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The Journal of Immunology, 2007, 178: 6148-6157.
Copyright © 2007 by The American Association of Immunologists, Inc.
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CD8 Blockade Promotes Antigen Responsiveness to Nontolerizing Antigen in Tolerant Mice by Inhibiting Apoptosis of CD4+ T Cells1

Zhuangzhi Wang and Joanna D. Davies2

Torrey Pines Institute for Molecular Studies, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Using the DO11.10 CD4+ TCR-transgenic mouse system, we have recently shown that CD8 blockade promotes the expansion of Ag-specific regulatory CD4+ T cells in mice made tolerant to OVA with anti-CD4 mAb. We now show that CD8 blockade is also critical to promoting responses to nontolerizing Ag in anti-CD4 mAb-treated tolerant mice. Previously published work shows that treatment with anti-CD4 mAb without CD8 blockade induces Ag-specific tolerance. We now show that, in addition to inducing tolerance, anti-CD4 mAb treatment also significantly reduces responsiveness to irrelevant, nontolerizing Ag, and this unresponsiveness is associated with significant apoptosis of the CD4+ T cells. Anti-CD4 mAb-induced apoptosis is inhibited by cotreatment with anti-CD8 mAb and responsiveness to irrelevant Ag is restored, while Ag-specific tolerance is maintained. These data suggest that CD8 blockade promotes responsiveness to nontolerizing Ags in tolerant mice by inhibiting CD4+ T cell apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Costimulation and accessory molecule blockade with mAbs specific for CD40L (1) or CD28 (2) or CD4 (3, 4) results in Ag-specific transplantation tolerance. Long-term survival is improved in all of these systems by including a cotreatment that also targets CD8+ cells (5, 6, 7, 8, 9). It has been suggested that effector CD8+ T cell function is less affected by CD40-CD40L, CD28-B7, and CD4 blockade than CD4+ T cell function (5, 6, 7, 10, 11); therefore, an additional CD8-directed approach is necessary to control CD8 effector function. However, we have recently shown that blockade of CD8 promotes CD4+ T cell-mediated immune regulation by promoting proliferation of Ag-specific CD4+ regulatory T cells (12), suggesting an additional role of CD8 blockade in the development of long-term tolerance. The model used in this study involves the well-characterized nondepleting CD4-specific mAb, YTS 177, and the CD8-specific mAb, YTS 105. These mAbs induce Ag-specific tolerance to skin (4, 13), bone marrow (3), and cardiac allografts (14), and OVA (12). Transplantation tolerance is associated with the development of FOXP3+CD4+ regulatory cells (15) that are capable of preventing (suppressing) nontolerant cells from rejecting the allograft (10, 16). Using the OVA-specific CD4+ TCR-transgenic system, DO11.10 (17), we have recently shown that treatment with the anti-CD4/anti-CD8 mAb mixture promotes an increase in the number of CD4+ TCR-transgenic cells compared with treatment with OVA and the anti-CD4 mAb without the anti-CD8 mAb and that these cells show regulatory activity on transfer into new recipients (12). Moreover, in the absence of anti-CD8 mAb, Ag-specific CD4+ T cells in anti-CD4 mAb-treated mice fail to proliferate in response to Ag in vivo but proliferation is significantly increased in mice cotreated with the anti-CD8 mAb (12). Using the same model system, the current study was set up to determine whether CD8 blockade also promotes the proliferation of non-TCR-transgenic CD4+ T cells in tolerant mice or whether the effect is restricted to Ag-specific regulatory cells.

