Ability of a Nondepleting Anti-CD4 to Inhibit Th2 Responses and Allergic Lung Is Independent of Coreceptor Function This information is current as of September 25, 2021. Li Li, Mary Crowley, Andrea Nguyen and David Lo J Immunol 1999; 163:6557-6566; ; http://www.jimmunol.org/content/163/12/6557 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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

Nondepleting anti-CD4 Abs have been used in vivo to induce Ag-specific immunological tolerance in Th1 responses, including tissue allograft rejection and autoimmune . 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 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 Downloaded from absence of TCR triggering, suggesting that signaling through CD4 alone induces significant physiological changes in 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 . The Journal of Immunology, 1999, 163: 6557–6566.

D4 T cells are critical cells in directing effector function fused. The Ag-specific nature of this effect was clear, as it did not http://www.jimmunol.org/ in several kinds of immune responses, including the Ab interfere with subsequent priming to secondary Ags. It was sug- C response to viral infection, cellular immune responses to gested that the anti-CD4 treatment induced a dominant Ag-specific tissue allografts, delayed-type , and autoimmune tolerance, but the specific mechanism was not clearly defined. One diseases such as type 1 diabetes, thyroiditis, and . of the possible mechanisms is the induction of clonal anergy, as Therefore, therapeutic approaches in pathological syndromes are described for -reactive V␤6ϩ T cells in the bone mar- often aimed toward the modification or modulation of CD4 T cell row transplantation model (7). In this study, bone marrow trans- functions. For example, in the case of type 1 diabetes, this has been plantation with Mls3-1a-positive cells did not significantly deplete achieved through some general approaches such as the in vivo the Mls-reactive V␤6ϩ T cells, but proliferative responses to Mls

depletion of cells expressing ␣␤-TCR (1), CD3 (2), or CD4 (3). In were reduced. However, since not all V␤6 cells are superantigen by guest on September 25, 2021 similar fashion, anti-CD4 depletion has been shown to prevent reactive, alterations in the receptor repertoire in the chimeras might lung-allergic inflammation (4, 5). Unfortunately, these approaches also account for this effect. risk generalized immune suppression, predisposing the subjects to Another compelling possible explanation for the effects of the opportunistic infections. Recently, however, a more subtle ap- nondepleting anti-CD4 Ab comes from studies on the nonobese proach has been developed using Abs against CD4 that do not diabetic mouse model of spontaneous autoimmune diabetes. As deplete the T cell pool, but were still found to have significant with tissue allograft rejection, Th1 cells appear to be the main immunomodulatory effects. These effects were not due to gener- effector cells driving pathogenesis, although in this case they are alized immunosuppression, but depended on simultaneous expo- specific for islet ␤ cell Ags. Treatment of nonobese diabetic mice sure of CD4ϩ T cells to a specific Ag, providing for an Ag-specific with the anti-CD4 Ab was able to prevent diabetes in three differ- tolerance. ent situations, including 1) the spontaneous development of dis- In studies by the Waldmann group, an anti-CD4 Ab (YTS177.9) ease (8), 2) adoptive transfer into sublethally irradiated recipients was found to have striking effects in Ag-specific CD4 T cell re- (9), and 3) induction with high doses of cyclophosphamide (10). sponses, without causing a depletion of peripheral CD4 T cells This last observation is most relevant, as cyclophosphamide-in- (reviewed in Ref. 6). When mice were primed to specific Ag dur- duced diabetes has also been shown to correlate with increased ing anti-CD4 treatment, a longstanding Ag-specific tolerance was IFN-␥ production by T cells (11, 12), essentially a Th1 shift. More- induced that could not be broken even when naive cells were in- over, 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. Department of Immunology IMM-25, The Scripps Research Institute, La Jolla, Thus, the longstanding tolerance in anti-CD4-treated mice may be CA 92037 due to the development of Ag-specific suppressor effector CD4 T Received for publication May 18, 1999. Accepted for publication September cells, most likely with a Th2 phenotype. 