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The Journal of Immunology, 1999, 163: 6557-6566.
Copyright © 1999 by The American Association of Immunologists

Ability of a Nondepleting Anti-CD4 Antibody to Inhibit Th2 Responses and Allergic Lung Inflammation Is Independent of Coreceptor Function1

Li Li, Mary Crowley, Andrea Nguyen and David Lo2

Department of Immunology IMM-25, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nondepleting anti-CD4 Abs have been used in vivo to induce Ag-specific immunological tolerance in Th1 responses, including tissue allograft rejection and autoimmune diabetes. To examine whether this Ab (YTS177.9) acts by provoking a Th2 shift, we tested the effect in a mouse model of allergic lung inflammation. Interestingly, nondepleting anti-CD4 treatment induces tolerance to allergens as well, especially when given during initial priming. In vitro studies indicate that the effect of the Ab is independent of CD4 coreceptor function, as Ab treatment also inhibits proliferation and induces a persistent anergy in naive CD4 T cells stimulated by anti-CD3/CD28. Moreover, the Ab stimulated a distinct pattern of tyrosine phosphorylation in T cells even in the absence of TCR triggering, suggesting that signaling through CD4 alone induces significant physiological changes in T cell function. These results show that tolerance induced by anti-CD4 triggering is not a simple shift in Th1/Th2 effector function or depletion of Ag-specific cells, but may instead induce a persistent clonal anergy capable of blocking subsequent immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 T cells are critical cells in directing effector function in several kinds of immune responses, including the Ab response to viral infection, cellular immune responses to tissue allografts, delayed-type hypersensitivity, and autoimmune diseases such as type 1 diabetes, thyroiditis, and multiple sclerosis. Therefore, therapeutic approaches in pathological syndromes are often aimed toward the modification or modulation of CD4 T cell functions. For example, in the case of type 1 diabetes, this has been achieved through some general approaches such as the in vivo depletion of cells expressing {alpha}ß-TCR (1), CD3 (2), or CD4 (3). In similar fashion, anti-CD4 depletion has been shown to prevent lung-allergic inflammation (4, 5). Unfortunately, these approaches risk generalized immune suppression, predisposing the subjects to opportunistic infections. Recently, however, a more subtle approach has been developed using Abs against CD4 that do not deplete the T cell pool, but were still found to have significant immunomodulatory effects. These effects were not due to generalized immunosuppression, but depended on simultaneous exposure of CD4+ T cells to a specific Ag, providing for an Ag-specific tolerance.

In studies by the Waldmann group, an anti-CD4 Ab (YTS177.9) was found to have striking effects in Ag-specific CD4 T cell responses, without causing a depletion of peripheral CD4 T cells (reviewed in Ref. 6). When mice were primed to specific Ag during anti-CD4 treatment, a longstanding Ag-specific tolerance was induced that could not be broken even when naive cells were infused. The Ag-specific nature of this effect was clear, as it did not interfere with subsequent priming to secondary Ags. It was suggested that the anti-CD4 treatment induced a dominant Ag-specific tolerance, but the specific mechanism was not clearly defined. One of the possible mechanisms is the induction of clonal anergy, as described for superantigen-reactive Vß6+ T cells in the bone marrow transplantation model (7). In this study, bone marrow transplantation with Mls3-1a-positive cells did not significantly deplete the Mls-reactive Vß6+ T cells, but proliferative responses to Mls were reduced. However, since not all Vß6 cells are superantigen reactive, alterations in the receptor repertoire in the chimeras might also account for this effect.

Another compelling possible explanation for the effects of the nondepleting anti-CD4 Ab comes from studies on the nonobese diabetic mouse model of spontaneous autoimmune diabetes. As with tissue allograft rejection, Th1 cells appear to be the main effector cells driving pathogenesis, although in this case they are specific for islet ß cell Ags. Treatment of nonobese diabetic mice with the anti-CD4 Ab was able to prevent diabetes in three different situations, including 1) the spontaneous development of disease (8), 2) adoptive transfer into sublethally irradiated recipients (9), and 3) induction with high doses of cyclophosphamide (10). This last observation is most relevant, as cyclophosphamide-induced diabetes has also been shown to correlate with increased IFN-{gamma} production by T cells (11, 12), essentially a Th1 shift. Moreover, cyclophosphamide can abrogate the effects of anti-CD4 even in thymectomized mice, suggesting that these reagents have direct and reversible effects on the T cells and their effector function. Thus, the longstanding tolerance in anti-CD4-treated mice may be due to the development of Ag-specific suppressor effector CD4 T cells, most likely with a Th2 phenotype.

