intraperitoneally with 200 µg of heat-killed Corynebacterium parvum cells. After 48 h of sensitization, peritoneal exudate cells were collected by peritoneal lavage with 50 mL of Hanks’ balanced salt solution (GIBCO BRL, Gaithersburg, MD). Macrophages (M{phi}) were isolated by adherence to plastic, were rested for 5 days in cRPMI, and were then used in bioassays.

Measurement of antigen-dependent IL-2 production and NO production
T-APCs and responder T cells (2.5x105/mL each) were cultured for 3 days with or without peritoneal exudate cells in the presence or absence of 1 µM RMBP. Supernatants were then transferred into replicate plates for an IL-2 bioassay, and another aliquot was transferred to a second set of plates to measure nitric oxide (NO) production. To assay IL-2 bioactivity, CTLL cells (2.5x104/50 µL of cRPMI/well) were cultured with 100 µL of supernatant for 48 h, and 10 µL of an MTS [3-(4,5-dimethylthiazol-2-yl)-5-3-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] phenazine methosulfate solution [2.9 mg/mL of MTS (Promega, Madison, WI) and 0.1 mg/mL of phenazine methosulfate (Sigma)] were added to each well. Plates were read the next day at an optical density (OD) of 492 nm (OD492) on an enzyme-linked immunosorbent assay microplate reader. Production of NO was determined by measuring the formation of the stable decomposition product nitrite by mixing supernatant (100 µL) with an equal volume of Griess reagent (1% sulfanilamide–0.1% N-[1-naphthy] ethylenediamine in 2.5% phosphoric acid) [43 ]. After 10 min of incubation, the OD540 was measured in a microplate reader. Antigen-induced IL-2 production was measured as the mean OD values from MBP-stimulated cultures minus mean OD values from unstimulated control cultures. Assays were routinely performed with triplicate or quadruplet cultures per group.

Flow-cytometric analysis
T cells were stained with PKH67 (FL1) or PKH26 (FL2) lipophilic dye (Sigma) according to the manufacturer’s instructions. Washing and staining of T cells were performed at 4°C, and Fc receptors were blocked by the addition of heat-inactivated Lewis rat serum to incubation mixtures. The mAbs were added at a concentration of 2.5 µg/mL or a 1:20–1:50 dilution of a concentrated supernatant. T cells were incubated for 45 min with the primary mAb, were washed two times, and then were incubated for 45 min with a fluorescein isothiocyanate-conjugated F(ab')2 rat anti-mouse IgG (heavy plus light chains). Dead cells were excluded from analysis by forward versus side scatter profiles. Data were acquired with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and were analyzed with Lysis II and CellQuest software programs.


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RESULTS
 
Previous studies showed that RMBP antagonized GPMBP-stimulated proliferation and IL-2 production by 3H3 T cells whereas RMBP strongly induced both responses by R2.2F4 (2F4) T cells [36 , 37 ]. Nonetheless, RMBP had sufficient agonistic activity to elicit expression of I-A on 3H3 T cells and enabled presentation of RMBP to other T cells [38 ]. Paradoxically, recognition of antagonistic ligands resulted in stronger, more enduring T-APC activity than that elicited by recognition of agonistic ligands. To assess the longevity of T-APC activity, I-A+ 3H3 T cells were purified from a 3-day culture with RMBP and irradiated SPLs, were propagated in CM for 4 or 29 days, and then were tested for T-APC activity (Fig. 1 ). Because 3H3 T cells were not overtly activated by RMBP, these T cells remained quiescent, did not expand in CM, and retained low levels of I-A molecules (data not shown). Even after 29 days of culture in CM, 3H3 T-APCs retained sufficient stimulatory activity to drive proliferation of 2F4 responders. RMBP was added only during the initial 3-day culture and was added to neither the CM nor the bioassay. Previous studies have shown that T-APC activity is entirely dependent on presentation of antigen rather than on a nonspecific mitogenic activity [14 , 37 , 38 ]. These data indicate that T cells have the capacity to retain and present antagonistic ligands for prolonged periods.



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  		Figure 1. 3H3 T-APCs retain immunogenic antigen for a prolonged duration in vitro. 3H3 T cells (5x105/mL) were cultured for 3 days with irradiated SPL (5x106/mL) and 2 µM RMBP. On the last day, silica was added to cultures overnight to deplete phagocytic APCs. T cells were purified on discontinuous Percoll gradients and were cultured (105/mL) in CM for 4 or 29 days. 3H3 T cells were then irradiated and used as T-APCs (x-axis) to stimulate 2F4 responder T cells. These data are representative of two experiments.