27, 1999. In the studies on tissue allograft rejection and autoimmune di- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance abetes, the highly polyclonal nature of the responding T cell pop- with 18 U.S.C. Section 1734 solely to indicate this fact. ulation made it difficult to closely follow Ag-specific cells to de- 1 This work was supported by Grants AI29689 and AI31583 to D.L. from the National termine the effects of the anti-CD4 treatment. Thus, we have used Institutes of Health, and a grant from the Juvenile Diabetes Foundation International. 2 Address correspondence and reprint requests to Dr. David Lo, Department of Im- munology IMM-25, The Scripps Research Institute, 10550 North Torrey Pines Road, 3 Abbreviations used in this paper: Mls, minor stimulating; BAL, bron- La Jolla, CA 92037. E-mail address: [email protected] choalveolar lavage.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 6558 NONDEPLETING ANTI-CD4 AND ALLERGIC LUNG INFLAMMATION Downloaded from http://www.jimmunol.org/

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 peroxidase (EPO) activity for (ϫ200). by guest on September 25, 2021 TCR transgenic mice to provide more detailed in vitro information Research Institute), with normal rat Ig (Sigma, St. Louis, MO) as control on whether the Ab will have significant effects on Th2-mediated Ab. Human rIL-2 and mouse rIL-4 were obtained from Pepro Tech (Rocky immunity such as allergic asthma, and whether the primary effect 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, on Ag-specific T cells involves a Th1 to Th2 shift or clonal anergy. Philadelphia, PA). Anti-CD3, CD28, CD4, CD8␣, B220, and CD62L Abs Our results suggest that anti-CD4 treatment can have potent effects were all obtained from PharMingen (San Diego, CA), and mouse anti-rat Ј in blocking the development of allergic lung inflammation. Con- IgG polyclonal Ab (F(ab )2 fragment) was obtained from Jackson Immu- sistent with this effect, we find that in vitro the anti-CD4 inhibits noResearch (West Grove, PA). Abs and standards for ELISAs measuring and serum Ig were obtained from PharMingen. 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 sig- Induction of OVA-specific lung inflammation naling pathways. Thus, anti-CD4 therapy may have broader appli- Naive mice were immunized i.p. with 10 ␮g chicken egg OVA (Sigma) in cation than generally assumed, and may be an effective method for 100 ␮l of PBS mixed with the same volume of Imject Alum (Pierce, Rock- inducing anergy in the prevention of allergic diseases, including ford, IL); these mice were then boosted the same way in the following asthma. 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 Materials and Methods 30 ␮l 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 ven- Mice tricle using PBS until they had turned white, and bronchoalveolar lavage TCR-SFE mice (13, 14) are transgenic for a TCR specific for influenza PR8 (BAL) was collected by washing lung through the trachea three times using hemagglutinin peptide 110–119 (SFERFEIFPK) presented on I-Ed. The 1 ml of RPMI with 2% horse serum. Cytospins were prepared for BAL mice had been backcrossed to the BALB/c strain for more than 10 gener- cells from each mouse, and BAL fluids were frozen for detection. ations. AND mice (15) are transgenic for a TCR specific for moth cyto- The right lobes of the lung were fixed in Bouin’s for hematoxylin and eosin chrome c peptide presented on I-Eb. They were backcrossed more than 9 (H&E) staining. The left lungs were frozen in OCT compound (Miles, generations to the C.B10-H2b/LiMcdJ congenic mouse strain with the Elkhart, IN) for immunohistochemical staining. BALB/c background, and H-2b. Naive BALB/c mice (6–8 wk) were pro- vided by the Rodent Breeding Colony in the Scripps Research Institute (La Jolla, CA). All mice were maintained in the Scripps Research Institute Histology rodent colony under specific -free conditions, in accordance with BAL cells on cytospin slides were fixed with methanol and stained with National Institutes of Health and the Scripps Research Institute institutional eosin and methylene blue (Fisher, Pittsburgh, PA). Leukocytes were ana- guidelines. lyzed by differential count of total 200–300 cells on coded slides. Lung Abs and reagents was perfused, injected with OCT through the trachea, and frozen in OCT. Frozen lung sections were fixed with cold acetone with 1% formamide The nondepleting anti-CD4 Ab YTS177.1 (16Ͻzrefx.) was obtained as a (Fisher) and eosinophils were stained for cyanide-resistant eosinophil per- hybridoma cell line generously provided by Dr. J. Davies (The Scripps oxidase activity, as described (17). The Journal of Immunology 6559 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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 p Ͻ 0.02, compared with the control Ab-treated group. This figure represents two independent ,ء ;per slide. Each bar represents the mean Ϯ SD of four mice 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 Ϯ SD of four mice. C, Anti-CD4 Ab suppressed the up-regulation of serum Ig levels induced by OVA. Serum total IgE, IgG1, .p Ͻ 0.005. This figure represents two independent experiments ,ء ;and IgG2a levels were measured by ELISA. Each dot represents one mouse