In the studies on tissue allograft rejection and autoimmune diabetes, the highly polyclonal nature of the responding T cell population made it difficult to closely follow Ag-specific cells to determine the effects of the anti-CD4 treatment. Thus, we have used TCR transgenic mice to provide more detailed in vitro information on whether the Ab will have significant effects on Th2-mediated immunity such as allergic asthma, and whether the primary effect on Ag-specific T cells involves a Th1 to Th2 shift or clonal anergy. Our results suggest that anti-CD4 treatment can have potent effects in blocking the development of allergic lung inflammation. Consistent with this effect, we find that in vitro the anti-CD4 inhibits proliferation and induces a persistent anergy in primary CD4 T cells; moreover, the induction of anergy is independent of CD4 coreceptor function, and may involve the activation of novel signaling pathways. Thus, anti-CD4 therapy may have broader application than generally assumed, and may be an effective method for inducing anergy in the prevention of allergic diseases, including asthma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

TCR-SFE mice (13, 14) are transgenic for a TCR specific for influenza PR8 hemagglutinin peptide 110–119 (SFERFEIFPK) presented on I-Ed. The mice had been backcrossed to the BALB/c strain for more than 10 generations. AND mice (15) are transgenic for a TCR specific for moth cytochrome c peptide presented on I-Eb. They were backcrossed more than 9 generations to the C.B10-H2b/LiMcdJ congenic mouse strain with the BALB/c background, and H-2b. Naive BALB/c mice (6–8 wk) were provided by the Rodent Breeding Colony in the Scripps Research Institute (La Jolla, CA). All mice were maintained in the Scripps Research Institute rodent colony under specific pathogen-free conditions, in accordance with National Institutes of Health and the Scripps Research Institute institutional guidelines.

Abs and reagents

The nondepleting anti-CD4 Ab YTS177.1 (16) was obtained as a hybridoma cell line generously provided by Dr. J. Davies (The Scripps Research Institute), with normal rat Ig (Sigma, St. Louis, MO) as control Ab. Human rIL-2 and mouse rIL-4 were obtained from Pepro Tech (Rocky Hill, NJ). Anti-IL-12 was the monoclonal rat IgG clone C17.8.20, obtained as a hybridoma generously provided by Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA). Anti-CD3, CD28, CD4, CD8{alpha}, B220, and CD62L Abs were all obtained from PharMingen (San Diego, CA), and mouse anti-rat IgG polyclonal Ab (F(ab')2 fragment) was obtained from Jackson ImmunoResearch (West Grove, PA). Abs and standards for ELISAs measuring cytokines and serum Ig were obtained from PharMingen.

Induction of OVA-specific lung inflammation

Naive mice were immunized i.p. with 10 µg chicken egg OVA (Sigma) in 100 µl of PBS mixed with the same volume of Imject Alum (Pierce, Rockford, IL); these mice were then boosted the same way in the following weeks, as indicated in the figures. Anti-CD4 or control Abs were given 1 mg/mouse/time i.p. as indicated in the figures. Mice were challenged with 30 µl OVA (2 mg/ml) intranasally once per day for 3 days, and sacrificed 3 h after the last challenge. The lungs were perfused from the right ventricle using PBS until they had turned white, and bronchoalveolar lavage (BAL) was collected by washing lung through the trachea three times using 1 ml of RPMI with 2% horse serum. Cytospins were prepared for BAL cells from each mouse, and BAL fluids were frozen for cytokine detection. The right lobes of the lung were fixed in Bouin’s for hematoxylin and eosin (H&E) staining. The left lungs were frozen in OCT compound (Miles, Elkhart, IN) for immunohistochemical staining.

Histology

BAL cells on cytospin slides were fixed with methanol and stained with eosin and methylene blue (Fisher, Pittsburgh, PA). Leukocytes were analyzed by differential count of total 200–300 cells on coded slides. Lung was perfused, injected with OCT through the trachea, and frozen in OCT. Frozen lung sections were fixed with cold acetone with 1% formamide (Fisher) and eosinophils were stained for cyanide-resistant eosinophil peroxidase activity, as described (17).

In vitro assays.

On primary cells. TCR-SFE CD4+ T cells were purified from lymph node cells with magnetic beads by depleting with Abs to CD8+ and B220+ cells. A total of 5 x 105 CD4 T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 were used to coat the plates, 37°C for 1 h) in 200 µl media with 100 µg/ml of either anti-CD4 or control Abs. In addition, 3 x 105 CD4+ T cells were stimulated with 5 x 105 irradiated BALB/c spleen APC plus 1 µg/ml SFE peptide in the presence of either the anti-CD4 or control Abs. Media were changed every 2 days, and supernatants were collected at day 3 or day 5 for cytokine detection. IL-4, IL-5, and IFN-{gamma} were measured by ELISA. T cell proliferation was also tested on day 3 or day 5 by [3H]TdR incorporation. To test the persistence of anergy, stimulated cells were harvested on day 3 and cultured in media with 2 ng/ml IL-2 for another 3 days, then 5 x 105 of these cells were restimulated by anti-CD3 plus anti-CD28 or 3 x 105 T cells stimulated with 5 x 105 APCs plus SFE peptide in the absence of Abs. Cytokine production and cell proliferation were tested 3 days after the restimulation.