Consistent with the effect of the anti-CD4 and anti-CD8 mAbs on the OVA-specific CD4+ TCR-transgenic cells, our data show that there are significantly fewer nontransgenic CD4+ T cells in the spleens and lymph nodes of mice with OVA and anti-CD4 mAb plus isotype control than in mice treated with OVA and the anti-CD4/anti-CD8 mAb mixture. However, treatment with OVA and anti-CD4 mAb alone induces apoptosis of nontransgenic CD4+ T cells, and this is associated with unresponsiveness to both tolerizing and irrelevant (nontolerizing) Ag. Although this reduced responsiveness might suggest that the anti-CD4 mAb induces nonspecific immunosuppression in this model, we find that treatment with OVA and anti-CD4 mAb promotes a significant increase in Ag-specific CD4+FOXP3+ regulatory cells (18, 19, 20). Cotreatment with anti-CD8 mAb, in contrast, inhibits CD4+ T cell apoptosis and restores responsiveness to irrelevant Ag, but not to tolerizing Ag. The differences seen between the effects of the anti-CD4 and anti-CD8 mAbs on TCR-transgenic and nontransgenic CD4+ T cells is discussed.


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

Female BALB/cJ (BALB/c) and BALB/c DO11.10 TCR-transgenic (DO11.10) mice were purchased from The Jackson Laboratory and used at 8–12 wk of age. All mice were housed in specific pathogen-free conditions. All protocols used in this study were conducted according to institutional guidelines and approved by the Institutional Animal Care and Use Committee.

Transgenic mouse model system

In this study, we have analyzed the effect of OVA plus the anti-CD4/anti-CD8 mAb mixture on nontransgenic CD4+ T cells. Previously, we have published the effect of the same treatment on transgenic CD4+ T cells from the DO11.10 mouse strain. To directly compare the effect of this treatment on nontransgenic and transgenic CD4+ T cells, we have chosen to study the nontransgenic CD4+ T cells using the transgenic mouse model system previously used to study transgenic CD4+ T cells as follows: the BALB/c DO11.10 mouse strain is transgenic for a TCR that recognizes peptide 323–333 derived from OVA (17). The majority of CD4+ T cells in the resulting BALB/c DO11.10 TCR-transgenic mouse carry the transgenic TCR. To study transgenic T cells within a more physiological environment with respect to the presence of nontransgenic T cells, we have adopted the Jenkins method of transferring small numbers of splenocytes containing a defined number of transgenic CD4+ T cells from DO11.10 mice to BALB/c recipients (21, 22). Tolerance is induced in these mice by priming with Ag, OVA, in the presence of the tolerizing anti-CD4/anti-CD8 mAb mixture. The effect of Ag and mAb treatment on nontransgenic CD4+ T cells is analyzed by FACS.

Antibodies

In vivo treatment. The CD4-specific hybridoma, YTS 177.9.6.1 (4), and the CD8-specific hybridoma, YTS 105.18.10 (4), were a gift from H. Waldmann (Oxford University, Oxford, U.K.). The rat IgG2a isotype control HB-189 hybridoma was purchased from the American Type Culture Collection. A total of 50 mg of each mAb per kilogram of body weight was injected into mice on the same day as Ag treatment by i.p. injection. Additional doses were administered on days 2, 4, and 7 post-Ag treatment.

In vitro. Biotinylated KJ1–26 mAb was purchased from Caltag Laboratories. FITC-labeled FOXP3-specific mAb was purchased from eBioscience. Allophycocyanin-labeled CD4-specific mAb, PE-labeled streptavidin, and isotype control mAbs were purchased from BD Biosciences/BD Pharmingen. All reagents were used according to the manufacturers’ instructions.

Ag preparation

The Ags, OVA (Sigma-Aldrich) and beta-galactosidase (beta-gal)3 (Sigma- Aldrich), were aggregated using a modification of a method described by Weigle (23). Briefly, either OVA or beta-gal were heated for 25 min at 63°C at a concentration of 10 mg/ml. After heating, the Ag was placed on ice overnight and stored at –20°C.