In vitro assays. by stimulation in the presence of IL-12, and Th2 by stimulation in the ϫ 5 ϩ presence of IL-4 and neutralizing anti-IL-12. After 7 days, 2 10 of the On primary cells. TCR-SFE CD4 T cells were purified from lymph node ␮ ϩ ϩ Th1 or Th2 cells were restimulated with spleen APC plus 1 g/ml SFE cells with magnetic beads by depleting with Abs to CD8 and B220 cells. peptide in the presence of anti-CD4 or control Abs. Cytokine production ϫ 5 A total of 5 10 CD4 T cells were stimulated with plate-bound anti-CD3 and cell proliferation were tested 2 days after restimulation. and anti-CD28 (10 ␮g/ml anti-CD3 plus 1 ␮g/ml anti-CD28 were used to coat the plates, 37°C for 1 h) in 200 ␮l media with 100 ␮g/ml of either anti-CD4 or control Abs. In addition, 3 ϫ 105 CD4ϩ T cells were stimu- Effects of anti-CD4 Ab on signaling induced by stimulation 5 lated with 5 ϫ 10 irradiated BALB/c spleen APC plus 1 ␮g/ml SFE through TCR 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 Lymph node CD4 T cells were purified from AND transgenic mice. A total cytokine detection. IL-4, IL-5, and IFN-␥ were measured by ELISA. T cell of 2 ϫ 106 cells were stimulated with plate-bound anti-CD3 plus anti- proliferation was also tested on day 3 or day 5 by [3H]TdR incorporation. CD28 in the presence of 100 ␮g/ml anti-CD4 or control Abs. Cells were To test the persistence of anergy, stimulated cells were harvested on day 3 harvested at different time points, as indicated in the figure, and lysed with and cultured in media with 2 ng/ml IL-2 for another 3 days, then 5 ϫ 105 ice-cold lysis buffer consisting of 1% Triton X-100, 50 mM HEPES, 10% ϫ 5 of these cells were restimulated by anti-CD3 plus anti-CD28 or 3 10 T glycerol, 1.5 mM MgCl2, 100 mM NaF, 1 mM PMSF, and 1 mM NaVO4. cells stimulated with 5 ϫ 105 APCs plus SFE peptide in the absence of Soluble lysate proteins were separated on 9% SDS-PAGE and transferred Abs. Cytokine production and cell proliferation were tested 3 days after the to 0.22-␮m nitrocellulose membranes. The tyrosine-phosphorylated pro- restimulation. teins were detected by immunoblotting with anti-phosphotyrosine mAb On differentiated Th1 and Th2 cells. Th1 and Th2 cells were generated 4G10 (PharMingen), followed by HRP-conjugated sheep anti-mouse Ig from TCR-SFE cells, as described previously (17). Briefly, naive CD4 T (Amersham, Arlington Heights, IL). The bands were visualized with the cells (FACS sorted for CD62Lϩ cells) were induced to differentiate to Th1 ECL chemiluminescence system (Amersham). 6560 NONDEPLETING ANTI-CD4 AND ALLERGIC LUNG INFLAMMATION

tion/boost, intranasal challenge with OVA provoked only minimal lung inflammation, with few if any eosinophils detected in tissue or BAL (Fig. 2A). 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. 2B). Thus, as with Th1-medi- ated immune responses, anti-CD4 treatment was able to block the development of Th2-mediated allergic lung inflammation. Up-regulation of total serum IgE and IgG1 is another charac- teristic 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 in- creased levels of the Th1-associated IgG2a (Fig. 2C). 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 toler- ance induction is specific to the period of treatment, it was impor- tant to demonstrate that the effect of anti-CD4 treatment is tran- sient. Thus, to examine the kinetics of the anti-CD4 Ab on CD4 T Downloaded from 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. 3A). http://www.jimmunol.org/ 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 de- crease 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 to- 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 ward normal levels (Fig. 3B). Thus, the effect of the anti-CD4 days (days Ϫ1 and 0). On the next day, and for 36 days thereafter, PBL treatment may persist in vivo beyond the immediate effects during by guest on September 25, 2021 were analyzed for surface rat IgG on CD3ϩ T cells (A), and the cell surface Ag presentation. Alternatively, it is possible that extremely low levels of CD4 on the CD4ϩ T cell subset (B). On day ϩ1, cell surface rat levels of anti-CD4 Ab in vivo are sufficient to maintain dramatic ϩ IgG was detectable on a high percentage of