On differentiated Th1 and Th2 cells. Th1 and Th2 cells were generated from TCR-SFE cells, as described previously (17). Briefly, naive CD4 T cells (FACS sorted for CD62L+ cells) were induced to differentiate to Th1 by stimulation in the presence of IL-12, and Th2 by stimulation in the presence of IL-4 and neutralizing anti-IL-12. After 7 days, 2 x 105 of the Th1 or Th2 cells were restimulated with spleen APC plus 1 µg/ml SFE peptide in the presence of anti-CD4 or control Abs. Cytokine production and cell proliferation were tested 2 days after restimulation.

Effects of anti-CD4 Ab on signaling induced by stimulation through TCR

Lymph node CD4 T cells were purified from AND transgenic mice. A total of 2 x 106 cells were stimulated with plate-bound anti-CD3 plus anti-CD28 in the presence of 100 µg/ml anti-CD4 or control Abs. Cells were harvested at different time points, as indicated in the figure, and lysed with ice-cold lysis buffer consisting of 1% Triton X-100, 50 mM HEPES, 10% glycerol, 1.5 mM MgCl2, 100 mM NaF, 1 mM PMSF, and 1 mM NaVO4. Soluble lysate proteins were separated on 9% SDS-PAGE and transferred to 0.22-µm nitrocellulose membranes. The tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine mAb 4G10 (PharMingen), followed by HRP-conjugated sheep anti-mouse Ig (Amersham, Arlington Heights, IL). The bands were visualized with the ECL chemiluminescence system (Amersham).

Statistics

Two-tailed Student’s t test was used to analyze all data, except for Figs. 4GoB and 5B, with p < 0.05 considered significant. Figs. 4GoB and 5B were analyzed by one-tailed Student’s t test, and p < 0.05 was considered significant.



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  		FIGURE 4. Anti-CD4 Ab showed long lasting inhibitory effects on allergic lung responses. OVA immunization and Ab administration were done as indicated in the figure. A, Airway infiltration was analyzed by differential and total cell counts of BAL. Each bar represents the mean ± SD of six mice; *, p < 0.05 compared with the control Ab-treated group. This figure combined two independent experiments. B, Anti-CD4 Ab showed long lasting inhibitory effects on OVA-induced up-regulation of serum total IgE and IgG1. Serum total IgE, IgG1, and IgG2a levels were measured by ELISA. Each dot represents one mouse; *, p < 0.02, compared with the control Ab-treated group.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A nondepleting anti-CD4 Ab induces long lasting protection from OVA-specific allergic lung inflammation

To test the effects of a nondepleting anti-CD4 on Th2-mediated immune responses, we induced an OVA-specific allergic lung inflammation by immunizing and boosting mice i.p. with OVA/alum, followed by an intranasal OVA challenge. This protocol induces extensive lung inflammation characterized by perivascular and peribronchial infiltration with T cells, macrophages, eosinophils, and neutrophils (Fig. 1Go). Consistent with the allergic/Th2 nature of the inflammation, eosinophils comprised as much as 40% of BAL recovered from these mice (Fig. 2GoA). When mice were given anti-CD4 Abs 1 day before and after each OVA immunization/boost, intranasal challenge with OVA provoked only minimal lung inflammation, with few if any eosinophils detected in tissue or BAL (Fig. 2GoA). Similarly, BAL levels of the Th2 cytokines IL-4 and IL-5 were significantly increased in immunized controls, but not in anti-CD4-treated mice (Fig. 2GoB). Thus, as with Th1-mediated immune responses, anti-CD4 treatment was able to block the development of Th2-mediated allergic lung inflammation.



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  		FIGURE 1. Nondepleting anti-CD4 Ab administered during immunization inhibits allergic lung inflammation. Naive BALB/c mice were immunized and boosted with OVA/alum i.p. 1 wk apart, and the anti-CD4 Ab or normal rat Ig as control Ab were given i.p. 1 day before and 1 day after the immunization/boost. These mice were challenged with OVA intranasally in the third week once per day for 3 days, and were sacrificed 3 h after the last challenge. Their lungs were fixed with Bouin’s and stained with hematoxylin and eosin (HE), or embedded in OCT, and the frozen sections were stained for cyanide-resistant eosinophil peroxidase (EPO) activity for eosinophils (x200).

 


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  		FIGURE 2. Anti-CD4 Ab administered during immunization inhibits OVA-induced allergic lung inflammation. A, OVA-specific immunization and Ab administration were done as indicated, followed by bronchoalveolar lavage. Cytospins were analyzed for differential and total cell counts of 200–300 cells per slide. Each bar represents the mean ± SD of four mice; *, p < 0.02, compared with the control Ab-treated group. This figure represents two independent experiments. B, Anti-CD4 Ab blocks Th2 cytokine production induced by OVA in BAL fluid. Cytokines were detected from BAL fluid by ELISA. Each bar represents the mean ± SD of four mice. C, Anti-CD4 Ab suppressed the up-regulation of serum Ig levels induced by OVA. Serum total IgE, IgG1, and IgG2a levels were measured by ELISA. Each dot represents one mouse; *, p < 0.005. This figure represents two independent experiments.