Residual contamination by M{phi} or dendritic cells (DCs) in these experiments was discounted because of the following considerations: prior irradiation of SPLs (3,000 rads) reduced survival of I-A+ M{phi} and DCs during the initial 3-day culture. Purified M{phi}s and DCs did not persist in vitro in the absence of specific growth factors, particularly after irradiation (data not shown). Professional phagocytic APCs were further depleted at the end of the initial 3-day culture by the addition of silica. The possible persistence of contaminating professional APCs was also assessed by measurement of the M{phi}/DC product NO (Fig. 2 ). In these experiments, 3H3 T-APCs were generated in cultures of irradiated SPLs and RMBP, were purified, and were used to stimulate 2F4 responders. These 3H3 T-APCs stimulated IL-2 production but did not elicit detectable NO production. When added to these cultures, rested peritoneal M{phi} produced low levels of NO but were stimulated to produce high levels of NO in the presence of activated 2F4 T cells. M{phi} were more effective APCs than 3H3 T cells, presumably because M{phi} constitutively process antigen and express higher levels of costimulatory and adhesion molecules. Overall, these data demonstrated the lack of functional M{phi} and DCs in purified preparations of 3H3 T-APCs.



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  		Figure 2. T-cell-mediated antigen presentation elicits IL-2 production but not NO production. To prepare T-APCs, 3H3 T cells were cultured for 3 days with irradiated SPL in the presence [3H3(R)] or absence [3H3(-)] of 1 µM RMBP and were purified on a discontinuous gradient of Percoll. 3H3 T cells were then cultured for 3 days with or without 2F4 T cells (2.5x105/mL), rested peritoneal M{phi} (106/mL), or 1 µM RMBP. IL-2 production was measured by the CTLL assay. NO production was assessed by the Griess reaction. These data are representative of three experiments.

We concluded that a TCR antagonist promoted enduring T-APC activity (Fig. 1) . However, generation of 3H3 T-APCs in the presence of RMBP coupled with overt activation by conA or high concentrations of agonistic antigen resulted in the loss of T-APC activity during 3 days of expansion in CM (Fig. 3 ). The loss of T-APC activity by blastogenic T cells might in part reflect dilution of cell surface I-A during repeated cell divisions. The concentration of RMBP (2 µM) was chosen to optimize subsequent T-APC activity, whereas the concentration of GPMBP (1 µM) was chosen to overcome the antagonistic activity of RMBP and thereby ensure activation of the clone. Overall, these data indicate that TCR antagonists promote the generation of T-APC activity and that the maintenance of T-APC activity requires a lack of overt cellular activation.



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  		Figure 3. Overt activation of 3H3 T cells results in the loss of T-APC activity. 3H3 T cells (3.3x105/mL) were cultured for 3 days with irradiated SPL (2.5x106/mL) and 1 µM GPMBP, 2 µM RMBP, or 2.5 µg/mL con A. 3H3 T cells were purified on a discontinuous Percoll gradient and were cultured (105/mL) in CM for 3 days. These T cells were then washed, irradiated, and used as T-APCs (x-axis) to stimulate MBP-specific 2F4 responders. These data are representative of three experiments.

3H3 T-APCs that were cultured with cognate responders lost I-A expression (Table 2 ). That is, 3H3 T-APCs that presented RMBP to 2F4 responders during a 3-day culture lost both I-A and I-E glycoproteins. Presentation of conalbumin to conalbumin-specific CONAL.8D9 (8D9) responders also resulted in the loss of I-A and I-E from 3H3 T-APCs. The cognate interaction between T-APCs and responders was required, in that 3H3 T-APCs retained high levels of surface I-A in the absence of relevant responders. Loss of I-A from T-APCs correlated with acquisition of I-A molecules by responder T cells (data not shown) [28 , 29 ]. Various types of APCs shed intact I-A/peptide complexes that are acquired by responder T cells [28 , 29 ]. Intercellular transfer of integral I-A proteins might be mediated by APC-derived vesicles (i.e., exosomes) that are released from multivesicular MHC class II biosynthetic compartments and that are subsequently acquired by activated responder T cells.