CD3 T cells in the anti-CD4- modulation of surface CD4 levels. treated mice, decaying to near background levels by 36 days. In contrast, CD4 T cell recognition of Ag on APC is in part dependent on cell surface levels of CD4 (mean fluorescence intensity, or MFI) were the coreceptor function of the CD4 molecule, both in enhancing persistently modulated on CD4 T cells, with only a very slow recovery. TCR binding to the class II MHC molecule, and in the recruitment 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 of intracellular signaling kinases such as lck. Thus, one possible assays. The day 0 time point actually reflects baseline data taken on day effect of the anti-CD4 Ab in vivo is to simply block effective rec- Ϫ3. Similar results were also obtained in two other separate experiments. ognition 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 ab- Statistics sence of anti-CD4 Ab would result in normal allergic inflamma- tory responses. To test this, mice were given the standard protocol Two-tailed Student’s t test was used to analyze all data, except for Figs. 4B and 5B, with p Ͻ 0.05 considered significant. Figs. 4B and 5B were ana- of OVA immunization and boost in the presence of anti-CD4 or lyzed by one-tailed Student’s t test, and p Ͻ 0.05 was considered control Ab. Six weeks later, when the Ab appeared to have cleared significant. from the animals, a second set of OVA immunization and boost was given without any Ab treatment, followed by intranasal Ag Results challenge. A nondepleting anti-CD4 Ab induces long lasting protection In mice given only control Abs, intranasal challenge provoked from OVA-specific allergic lung inflammation the expected lung-allergic inflammation dominated by eosinophils. To test the effects of a nondepleting anti-CD4 on Th2-mediated In contrast, in mice treated with anti-CD4 during the first set of immune responses, we induced an OVA-specific allergic lung in- OVA immunizations, inflammation and eosinophilia were nearly flammation by immunizing and boosting mice i.p. with OVA/ absent (Fig. 4A). Moreover, increases in total serum IgE and IgG1 alum, followed by an intranasal OVA challenge. This protocol levels were also blocked, while IgG2a was not significantly af- induces extensive lung inflammation characterized by perivascular fected (Fig. 4B). Thus, in mice immunized in the presence of the and peribronchial infiltration with T cells, , eosino- anti-CD4 Ab, a persistent tolerance to the was established phils, and (Fig. 1). Consistent with the allergic/Th2 that could not be broken by later immunizations. This induced nature of the inflammation, eosinophils comprised as much as 40% tolerance appears to be active, and not through simple prevention of BAL recovered from these mice (Fig. 2A). When mice were of immunization. However, as noted above, a persistent modula- given anti-CD4 Abs 1 day before and after each OVA immuniza- tion of cell surface CD4 may also influence responses to the The Journal of Immunology 6561

FIGURE 4. Anti-CD4 Ab showed long lasting inhib- itory effects on allergic lung responses. OVA immuni- zation and Ab administration were done as indicated in Downloaded from the figure. A, Airway infiltration was analyzed by dif- ferential and total cell counts of BAL. Each bar repre- p Ͻ 0.05 compared ,ء ;sents the mean Ϯ SD of six mice 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-reg- ulation of serum total IgE and IgG1. Serum total IgE, http://www.jimmunol.org/ IgG1, and IgG2a levels were measured by ELISA. Each p Ͻ 0.02, compared with ,ء ;dot represents one mouse the control Ab-treated group. by guest on September 25, 2021

second priming. Although not formally demonstrated in this study, anti-CD4-treated mice, but the proportions of eosinophils among it is likely that this tolerance is OVA specific, as studies by the the BAL cells were not greatly reduced (Fig. 5A). Interestingly, Waldmann group have shown that mice given similar anti-CD4 serum IgE and IgG1 titers were lower among anti-CD4-treated treatments remain competent to respond to subsequent priming to mice, but IgG2a titers were similar in both control and treated second party Ags (6). groups (Fig. 5B). Thus, the late treatment with anti-CD4 in allergic lung inflammation was able to significantly reduce the overall in- Moderate inhibition of allergic inflammation by anti-CD4 tensity of the inflammatory response (both BAL counts and IgE/ treatment during intranasal Ag challenge IgG1 elevation), but the essential Th2 character of the response to In most clinical situations, treatment can only be initiated after OVA challenge was not changed. allergen immunization has