The transgenic mouse model

Splenocytes were isolated from DO11.10 mice and an aliquot of 2 x 105 cells were incubated with allophycocyanin-labeled anti-CD4 and either biotinylated KJ1–26 mAb (mouse IgG2a specific for the transgenic TCR) or mouse IgG2a isotype control, followed by PE-labeled streptavidin. After washing, the percent of transgenic CD4+KJ1–26+ cells was determined by FACS. Using this information, BALB/c mice were injected with DO11.10 splenocytes containing 2.5 x 106 transgenic CD4+KJ1–26+ cells. For the remainder of this manuscript, DO11.10 cells will be termed CD4+KJ1–26+ cells.

Ag priming and boosting

On the same day as mice were injected with CD4+ KJ1–26+ cells, the mice were injected i.v. with either 25 mg/kg aggregated OVA or with 2.5 mg/kg aggregated beta-gal. Mice were boosted at 6 wk postpriming with the same dose of Ag as used for priming but boosting Ag was administered i.p. This protocol has been shown previously to induce immunity and not tolerance (24, 25).

OVA- and beta-gal-specific IgG measurements

Mice were bled from the tail vein at the times indicated for each experiment. Serum was isolated and stored at –20°C. Sera were diluted at 1/100 in blocking buffer, and serum OVA and beta-gal-specific Abs were measured by ELISA. Briefly, plates were coated with either 20 µg/ml OVA or 20 µg/ml beta-gal for 2 h at 37°C. After blocking, sera were added, and the plates were incubated overnight at 4°C. After washing, biotinylated goat anti-mouse IgG (Sigma-Aldrich) was added followed by extravidin peroxidase (Sigma-Aldrich). O-phenylenediamine dihydrochloride/urea hydrogen peroxidase detection reagents (Sigma-Aldrich) were measured at 490 nm. Standard control anti-OVA and anti-beta-gal Ab was made by priming and boosting mice at 6 and 10 wk postpriming, as described above. Mice were bled and serum isolated 10 days after the last boost. For the purpose of these experiments, standard serum at 1/500 dilution contained 500 U/ml anti-OVA or anti-beta-gal Ab.

T cell percentage, number, proliferation, and apoptosis determination

Splenocytes and lymph nodes (pooled cervical, axillary, brachial, mesenteric, inguinal) were isolated; the viable cells were counted, using trypan blue and a hemocytometer, and incubated with CD4-specific mAb, and, in some experiments, CD8-specific mAb. The percentage and total number of CD4+ and CD8+ cells in spleens and lymph nodes were determined by FACS. Proliferation and apoptosis of CD4+ cells were determined using a FITC BrdU Flow kit for proliferation (BD Biosciences/BD Pharmingen), and Annexin V FITC Apoptosis Detection kit for apoptosis (BD Biosciences/BD Pharmingen) after cell surface staining with CD4-specific mAb. Isotype controls were included for cell surface and intracellular stains. A total of 2–5000 CD4+ cells were acquired using a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest software version 3.2 (BD Biosciences). Samples were gated on the CD4+ cells to determine the expression of the second marker.

Statistical analysis

The statistical significance of data shown was assessed using the Mann-Whitney U test (26) and the Student t test (27) as indicated for each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The anti-CD4/anti-CD8 mAb mixture induces tolerance to OVA

BALB/c mice were infused with 2.5 x 106 CD4+ KJ1–26+ cells and on the same day were treated with either OVA alone, OVA with the anti-CD4/anti-CD8 mAb mixture, or with the anti-CD4/anti-CD8 mAb mixture alone. After 6 wk, all mice were boosted with OVA, and after an additional 10 days, the mice were bled and OVA-specific Ab was measured in the serum. OVA induces a strong anti-OVA Ab response in mice pretreated with OVA alone and this response was significantly inhibited in mice treated with OVA in combination with the anti-CD4/anti-CD8 mAb mixture, but not in mice pretreated with the anti-CD4/anti-CD8 mAb mixture alone (Fig. 1). The substantial response in mice treated with the anti-CD4/anti-CD8 mAb mixture without OVA indicates the requirement for OVA in inducing unresponsiveness. In addition, these data also indicate that the lack of response in mice treated with the anti-CD4/anti-CD8 mAb mixture with OVA was not due to a residual effect of the mAb mixture 6 wk after treatment. Because unresponsiveness to OVA required a previous encounter with that Ag, we conclude that the anti-CD4/anti-CD8 mAb mixture induces tolerance to OVA.