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  		Table 2. Loss of MHC Class II Molecules From T-APC During Interaction With Responders

The interaction of T-APCs with responder T cells induced proliferation of both populations (Fig. 4 ) even though RMBP was insufficient as a stimulus for 3H3 T-cell proliferative responses [36 , 38 ]. When generated in the presence of RMBP, irradiated T-APCs stimulated responders in accordance with responder reactivity to RMBP (2F4>GP2.5F3>3H3, 8D9 T cells=0) (Fig. 4A) . When generated in the presence of both RMBP and conalbumin, irradiated T-APCs stimulated both RMBP-specific and conalbumin-specific responders (Fig. 4B) . Conversely, cultures of T-APCs and irradiated responders revealed extensive reciprocal activation and proliferation of T-APCs. Reciprocal activation of T-APCs required specific presentation of cognate antigen to responders (Fig. 4C and 4D) . For example, 3H3 T-APCs generated in the presence of RMBP exhibited activation when cultured with RMBP-specific responders but not when cultured with conalbumin-specific 8D9 T cells. When generated in the presence of both RMBP and conalbumin, 3H3 T-APCs exhibited proliferation when cultured with either responder.



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  		Figure 4. T-APC interaction with responders elicits proliferation of T-APCs. 3H3 T cells (5x105/mL) and irradiated SPL (5x106/mL) were cultured for 3 days with no antigen [3H3(-)] or 2 µM RMBP in the absence [3H3(R)] or presence of 100 µg/mL conalbumin [3H3(R+C)]. These T cells were then purified on Percoll gradients, were propagated in CM for 1 day, and were used as T-APCs to stimulate 2F4, GP2.5F3, 8D9, or 3H3 responder T cells. Irradiated [3H3(R)] T-APCs (A) or irradiated [3H3(R+C)] T-APCs (B) were cultured at designated densities (x-axis) with responders (2.5x104/well). Viable T-APCs (2x104/well) were cultured with designated densities (x-axis) of irradiated 2F4 (C) or irradiated 8D9 responders (D). These data are representative of four experiments.

This reciprocal mechanism of T-APC/responder activation was also assayed by flow cytometry (Fig. 5 ). 3H3 T-APCs that presented RMBP [3H3(R)+2F4] to 2F4 T cells exhibited right-shifted forward-scatter profiles, whereas T-APCs [3H3(R) or 3H3(-)] that were cultured without responders did not show blastogenesis (Fig. 5A) . Presentation of RMBP by 3H3 T-APCs to 2F4 responders also induced up-regulation of activation markers on the T-APCs, including the IL-2 receptor (Fig. 5B) and the OX40 marker (Table 3 ). Induction of activation markers on T-APCs was dependent on the strength of antigenic interactions with responders. For example, induction of OX40 on T-APCs reflected the anti-RMBP reactivity of the respective responder (2F4>GP2.5F3>GP2.4E5). The intensity of activation was less marked in T-APCs than in responders (Table 3 and Fig. 4 and 5 ). Hence, reciprocal activation of T-APCs caused a partially activated phenotype together with clonal expansion but did not elicit full cellular activation as was observed in responder T cells (Fig. 5B and 5C) .



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  		Figure 5. Antigen-dependent T-APC-responder interactions cause T-APC activation. 3H3 T cells were labeled with PKH26 and were then cultured (5x105/mL) with irradiated SPL (5x106/mL) in the presence or absence of 2 µM RMBP [3H3(R) or 3H3(-), respectively] for 3 days. Silica (500 µg/mL) was added during the last 2 h of culture. T-APCs were then purified on discontinuous Percoll gradients and were cultured with or without 2F4 responders for 3 days. T cells were labeled with OX39 (anti-IL-2 receptor mAbs) and a fluorescein isothiocyanate-conjugated rat anti-mouse secondary antibody. (A) Histograms show forward-scatter profiles of 3H3 T cells. Note that the three left-most histograms are overlapping. (B and C) Histograms show expression of IL-2 receptor (OX39 mAb) of PKH26+ gated 3H3 T cells (B) or PKH26- 2F4 responders (C). Shaded histograms represent T cells stained without primary mAb but with the secondary antibody. These data are representative of three experiments.