already occurred. To determine the ef- To summarize the effects of anti-CD4 treatment on allergic lung fect of anti-CD4 Ab during the effector phase of allergic immune inflammation in vivo, we found that the treatment has dramatic responses, mice were immunized and boosted according to the inhibitory effects when given during initial immunization, and it standard protocol to establish allergic immune responses, but they induces a long lasting tolerance to the allergen even in the face of were subsequently given two additional boosts 1 wk apart in the additional priming. However, this persistent tolerance was not presence of anti-CD4 or control Ab. In this case, the total numbers associated with a shift toward Th1-associated responses such as of inflammatory cells in BAL were significantly reduced among increased IFN-␥ levels in BAL, nor increases in serum IgG2a 6562 NONDEPLETING ANTI-CD4 AND ALLERGIC LUNG INFLAMMATION

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-spe- cific cells, peripheral deletion, or development of a novel pheno- type. 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 dif- ferentiated effector Th1/Th2 cells separately. In the case of the TCR transgenic (TCR-SFE) specific for the I-Ed-restricted hem- agglutinin 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 Downloaded from 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 http://www.jimmunol.org/ 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 ex- amined the ability of Ag-specific naive T cells to respond to stim- ulation 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 by guest on September 25, 2021 were also treated with anti-CD4 Ab or control Ab, and T cell proliferation and cytokine production were measured (Fig. 6). 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 pro- liferation to peptide/APC as expected, it also dramatically inhib- ited the response to anti-CD3/CD28 (Fig. 6A), showing that the effect of the Ab could be independent of the class II-binding co- receptor function of CD4. As with proliferative responses, treat- FIGURE 5. Anti-CD4 Ab administered at the effector stage inhibited ment with anti-CD4 in vitro also inhibited the production of cy- the intensity of OVA-induced asthma inflammation. OVA-specific asthma tokines in primary stimulation. The inhibition affected all induction and Ab administration were done as indicated. A, Airway-infil- cytokines tested, including IL-2, the Th1 cytokine IFN-␥, and the trating cells were analyzed by differential and total cell counts of BAL. Th2 cytokines IL-4 and IL-5 (Fig. 6B). p Ͻ 0.02, compared with the control In vivo, anti-CD4 treatment was able to induce a tolerance that ,ء ;Each dot represents one mouse Ab-treated group. This figure combined three independent experiments. B, persisted over several weeks. To determine whether this effect Anti-CD4 Ab given at the effector stage inhibited OVA-induced up-regu- could also be demonstrated in vitro, we took CD4 T cells treated lation of serum total IgE and IgG1 levels. Serum total IgE, IgG1, and as above in vitro for 3 days, then washed and cultured for an IgG2a levels were measured by ELISA. Each dot represents one mouse; -p Ͻ 0.05, compared with the control Ab-treated group. This figure rep- additional 3 days in IL-2-containing media to insure viability. Af ,ء resents two independent experiments. ter 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 levels. Moreover, when treatment was given late in the response, stimulation of anti-CD4-treated cultures was also seen in the re- the intensity of the response could be reduced without altering the stimulated cells (Fig. 6C). The persistent nonresponsiveness was Th2 skewed pattern in the immune response. Thus, the anti-CD4 most evident in the cells restimulated with peptide/APC. Thus, at treatment clearly did not augment Th2 responses, as might be pre- least for the 6-day period studied, the anti-CD4 appeared to induce dicted from earlier studies on Th1-mediated disease, and indeed a persistent clonal anergy. It is not clear whether this effect would appeared to have no specific influence on the Th1/Th2 balance also apply in vivo for longer periods; documentation of this in vivo in vivo. will clearly require further studies on adoptively transferred cells. The Journal of Immunology 6563 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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 ␮g/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 Ϯ 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 ␮g/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 Ϯ SD of three wells. This figure represents three independent experiments.