 
Up-regulation of total serum IgE and IgG1 is another characteristic of allergic responses, related to the development of Th2 effector cells. Thus, in mice immunized and challenged with OVA, high levels of serum IgE and IgG1 were detected, along with increased levels of the Th1-associated isotype IgG2a (Fig. 2GoC). Anti-CD4 Ab treatment during immunization blocked the increases in IgE and IgG1, although increases in IgG2a could still be seen under these conditions.

Because one goal of anti-CD4 treatment is to insure that tolerance induction is specific to the period of treatment, it was important to demonstrate that the effect of anti-CD4 treatment is transient. Thus, to examine the kinetics of the anti-CD4 Ab on CD4 T cells in vivo, the persistence of the anti-CD4 Ab was assessed by serial determinations of staining for surface rat IgG on peripheral blood T cells. Although control rat IgG showed no detectable T cell surface binding, anti-CD4 treatment caused an early peak in detectable cell surface rat IgG that decayed to near background levels by 6 wk postinjection (Fig. 3GoA).



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  		FIGURE 3. Kinetics of anti-CD4 activity in vivo. Naive BALB/c mice were given i.p. injections of 1 mg anti-CD4 Ab or rat IgG on 2 consecutive days (days -1 and 0). On the next day, and for 36 days thereafter, PBL were analyzed for surface rat IgG on CD3+ T cells (A), and the cell surface levels of CD4 on the CD4+ T cell subset (B). On day +1, cell surface rat IgG was detectable on a high percentage of CD3+ T cells in the anti-CD4-treated mice, decaying to near background levels by 36 days. In contrast, cell surface levels of CD4 (mean fluorescence intensity, or MFI) were persistently modulated on CD4 T cells, with only a very slow recovery. Data show two mice treated with anti-CD4, and two mice treated with rat IgG, with the same mice providing data for both surface rat IgG and CD4 assays. The day 0 time point actually reflects baseline data taken on day -3. Similar results were also obtained in two other separate experiments.

 
As noted previously, CD4 T cells were not eliminated by the Ab treatment, although there was a variable decrease in the numbers of CD4 T cells. The remaining CD4 T cells showed a striking decrease in cell surface levels of CD4. Curiously, in some cases, this modulation was persistent beyond the point in which cell surface rat IgG was detectable, showing only a very gradual recovery toward normal levels (Fig. 3GoB). Thus, the effect of the anti-CD4 treatment may persist in vivo beyond the immediate effects during Ag presentation. Alternatively, it is possible that extremely low levels of anti-CD4 Ab in vivo are sufficient to maintain dramatic modulation of surface CD4 levels.

CD4 T cell recognition of Ag on APC is in part dependent on the coreceptor function of the CD4 molecule, both in enhancing TCR binding to the class II MHC molecule, and in the recruitment of intracellular signaling kinases such as lck. Thus, one possible effect of the anti-CD4 Ab in vivo is to simply block effective recognition of the TCR ligand, and prevent Ag-specific priming. If this were the case, then the anti-CD4-treated mice would remain naive to the immunizing Ag, and subsequent priming in the absence of anti-CD4 Ab would result in normal allergic inflammatory responses. To test this, mice were given the standard protocol of OVA immunization and boost in the presence of anti-CD4 or control Ab. Six weeks later, when the Ab appeared to have cleared from the animals, a second set of OVA immunization and boost was given without any Ab treatment, followed by intranasal Ag challenge.

In mice given only control Abs, intranasal challenge provoked the expected lung-allergic inflammation dominated by eosinophils. In contrast, in mice treated with anti-CD4 during the first set of OVA immunizations, inflammation and eosinophilia were nearly absent (Fig. 4GoA). Moreover, increases in total serum IgE and IgG1 levels were also blocked, while IgG2a was not significantly affected (Fig. 4GoB). Thus, in mice immunized in the presence of the anti-CD4 Ab, a persistent tolerance to the allergen was established that could not be broken by later immunizations. This induced tolerance appears to be active, and not through simple prevention of immunization. However, as noted above, a persistent modulation of cell surface CD4 may also influence responses to the second priming. Although not formally demonstrated in this study, it is likely that this tolerance is OVA specific, as studies by the Waldmann group have shown that mice given similar anti-CD4 treatments remain competent to respond to subsequent priming to second party Ags (6).

Moderate inhibition of allergic inflammation by anti-CD4 treatment during intranasal Ag challenge

In most clinical situations, treatment can only be initiated after allergen immunization has already occurred. To determine the effect of anti-CD4 Ab during the effector phase of allergic immune responses, mice were immunized and boosted according to the standard protocol to establish allergic immune responses, but they were subsequently given two additional boosts 1 wk apart in the presence of anti-CD4 or control Ab. In this case, the total numbers of inflammatory cells in BAL were significantly reduced among anti-CD4-treated mice, but the proportions of eosinophils among the BAL cells were not greatly reduced (Fig. 5GoA). Interestingly, serum IgE and IgG1 titers were lower among anti-CD4-treated mice, but IgG2a titers were similar in both control and treated groups (Fig. 5GoB). Thus, the late treatment with anti-CD4 in allergic lung inflammation was able to significantly reduce the overall intensity of the inflammatory response (both BAL counts and IgE/IgG1 elevation), but the essential Th2 character of the response to OVA challenge was not changed.