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  		Table 3. Antigen-Specific Interactions of T-APC and Responders Result in Mutual Induction of OX40

To assess the specificity of reciprocal activation, 3H3 T-APCs were cultured with responder 2F4 T cells in the presence of bystander conalbumin-specific 8D9 T cells (Fig. 6 ). 3H3 T cells were prepared in the presence or absence of RMBP [3H3(R) or 3H3(-)], were labeled with PKH67 (FL1), and were cultured with 2F4 responders together with biotinylated 8D9 bystander T cells. Presentation of RMBP (first row) caused up-regulation of OX40 on both 3H3 T-APCs and 2F4 responders but did not elicit up-regulation of OX40 on bystander 8D9 T cells. In the absence of RMBP (second row), T cells exhibited basal levels of OX40. That is, 3H3 T cells expressed low levels of OX40, 2F4 cells exhibited a bimodal distribution of OX40 expression, and 8D9 bystanders did not express OX40. RMBP-induced up-regulation of OX40 on 3H3 T cells was dependent on 2F4 responders and therefore was not attributed to RMBP-mediated stimulation alone. These data indicate that responder-mediated activation of T-APCs might elicit a focused expansion of T-APCs. Particularly in vivo, T-APC-responder interactions probably would not drive bystander T cells because the latter population would be largely quiescent T cells lacking high levels of cytokine responsiveness.



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  		Figure 6. Mutual activation of T-APC/responder T cells does not activate quiescent bystander T cells. 3H3 T cells were cultured (5x105/mL) with irradiated SPL (5x106/mL) in the presence or absence of 2 µM RMBP [3H3(R) or 3H3(-)] for 2 days and were then purified on Percoll discontinuous gradients and were labeled with PKH67. These T-APCs were cultured with or without unlabeled 2F4 responders together with biotinylated conalbumin-specific 8D9 T cells (105/mL each), as designated, for 2 days. Histograms show PKH67-labeled 3H3 T-APCs (left), unlabeled 2F4 responders (middle), and biotinylated 8D9 bystanders (right) after staining with no mAb (shaded) or the anti-OX40 mAb (bold lines) and then with a goat anti-mouse secondary antibody and Red670-streptavidin. These data are representative of three experiments.

RMBP also elicited selective enlargement of 3H3 T-APCs compared with bystander 8D9 T cells. That is, basal forward-scatter profiles (mean±SD) for 3H3, 2F4, and 8D9 T cells (306±2, 299±8, and 271±1, respectively) increased in these cocultures by 1.47 ± 0.02, 1.78 ± 0.06, and 1.27 ± 0.04-fold when 3H3 T-APCs presented RMBP. Size enlargement by all three clones might be a response to the elaboration of IL-2 in activated cultures. These three clones expressed high levels of the IL-2 receptor, as is typical of clones that have been derived by long-term propagation in IL-2, and size enlargement is a typical response of IL-2-dependent T cells to IL-2.

Hence, cytokines, particularly IL-2, might mediate important roles in the reciprocal activation of 3H3 T-APCs. IL-2 is produced and rapidly consumed during RMBP-dependent interactions between 3H3 and responder T cells (data not shown). To assess whether IL-2 and activated responder T cells had common capacities to elicit parameters of T-APC activation, 3H3 T-APCs that presented RMBP [3H3(R)] or RMBP and conalbumin [3H3(R+C)] were generated. These T-APCs were cultured with irradiated conalbumin-specific responders (Fig. 7A ). Exogenous IL-2 elicited proliferative responses by 3H3 T cells that were similar in magnitude to those elicited by irradiated conalbumin-specific responders. The observation that IL-2 and responder-mediated activation induced nonadditive responses by T-APCs suggested that feedback activationmight be mediated, at least in part, by IL-2. IL-2 and activated responders also elicited similar levels of B7.1 on T-APCs, whereas IL-2 did not fully mimic responder-mediated induction of the IL-2 receptor on 3H3 T cells (Fig. 7B) . Hence, feedback activation of T-APCs might involve a number of pathways, some of which might involve IL-2 as a central mediator. Interpretation of these data might be qualified by the fact that exogenous IL-2 was added at substantially higher concentrations than what was detectably produced by responder T cells.