Cytokine production from the restimulated cells showed a cu- inflammation. This suggested that established Th2 skewed effector rious alteration in the pattern of Th1 vs Th2 cytokines (Fig. 6D). responses are resistant to further modification by anti-CD4, al- Although control cells stimulated under these neutral conditions though recruitment or amplification of their responses could still (immobilized anti-CD3/CD28) consistently showed increased pro- be affected. To examine this in vitro, we generated differentiated duction of Th2 cytokines and reduced production of IFN-␥ (rela- Th1 and Th2 T cells using TCR transgenic cells and primary stim- tive to controls) on restimulation, the anti-CD4-treated cells ulation in vitro under skewing conditions. As reported previously, showed persistently reduced production of IL-4 and IL-5, but in- our protocols give rise to differentiated Th1 and Th2 effector cells creased production of IFN-␥. This appears to comprise a signifi- that are capable of mediating Ag-specific responses in vivo with cant shift toward the Th1 phenotype, but this is also in the context characteristic patterns of inflammation (17). In concordance with of greatly reduced T cell proliferation, so the production of IFN-␥ the in vivo results above, we found that differentiated Th1 and Th2 overall may still remain low with respect to the original input T cells were entirely resistant to the anti-CD4 Ab treatment (Fig. 7). cell numbers. Thus, proliferative responses were unaffected when both Th1 and As shown above, the treatment of OVA-immunized mice with Th2 cells were restimulated in the presence of anti-CD4. More im- anti-CD4 in the weeks after initial immunization and boosting ef- portantly, the patterns and amounts of Th1 and Th2 cytokines were fected only a moderate reduction in the intensity of the lung in- also unaffected, confirming the notion that anti-CD4 treatment cannot flammation without significantly altering the allergic nature of the alter the character of established skewed immune responses. 6564 NONDEPLETING ANTI-CD4 AND ALLERGIC LUNG INFLAMMATION

FIGURE 7. Anti-CD4 Ab fails to suppress the re- sponse of previously differentiated Th1 or Th2 cells. TCR-SFE transgenic Th1 and Th2 cells were differ- entiated as indicated in Materials and Methods. Cells were restimulated by spleen APC with SFE peptides in the presence of 100 ␮g/ml anti-CD4 or control Abs. Cell proliferation was measured by [3H]TdR incorporation. Cytokines from the super- natants were measured by ELISA. Each bar repre- sents the mean Ϯ SD of three wells. This figure rep- resents two independent experiments.

Anti-CD4 treatment of naive T cells induces tyrosine More interestingly, anti-CD4 treatment also resulted in the ap- Downloaded from phosphorylation of novel intracellular substrates pearance of at least two tyrosine-phosphorylated proteins, p50 and Stimulation of naive T cells in vivo and in vitro in the presence of p54, which were not present in control cells treated with rat Ig. anti-CD4 induced a long lasting tolerance that was not due to a Furthermore, the sustained phosphorylation of these two substrates simple blockade of T cell activation. This suggested that the was not altered by stimulation by anti-CD3/CD28, suggesting that treated T cells were actively altered by the Ab treatment, causing the phosphorylation of these proteins was independent of TCR- the persistent anergic phenotype. To determine whether this was mediated activation. At this point, it is not clear whether phos- http://www.jimmunol.org/ induced through novel signaling pathways, we examined tyrosine phorylation of these substrates is directly responsible for the al- phosphorylation patterns in naive CD4 T cells stimulated in the tered responsiveness in the T cells, or whether this is only a presence and absence of anti-CD4. In these studies, to insure that functionally unlinked correlation. Studies have shown that Ag- the population of CD4 T cells was naive, we used a TCR transgene specific tolerance induced in vivo is dependent on simultaneous (TCR-AND) specific for a moth cytochrome c peptide presented exposure to both anti-CD4 and Ag (4). Thus, these phosphoryla- on I-Eb. Because thymic selection and export of these T cells occur tion substrates, if relevant to the anergic phenotype, may interact in H-2b mice lacking I-E expression, the transgenic T cells can with other molecules dependent on TCR activation. develop in these animals lacking the restricting I-E molecule and by guest on September 25, 2021 the target ligand, and remain truly naive. Discussion Stimulation of purified TCR-AND cells with immobilized anti- The development of effective Ag-specific immunotherapies is very CD3/CD28 induced a rapid tyrosine phosphorylation of a number much dependent on identifying regulatory mechanisms already ac- of cellular substrates, the most obvious ones having apparent mo- tive in normal immune responses. Although a number of reagents lecular mass of 160, 130, 105, 85, 73, 58, 52, and 