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  		FIGURE 5. Anti-CD4 Ab administered at the effector stage inhibited the intensity of OVA-induced asthma inflammation. OVA-specific asthma induction and Ab administration were done as indicated. A, Airway-infiltrating cells were analyzed by differential and total cell counts of BAL. Each dot represents one mouse; *, p < 0.02, compared with the control Ab-treated group. This figure combined three independent experiments. B, Anti-CD4 Ab given at the effector stage inhibited OVA-induced up-regulation of serum total IgE and IgG1 levels. Serum total IgE, IgG1, and IgG2a levels were measured by ELISA. Each dot represents one mouse; *, p < 0.05, compared with the control Ab-treated group. This figure represents two independent experiments.

 
To summarize the effects of anti-CD4 treatment on allergic lung inflammation in vivo, we found that the treatment has dramatic inhibitory effects when given during initial immunization, and it induces a long lasting tolerance to the allergen even in the face of additional priming. However, this persistent tolerance was not associated with a shift toward Th1-associated responses such as increased IFN-{gamma} levels in BAL, nor increases in serum IgG2a levels. Moreover, when treatment was given late in the response, the intensity of the response could be reduced without altering the Th2 skewed pattern in the immune response. Thus, the anti-CD4 treatment clearly did not augment Th2 responses, as might be predicted from earlier studies on Th1-mediated disease, and indeed appeared to have no specific influence on the Th1/Th2 balance in vivo.

Anti-CD4 treatment of naive but not skewed CD4 T cells in vitro shows coreceptor-independent inhibition of proliferation and cytokine production

The tolerance induced by in vivo treatment with nondepleting anti-CD4 could reflect induction of a persistent anergy in the Ag-specific cells, peripheral deletion, or development of a novel phenotype. To distinguish among these possibilities, and provide more detailed information on the specific effects of anti-CD4 on Ag-specific T cells, we began studies using CD4 T cells isolated from TCR transgenic mice specific for class II-restricted peptides. With these T cells, we have the advantage of working with relatively homogeneous populations of T cells specific for the same target Ag. In addition, in vitro studies allow us to study naive and differentiated effector Th1/Th2 cells separately. In the case of the TCR transgenic (TCR-SFE) specific for the I-Ed-restricted hemagglutinin peptide, we also have shown that in vitro generation of Th2 cells from TCR-SFE cells can adoptively transfer a dramatic allergic lung inflammation in normal mice on peptide challenge in vivo (17).

Our studies on the blockade of allergic inflammation to OVA priming in vivo suggested that the anti-CD4 did not simply prevent priming to allergen, but instead may have given rise to a persistent population of Ag-specific regulatory T cells. Yet, because most incarnations of regulatory or suppressor cells involve Th2 cells and associated cytokines, this mechanism seems to be incompatible with the observed effects on allergic immune responses. However, since it was clear that Ag-specific (albeit inhibitory) responses could still be generated in the presence of anti-CD4 Ab, we examined the ability of Ag-specific naive T cells to respond to stimulation in vitro in the presence of anti-CD4 Ab.

Purified lymph node CD4 T cells from TCR-SFE mice were stimulated in vitro using either specific SFE peptide presented on spleen APC, or immobilized anti-CD3 plus anti-CD28. Cultures were also treated with anti-CD4 Ab or control Ab, and T cell proliferation and cytokine production were measured (Fig. 6Go). In response to these two stimuli, control CD4 T cell preparations generally show optimal proliferative responses to peptide/APC, but maximal cytokine production in response to anti-CD3/CD28. Although the presence of anti-CD4 Ab significantly inhibited proliferation to peptide/APC as expected, it also dramatically inhibited the response to anti-CD3/CD28 (Fig. 6GoA), showing that the effect of the Ab could be independent of the class II-binding coreceptor function of CD4. As with proliferative responses, treatment with anti-CD4 in vitro also inhibited the production of cytokines in primary stimulation. The inhibition affected all cytokines tested, including IL-2, the Th1 cytokine IFN-{gamma}, and the Th2 cytokines IL-4 and IL-5 (Fig. 6GoB).



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  		FIGURE 6. Anti-CD4 Ab-inhibited proliferation and cytokine production of CD4 T cells in vitro. A, SFE-TCR transgenic CD4 T cells were stimulated either with immobilized anti-CD3/CD28 or spleen APC and SFE peptide in the presence of 100 µg/ml anti-CD4 or control Abs. Cell proliferation was measured by [3H]TdR incorporation. B, Cytokines from the supernatants of anti-CD3/CD28 cultures were measured by ELISA. Each bar represents the mean ± SD of three wells. This figure represents three independent experiments. C, Anti-CD4 Ab treatment during primary stimulation causes a persistent inhibition of proliferation and Th2 cytokine production even on secondary stimulation. TCR-SFE transgenic CD4 T cells were stimulated with immobilized anti-CD3/CD28 peptides in the presence of 100 µg/ml anti-CD4 or control Abs for 3 days. Cells were then washed and rested in IL-2-containing media for another 3 days and restimulated with immobilized anti-CD3/CD28 or spleen APC and SFE peptides in the absence of Abs. Cell proliferation was measured by [3H]TdR incorporation. D, Cytokines from the supernatants of secondary anti-CD3/CD28 cultures were measured by ELISA. Each bar represents the mean ± SD of three wells. This figure represents three independent experiments.