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  		Figure 7. IL-2 may account, in part, for responder-mediated activation of T-APCs. (A) 3H3 T cells were cultured (5x105/mL) for 2 days with irradiated SPL (5x106/mL) and 2 µM RMBP in the presence or absence of 100 µg/mL of conalbumin [3H3(R+C) or 3H3(R)]. 3H3 T-APCs were purified on a Percoll discontinuous gradient, were cultured in CM for 4 days, and then were cultured with designated densities (x-axis) of irradiated conalbumin-specific responders in the presence or absence of recombinant rat IL-2 (~100 U/mL) in a 3-day proliferative assay. (B) 3H3 T-APCs were cultured for 2 days with irradiated SPL in the presence or absence of RMBP [3H3(R) or 3H3(-)] and were purified on a Percoll gradient. 3H3 T-APCs were cultured for 2 days with PKH67-labeled 2F4 responder T cells in the presence or absence of recombinant IL-2. Histograms show 3H3 T-APCs that were stained with no mAb (shaded), anti-IL-2R OX39 mAb, or anti-B7.1 mAb and a phycoerythrin-conjugated goat anti-mouse secondary. These data are representative of three experiments.

T cells that recognize antagonistic ligands and that expand after an agonist-driven response might mediate regulatory functions. As shown in Figure 8A (bottom) and in previous studies [36 , 38 ], presentation of RMBP by irradiated 3H3 T-APCs was stimulatory when assayed with rested responders. However, presentation of RMBP to preactivated responders inhibited IL-2-dependent growth of those responders (Fig. 8A , top). The inhibitory activity of 3H3 T-APCs was blocked by anti-I-A OX6 mAbs. Activated responder T cells were also I-A+, and they mediated inhibitory cross-presentation of RMBP by a mechanism that was blocked by OX6 mAbs (compare bars 1 and 3 in Fig. 8A ). These data indicate that the functional consequence of T-APC-responder interactions might be related to the activation status of the respective T cells.



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  		Figure 8. Presentation of RMBP by 3H3 T-APCs inhibits IL-2-dependent growth of preactivated responders. (A) Preactivated RsL.11 responders were generated in a 3-day culture with irradiated SPL and 2 µM GPMBP and were purified on a discontinuous Ficoll/Hypaque gradient. To generate 3H3 T-APCs, 3H3 T cells (5x105/mL) were cultured for 3 days with irradiated SPL (5x106/mL) and 2 µM RMBP. Silica was added to the culture during the last day, and 3H3 T cells were purified on consecutive discontinuous Percoll and Ficoll/Hypaque gradients. Preactivated (top) or rested (bottom) RsL.11 responder T cells (104/well) were stimulated with recombinant IL-2 in the presence or absence of irradiated 3H3 T-APCs (2.5x104/well). The anti-I-A mAb OX6 (1:20 dilution) was or was not added to these cultures at the initiation of a 6-day proliferative assay. Maximal proliferative responses were 103,918 cpm (top) and 16,029 cpm (bottom). (B) Designated numbers of R1-trans T cells (legend) were cultured with different densities of irradiated 2F4 responders (x-axis) in the presence of 2 µM GPMBP. Maximal proliferative responses for low, medium, and high densities of R1-trans T cells were: 6,438, 36,588, and 62,532 cpm, respectively. Parallel cultures without GPMBP exhibited background proliferation of <1,000, <2,300, and <4,000 cpm, respectively. These data are representative of three experiments.

3H3 T-APCs were activated partially during interactions with responders and therefore were spared activation-induced cell death. However, use of a different experimental system revealed that fully activated T-APCs were stimulated or inhibited based on the density of irradiated responders (Fig. 8B) . When a constitutively active T-APC clone (i.e., R1-trans) was used in place of 3H3 T-APCs, relatively low densities of irradiated responders stimulated growth of R1-trans T cells, whereas progressively higher responder/T-APC ratios inhibited proliferation of R1-trans T cells (Fig. 8B) . Previous studies have shown that R1-trans T cells constitutively express a blastogenic phenotype marked by high levels of class II MHC and B7 glycoproteins [14 , 28 , 29 , 37 ]. The inhibitory mechanism that was observed in the presence of high responder cell densities depended on R1-trans presentation of MBP, was blocked by anti-I-A mAb OX6, and resulted from death of R1-trans T-APCs (data not shown). Overall, these data indicate that the relative expansion of interacting T-APC-responder populations depends on an interplay of positive influences, including reciprocal feedback activation of T-APCs and overt activation of responders balanced by inhibitory influences such as activation-induced cell death.