40 kDa, although can block T lymphocyte activation or induce depletion of T lym- the 40-kDa band was often undetectable (Fig. 8). Stimulation in phocytes (4, 5), they can at best only confirm the requirement for the presence of anti-CD4 Ab resulted in a moderate reduction of CD4 T cells in immune effector function; therapeutically, they tyrosine phosphorylation of a number of these proteins. Tyrosine only induce an undesirable generalized immunosuppression. The phosphorylation of p52, p73, p85, p130, and p160 exhibited re- discovery that nondepleting anti-CD4 Ab can induce tolerance in duced phosphotyrosine content at all time points, and reduced a number of situations is an important advance in the development phosphorylation of p58 and p105 was evident at later time points. of more sophisticated immunomodulatory therapies. However, it Thus, the treatment with the anti-CD4 Ab appears to directly sup- remains to be determined how this Ab mediates its effects. As- press T cell activation. Moreover, since the stimulation of the T suming that intrinsic immunoregulatory mechanisms are being cells in these studies did not involve class II-positive APC, this adapted by the Ab treatment, which possible mechanisms are in- suppression was independent of any blockade of CD4-class II volved? Over the past several years, two candidate mechanisms binding. have been identified that may provide natural peripheral immune

FIGURE 8. Anti-CD4 Ab affects tyrosine phos- phorylation of intracellular proteins in stimulated cells. AND-TCR transgenic CD4 T cells were stim- ulated with immobilized anti-CD3/CD28 in the pres- ence of 100 ␮g/ml anti-CD4 or control Abs. Cul- tures were stopped at indicated time points. Tyrosine phosphorylation of intracellular proteins was de- tected in cell lysates by Western blot. Phosphoryla- tion substrates specific to CD4 treatment are identi- This figure represents three .(ء) fied with an asterisk independent experiments. The Journal of Immunology 6565 tolerance among CD4 T cells: immune deviation/clonal diversion ulation (36–39). It should be noted that CD4 coreceptor function and clonal anergy. refers primarily to its ability to physically bind class II directly, Immune deviation/clonal diversion had been observed a long enhancing the avidity of the TCR complex binding to class II on time ago in situations in which animals appeared to develop strong APC. Thus, the highest affinity TCRs can appear to be CD4 inde- immune responses among T cells (delayed-type hypersensitivity) pendent in their recognition of ligand. Coreceptor function can or among B cells (), but not both simultaneously therefore be distinguished from the signaling function (positive or (18). More recently, this has been attributed in part to the obser- negative) of CD4, which is related instead to its interaction with vation that CD4 effector cells could develop into Th1 cells (capa- the intracellular signaling kinase lck (40). ble of mediating cellular immune responses such as delayed-type In our studies, we now provide new evidence that the direct hypersensitivity, allograft rejection, and ), or negative effect of anti-CD4 may be related to the observed tyrosine Th2 cells (capable of driving differentiation and isotype phosphorylation of novel substrates. However, the in vivo studies switching toward IgG1 and IgE). Because Th2 cytokines such as by Waldmann and colleagues indicate that the induction of long- IL-4 and IL-10 also have some inhibitory or anti-inflammatory standing specific tolerance requires a simultaneous exposure to activities, Th2 cells have also been proposed as regulatory cells, both the anti-CD4 and the target Ag (6). Because the phosphory- especially in diseases such as autoimmune diabetes (19) and in- lation of the new p50 and p54 substrates appears to be independent flammatory bowel disease (20). of TCR and CD28 triggering, it is not yet clear what specific in- Clonal anergy was first described as a nonresponsiveness in tracellular signaling mechanisms are responsible for inducing the CD4 T cells provided with a TCR signal in the absence of co- physiological changes in T cell function. In a recent report by stimulation through CD28 (21). An anergic phenotype has now Chirmule et al. (41), pretreatment of T cells with YTS177 inhibited Downloaded from been described in an impressive variety of situations, including CD3-mediated activation of NF-AT, AP-1, as well as Erk2, but stimulation by ligand on activated T cells (22), chemically fixed here too it is not clear how this inhibition was mediated. Addition APC (23), presentation on resting B cells (24), overstimulation by of costimulation (anti-CD28 Ab) appeared to overcome the effects superantigen (25), closely repeated stimulation (26), and altered of the YTS177 treatment, but this result contrasts with our own peptide ligands (27). In vivo, a few phenotypic markers appear to studies in which costimulation was always applied either as anti-