 
In vivo, anti-CD4 treatment was able to induce a tolerance that persisted over several weeks. To determine whether this effect could also be demonstrated in vitro, we took CD4 T cells treated as above in vitro for 3 days, then washed and cultured for an additional 3 days in IL-2-containing media to insure viability. After this rest period, cells were restimulated with either peptide/APC or immobilized anti-CD3/CD28 in the absence of any further Ab treatment. Interestingly, the reduced response seen in the primary stimulation of anti-CD4-treated cultures was also seen in the restimulated cells (Fig. 6GoC). The persistent nonresponsiveness was most evident in the cells restimulated with peptide/APC. Thus, at least for the 6-day period studied, the anti-CD4 appeared to induce a persistent clonal anergy. It is not clear whether this effect would also apply in vivo for longer periods; documentation of this in vivo will clearly require further studies on adoptively transferred cells.

Cytokine production from the restimulated cells showed a curious alteration in the pattern of Th1 vs Th2 cytokines (Fig. 6GoD). Although control cells stimulated under these neutral conditions (immobilized anti-CD3/CD28) consistently showed increased production of Th2 cytokines and reduced production of IFN-{gamma} (relative to controls) on restimulation, the anti-CD4-treated cells showed persistently reduced production of IL-4 and IL-5, but increased production of IFN-{gamma}. This appears to comprise a significant shift toward the Th1 phenotype, but this is also in the context of greatly reduced T cell proliferation, so the production of IFN-{gamma} overall may still remain low with respect to the original input T cell numbers.

As shown above, the treatment of OVA-immunized mice with anti-CD4 in the weeks after initial immunization and boosting effected only a moderate reduction in the intensity of the lung inflammation without significantly altering the allergic nature of the inflammation. This suggested that established Th2 skewed effector responses are resistant to further modification by anti-CD4, although recruitment or amplification of their responses could still be affected. To examine this in vitro, we generated differentiated Th1 and Th2 T cells using TCR transgenic cells and primary stimulation in vitro under skewing conditions. As reported previously, our protocols give rise to differentiated Th1 and Th2 effector cells that are capable of mediating Ag-specific responses in vivo with characteristic patterns of inflammation (17). In concordance with the in vivo results above, we found that differentiated Th1 and Th2 cells were entirely resistant to the anti-CD4 Ab treatment (Fig. 7Go). Thus, proliferative responses were unaffected when both Th1 and Th2 cells were restimulated in the presence of anti-CD4. More importantly, the patterns and amounts of Th1 and Th2 cytokines were also unaffected, confirming the notion that anti-CD4 treatment cannot alter the character of established skewed immune responses.



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  		FIGURE 7. Anti-CD4 Ab fails to suppress the response of previously differentiated Th1 or Th2 cells. TCR-SFE transgenic Th1 and Th2 cells were differentiated as indicated in Materials and Methods. Cells were restimulated by spleen APC with SFE peptides in the presence of 100 µg/ml anti-CD4 or control Abs. Cell proliferation was measured by [3H]TdR incorporation. Cytokines from the supernatants were measured by ELISA. Each bar represents the mean ± SD of three wells. This figure represents two independent experiments.

 
Anti-CD4 treatment of naive T cells induces tyrosine phosphorylation of novel intracellular substrates

Stimulation of naive T cells in vivo and in vitro in the presence of anti-CD4 induced a long lasting tolerance that was not due to a simple blockade of T cell activation. This suggested that the treated T cells were actively altered by the Ab treatment, causing the persistent anergic phenotype. To determine whether this was induced through novel signaling pathways, we examined tyrosine phosphorylation patterns in naive CD4 T cells stimulated in the presence and absence of anti-CD4. In these studies, to insure that the population of CD4 T cells was naive, we used a TCR transgene (TCR-AND) specific for a moth cytochrome c peptide presented on I-Eb. Because thymic selection and export of these T cells occur in H-2b mice lacking I-E expression, the transgenic T cells can develop in these animals lacking the restricting I-E molecule and the target ligand, and remain truly naive.