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DISCUSSION
 
This study indicates that T-APCs directly activate responder T cells, which then reciprocally activate T-APCs. This mechanism is reminiscent of the established model of B-cell/T-cell collaboration [44 , 45 ]. B cells use surface Ig receptors to concentrate specific antigen within the class II antigen-processing pathway for subsequent presentation to clonotypic responder T cells, which then provide feedback signals to activate B-cell antibody production. Likewise, the clonotypic specificity of TCRs strongly promotes acquisition of MHC II glycoproteins from professional APCs so that the T-APCs present only those antigens that are acquired with the cognate antigen [14 , 34 , 36 37 38 ]. These T-APCs then present class II MHC ligands to responder T cells, which, in turn, cause activation and proliferation of T-APCs. This mechanism might couple the clonal expansion of pathogenic T cells that strongly recognize a self-MHC ligand with the clonal expansion of regulatory T cells that recognize the same self-MHC ligand as an antagonist.

The mechanism by which responders elicit partial activation of T-APCs has not been determined. Responder-mediated activation of 3H3 T-APCs did not induce activation of bystander T cells. Hence, antigen-specific interactions appeared important for activation of T-APCs. High concentrations of IL-2 mimicked the action of responder T cells by promoting certain parameters of T-APC activation. This latter finding suggested that production of IL-2 by responders might strongly promote activation of T-APCs. Reciprocal activation of T-APCs resulted in the induction of B7 costimulatory molecules on T-APCs. Up-regulation of costimulatory molecules on T-APCs would presumably in turn facilitate IL-2 production by responders and thereby promote activation and growth of both T-APCs and responders. Activated responder T cells might release IL-2 and other cytokines directionally onto T-APCs and might also up-regulate cytokine responsiveness. The action of IL-2 in vivo is specific because of the activation-dependent expression of the IL-2 receptor. IL-2 also induces a fas/fas ligand pathway and might prime chronically activated T cells for activation-induced cell death. Hence, T-APC-responder interactions might be a key regulatory interaction in the control of cell-mediated immunity.

One of the central mechanisms by which T-helper cells may acquire class II MHC/peptide complexes is by acquisition of complexes assembled and shed by professional APCs [29 ] in the form of vesicles or exosomes [28 ]. The required role of the TCR is to catalyze T-cell activation, but, thereafter, the TCR is dispensable for the acquisition of MHC class II/peptide complexes. That is, preactivation of responders obviates the need for TCR recognition and enables responders to acquire allogeneic or xenogeneic MHC molecules from APCs [29 ]. Physiologically, TCR recognition represents a necessary step in acquisition of MHC class II glycoproteins because TCR ligation is required for activation and activation is required for acquisition of MHC class II glycoproteins. TCR recognition events required for acquisition of MHC class II ligands are initiated even by antigens that exhibit low levels of agonistic activity in other assays. For example, RMBP is a TCR antagonist of 3H3 T cells in assays of IL-2 production or proliferation, but RMBP nonetheless has sufficient agonistic strength to promote acquisition of MHC class II glycoproteins [38 ]. Thus, even inefficient cognate interactions are sufficient to catalyze intercellular transfer of class II MHC glycoproteins, and the threshold required for induction of an I-A+ phenotype on T-APCs is substantially lower than that required for overt T-cell activation.

Positive thymic selection promotes differentiation of T-helper cells that recognize specific class II MHC/self-peptide complexes as inefficient ligands [46 47 48 49 ]. Thus, the repertoire of naïve T-cells is selected to recognize self-MHC ligands in peripheral tissues and appears to depend on the continued inefficient recognition of self for survival [50 , 51 ]. Thus, the homeostatic self-recognition events necessary for T-cell survival in vivo might also be sufficient to promote acquisition of MHC class II/peptide complexes. This postulate is supported by the observation that naïve splenic and thymic T cells express low levels of MHC class II glycoproteins [29 ]. Normal T cells might present those self-MHC class II complexes to any responder T cell able to recognize those self-peptides as strongly agonistic ligands. If such T-cell/T-cell interactions were sufficiently tolerogenic, this mechanism would continually purge the mature repertoire of highly autoreactive T cells. Given that T-to-T interactions cause clonal expansion of T-APCs, this mechanism would also reinforce the contingent of mature T cells prone to presenting that self-antigen in proportion to the frequency of highly autoreactive (i.e., pathogenic) T cells.


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ACKNOWLEDGEMENTS
 
This study was supported by a research grant from the National Multiple Sclerosis Society.

Received November 20, 2000; revised March 31, 2001; accepted April 5, 2001.


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