be associated with a naturally occurring population of anergic reg- CD28 or as normal APC in vivo. http://www.jimmunol.org/ ulatory cells, including CD38 (28) and CD25 (29). Although the negative regulatory effects are clearly important in For both immune deviation and clonal anergy, Ag-specific T the induction of the persistent tolerance, our studies also suggest a cells may regulate immune responses, but their mechanisms of complementary role for the blockade of the coreceptor function in action differ significantly. Regulatory cells with a Th2 phenotype vivo. This would be especially true in situations in which estab- act in part by conversion or recruitment of potentially pathogenic lished skewed immune responses were already present before T cells into becoming nonpathogenic Th2 cells, and countering the treatment. For example, when anti-CD4 was added after initial inflammatory actions of Th1 cytokines with the suppressive effects immunization and boosting, the therapeutic effects of the Ab of IL-4 and IL-10. By contrast, the action of anergic T cells is more seemed to be mainly dependent on its ability to block coreceptor dependent on their inability to act; thus, they may interfere with function, reducing the intensity of the T cell response without al- by guest on September 25, 2021 immune responses by nonproductively occupying a limiting num- tering the character of the response. This reduction could be crit- ber of APC (30, 31), or by interfering with local cytokine usage ical in minimizing the recruitment of additional naive T cells in the (28, 32) or production (29). These two distinct mechanisms are not perpetuation of allergic responses. With continued exposure to al- necessarily mutually exclusive; however, it has been suggested lergen in the presence of anti-CD4, dominant regulatory cells that clonal anergy might only be inducible in Th1 cells and not in might eventually develop from the naive population, reversing the Th2 cells (33). course of disease. The studies presented in this work suggest that the anti-CD4 Ab As reported in other applications of the anti-CD4 Ab in the YTS177 acts by inducing anergy in T cells triggered by both the induction of tolerance, the tolerant state is infectious in the sense anti-CD4 and the Ag receptor. However, as noted above, the va- that new thymic emigrants also acquire the tolerant phenotype, and riety of situations associated with T cell anergy implies that there adoptive transfer of tolerant cells can prevent immunity in host may be several distinct anergic phenotypes. For example, while the cells (6). Our studies provide direct evidence that the tolerant phe- present studies appear to rule out a specific shift toward Th2 re- notype involves anergy rather than an immune deviation, with sig- sponses, they do not rule out a shift toward a Tr1 phenotype de- nificant implications for the mechanism of infectious tolerance. scribed by Groux et al. (34). The production of high levels of IL-10 Because the anergic cells produce little if any cytokine, including by such cells has been proposed to induce a functional nonrespon- suppressive cytokine, their action is more likely to be through the siveness by both T and B cells (35), which may be mistaken for physical interference of Ag presentation to naive cells (30, 31), and clonal anergy. Another example of a distinct anergic phenotype is absorption of local cytokines necessary for the amplification of the CD4ϩ CD38ϩ CD45RBlow cell described by Read et al. (28). immune responses (32). However, further studies will be necessary This Ag-specific cell is able to suppress proliferation by CD38Ϫ to directly demonstrate this effect in vivo. cells, but by an unknown mechanism. Similarly, Thornton and In sum, our results show that anti-CD4-induced negative regu- Shevach (29) have described a population of CD4ϩ CD25ϩ cells latory effects can extend well beyond the initial stimulation to pro- that also can suppress activation by CD25Ϫ cells, again by an vide a persistent anergic state, a phenotype that provides an im- unknown mechanism. In all of these cases, the generation of these portant explanation for the persistent tolerance observed in vivo. suppressor phenotypes in vivo is not well understood, nor is it Moreover, while early studies on the effects of anti-CD4 were known whether anti-CD4 treatment can induce the development of primarily on Th1-mediated responses, our studies show an equally these cells. effective application to treatment of Th2-mediated diseases such as Previous studies have shown inhibitory effects of anti-CD4 re- allergic lung inflammation. Previous studies suggested that the anti- lated to its coreceptor function in the recognition of the class II CD4-induced tolerance was infectious in the sense that the tolerant ligand on APC, but in addition, it was clear that anti-CD4 could phenotype could be transferred to naive animals converting the have direct negative regulatory effects as well during primary stim- recipient to tolerance as well. 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