Stimulation of purified TCR-AND cells with immobilized anti-CD3/CD28 induced a rapid tyrosine phosphorylation of a number of cellular substrates, the most obvious ones having apparent molecular mass of 160, 130, 105, 85, 73, 58, 52, and 40 kDa, although the 40-kDa band was often undetectable (Fig. 8Go). Stimulation in the presence of anti-CD4 Ab resulted in a moderate reduction of tyrosine phosphorylation of a number of these proteins. Tyrosine phosphorylation of p52, p73, p85, p130, and p160 exhibited reduced phosphotyrosine content at all time points, and reduced phosphorylation of p58 and p105 was evident at later time points. Thus, the treatment with the anti-CD4 Ab appears to directly suppress T cell activation. Moreover, since the stimulation of the T cells in these studies did not involve class II-positive APC, this suppression was independent of any blockade of CD4-class II binding.



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  		FIGURE 8. Anti-CD4 Ab affects tyrosine phosphorylation of intracellular proteins in stimulated cells. AND-TCR transgenic CD4 T cells were stimulated with immobilized anti-CD3/CD28 in the presence of 100 µg/ml anti-CD4 or control Abs. Cultures were stopped at indicated time points. Tyrosine phosphorylation of intracellular proteins was detected in cell lysates by Western blot. Phosphorylation substrates specific to CD4 treatment are identified with an asterisk (*). This figure represents three independent experiments.

 
More interestingly, anti-CD4 treatment also resulted in the appearance of at least two tyrosine-phosphorylated proteins, p50 and p54, which were not present in control cells treated with rat Ig. Furthermore, the sustained phosphorylation of these two substrates was not altered by stimulation by anti-CD3/CD28, suggesting that the phosphorylation of these proteins was independent of TCR-mediated activation. At this point, it is not clear whether phosphorylation of these substrates is directly responsible for the altered responsiveness in the T cells, or whether this is only a functionally unlinked correlation. Studies have shown that Ag-specific tolerance induced in vivo is dependent on simultaneous exposure to both anti-CD4 and Ag (4). Thus, these phosphorylation substrates, if relevant to the anergic phenotype, may interact with other molecules dependent on TCR activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of effective Ag-specific immunotherapies is very much dependent on identifying regulatory mechanisms already active in normal immune responses. Although a number of reagents can block T lymphocyte activation or induce depletion of T lymphocytes (4, 5), they can at best only confirm the requirement for CD4 T cells in immune effector function; therapeutically, they only induce an undesirable generalized immunosuppression. The discovery that nondepleting anti-CD4 Ab can induce tolerance in a number of situations is an important advance in the development of more sophisticated immunomodulatory therapies. However, it remains to be determined how this Ab mediates its effects. Assuming that intrinsic immunoregulatory mechanisms are being adapted by the Ab treatment, which possible mechanisms are involved? Over the past several years, two candidate mechanisms have been identified that may provide natural peripheral immune tolerance among CD4 T cells: immune deviation/clonal diversion and clonal anergy.

Immune deviation/clonal diversion had been observed a long time ago in situations in which animals appeared to develop strong immune responses among T cells (delayed-type hypersensitivity) or among B cells (humoral immunity), but not both simultaneously (18). More recently, this has been attributed in part to the observation that CD4 effector cells could develop into Th1 cells (capable of mediating cellular immune responses such as delayed-type hypersensitivity, allograft rejection, and autoimmune disease), or Th2 cells (capable of driving B cell differentiation and isotype switching toward IgG1 and IgE). Because Th2 cytokines such as IL-4 and IL-10 also have some inhibitory or anti-inflammatory activities, Th2 cells have also been proposed as regulatory cells, especially in diseases such as autoimmune diabetes (19) and inflammatory bowel disease (20).

Clonal anergy was first described as a nonresponsiveness in CD4 T cells provided with a TCR signal in the absence of costimulation through CD28 (21). An anergic phenotype has now been described in an impressive variety of situations, including stimulation by ligand on activated T cells (22), chemically fixed APC (23), presentation on resting B cells (24), overstimulation by superantigen (25), closely repeated stimulation (26), and altered peptide ligands (27). In vivo, a few phenotypic markers appear to be associated with a naturally occurring population of anergic regulatory cells, including CD38 (28) and CD25 (29).

For both immune deviation and clonal anergy, Ag-specific T cells may regulate immune responses, but their mechanisms of action differ significantly. Regulatory cells with a Th2 phenotype act in part by conversion or recruitment of potentially pathogenic T cells into becoming nonpathogenic Th2 cells, and countering the inflammatory actions of Th1 cytokines with the suppressive effects of IL-4 and IL-10. By contrast, the action of anergic T cells is more dependent on their inability to act; thus, they may interfere with immune responses by nonproductively occupying a limiting number of APC (30, 31), or by interfering with local cytokine usage (28, 32) or production (29). These two distinct mechanisms are not necessarily mutually exclusive; however, it has been suggested that clonal anergy might only be inducible in Th1 cells and not in Th2 cells (33).

The studies presented in this work suggest that the anti-CD4 Ab YTS177 acts by inducing anergy in T cells triggered by both the anti-CD4 and the Ag receptor. However, as noted above, the variety of situations associated with T cell anergy implies that there may be several distinct anergic phenotypes. For example, while the present studies appear to rule out a specific shift toward Th2 responses, they do not rule out a shift toward a Tr1 phenotype described by Groux et al. (34). The production of high levels of IL-10 by such cells has been proposed to induce a functional nonresponsiveness by both T and B cells (35), which may be mistaken for clonal anergy. Another example of a distinct anergic phenotype is the CD4+ CD38+ CD45RBlow cell described by Read et al. (28). This Ag-specific cell is able to suppress proliferation by CD38- cells, but by an unknown mechanism. Similarly, Thornton and Shevach (29) have described a population of CD4+ CD25+ cells that also can suppress activation by CD25- cells, again by an unknown mechanism. In all of these cases, the generation of these suppressor phenotypes in vivo is not well understood, nor is it known whether anti-CD4 treatment can induce the development of these cells.

Previous studies have shown inhibitory effects of anti-CD4 related to its coreceptor function in the recognition of the class II ligand on APC, but in addition, it was clear that anti-CD4 could have direct negative regulatory effects as well during primary stimulation (36, 37, 38, 39). It should be noted that CD4 coreceptor function refers primarily to its ability to physically bind class II directly, enhancing the avidity of the TCR complex binding to class II on APC. Thus, the highest affinity TCRs can appear to be CD4 independent in their recognition of ligand. Coreceptor function can therefore be distinguished from the signaling function (positive or negative) of CD4, which is related instead to its interaction with the intracellular signaling kinase lck (40).

In our studies, we now provide new evidence that the direct negative effect of anti-CD4 may be related to the observed tyrosine phosphorylation of novel substrates. However, the in vivo studies by Waldmann and colleagues indicate that the induction of longstanding specific tolerance requires a simultaneous exposure to both the anti-CD4 and the target Ag (6). Because the phosphorylation of the new p50 and p54 substrates appears to be independent of TCR and CD28 triggering, it is not yet clear what specific intracellular signaling mechanisms are responsible for inducing the physiological changes in T cell function. In a recent report by Chirmule et al. (41), pretreatment of T cells with YTS177 inhibited CD3-mediated activation of NF-AT, AP-1, as well as Erk2, but here too it is not clear how this inhibition was mediated. Addition of costimulation (anti-CD28 Ab) appeared to overcome the effects of the YTS177 treatment, but this result contrasts with our own studies in which costimulation was always applied either as anti-CD28 or as normal APC in vivo.

Although the negative regulatory effects are clearly important in the induction of the persistent tolerance, our studies also suggest a complementary role for the blockade of the coreceptor function in vivo. This would be especially true in situations in which established skewed immune responses were already present before treatment. For example, when anti-CD4 was added after initial immunization and boosting, the therapeutic effects of the Ab seemed to be mainly dependent on its ability to block coreceptor function, reducing the intensity of the T cell response without altering the character of the response. This reduction could be critical in minimizing the recruitment of additional naive T cells in the perpetuation of allergic responses. With continued exposure to allergen in the presence of anti-CD4, dominant regulatory cells might eventually develop from the naive population, reversing the course of disease.

As reported in other applications of the anti-CD4 Ab in the induction of tolerance, the tolerant state is infectious in the sense that new thymic emigrants also acquire the tolerant phenotype, and adoptive transfer of tolerant cells can prevent immunity in host cells (6). Our studies provide direct evidence that the tolerant phenotype involves anergy rather than an immune deviation, with significant implications for the mechanism of infectious tolerance. Because the anergic cells produce little if any cytokine, including suppressive cytokine, their action is more likely to be through the physical interference of Ag presentation to naive cells (30, 31), and absorption of local cytokines necessary for the amplification of immune responses (32). However, further studies will be necessary to directly demonstrate this effect in vivo.

In sum, our results show that anti-CD4-induced negative regulatory effects can extend well beyond the initial stimulation to provide a persistent anergic state, a phenotype that provides an important explanation for the persistent tolerance observed in vivo. Moreover, while early studies on the effects of anti-CD4 were primarily on Th1-mediated responses, our studies show an equally effective application to treatment of Th2-mediated diseases such as allergic lung inflammation. Previous studies suggested that the anti-CD4-induced tolerance was infectious in the sense that the tolerant phenotype could be transferred to naive animals converting the recipient immune system to tolerance as well. Although the precise details of this conversion mechanism are not yet clear, the present studies now raise the exciting prospect of adapting these mechanisms to treatment of allergic diseases.


   Acknowledgments
 
We thank Drs. J. Davies and G. Trinchieri for their generous gifts of hybridomas and Christina Reilly for technical assistance. This is manuscript 12290-IMM from The Scripps Research Institute.


   Footnotes
 
1 This work was supported by Grants AI29689 and AI31583 to D.L. from the National Institutes of Health, and a grant from the Juvenile Diabetes Foundation International. Back

2 Address correspondence and reprint requests to Dr. David Lo, Department of Immunology IMM-25, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: Back

3 Abbreviations used in this paper: Mls, minor lymphocyte stimulating; BAL, bronchoalveolar lavage. Back

Received for publication May 18, 1999. Accepted for publication September 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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