Figure 1
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  		FIGURE 1. The anti-CD4/anti-CD8 mAb mixture induces tolerance to OVA. BALB/c mice were infused with 2.5 x 106 CD4+ KJ1–26+ cells isolated from BALB/c DO11.10 mice and either OVA and the anti-CD4/anti-CD8 mAb mixture (group 1), or with OVA alone (group 2), or with the anti-CD4/anti-CD8 mAb mixture alone (group 3). Six weeks later, all mice were boosted with OVA and again 10 days later. After a further 10 days, OVA-specific Ab was measured in the serum by ELISA. Data shown are mean ± SEM and are representative of three experiments. **, Statistical significance to 0.009–0.001. The Mann-Whitney U test was used to determine statistical significance; n = 6/group.

 
Treatment with anti-CD4 mAb alone induces Ag unresponsiveness

BALB/c mice were injected with CD4+KJ1–26+ cells and various combinations of OVA, the anti-CD4/anti-CD8 mAb mixture, and the anti-CD4/anti-CD8 mAb mixture as indicated, and boosted at 6 wk in the usual way. Anti-OVA Ab was measured. Mice treated with ether OVA plus anti-CD4 mAb, or anti-CD4 mAb alone, were equally unresponsive to a later challenge with OVA (Fig. 2), indicating that treatment with OVA was not necessary to inhibit the response to OVA in mice treated with anti-CD4 mAb.


Figure 2
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  		FIGURE 2. The anti-CD4/anti-CD8 mAb mixture induces tolerance to OVA whereas the anti-CD4 mAb alone induces immunosuppression. BALB/c mice were injected with 2.5 x 106 CD4+ KJ1–26+ cells isolated from DO11.10 mice. On the same day, the mice were primed with and without OVA and treated with either the anti-CD4/anti-CD8 mAb mixture, or with anti-CD4 mAb plus isotype control, or without mAb. All mice were boosted with OVA on 6 wk postpriming and anti-OVA Ab was measured 7, 10, and 14 days later. Day 10 measurements are shown because the trend is the same for all days. Data shown are the mean ± SD and represent two experiments. **, Statistical significance to 0.009–0.001. ***, Statistical significance to 0.0009–0.0001. The Student t test was used to determine statistical significance; n = 3/group.

 
Unresponsiveness induced with OVA and anti-CD4 mAb is not Ag specific

The finding that mice treated with anti-CD4 mAb alone are unresponsive to a later challenge with OVA (Fig. 2) would suggest that unresponsiveness induced in mice with anti-CD4 mAb in combination with OVA is not Ag specific, but rather due to nonspecific unresponsiveness. To test this, mice were treated with either anti-CD4 mAb plus OVA or with anti-CD4 mAb plus beta-gal. Additional mice were left untreated. After 6 wk, half of the mice in each group were boosted with OVA while the remaining half were boosted with beta-gal. Control groups were primed and boosted with either OVA or beta-gal. Ten days after boosting, mice were bled and OVA- and beta-gal-specific Abs were measured. The only mice that made a strong OVA-specific Ab response were those that were either primed and boosted with OVA, or that were given no treatment on day 0, and then OVA at the time of boosting (Fig. 3a). Mice that were primed with beta-gal and anti-CD4 and then boosted with OVA did not make a strong anti-OVA response suggesting that treatment with anti-CD4 mAb induced nonspecific suppression rather than Ag-specific unresponsiveness. Similarly, mice treated with OVA and anti-CD4 mAb and then boosted with beta-gal did not make a strong beta-gal response (Fig. 3b), confirming that anti-CD4 mAb induces nonspecific unresponsiveness.


Figure 3
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  		FIGURE 3. Unresponsiveness induced with OVA and anti-CD4 mAb is not Ag specific. BALB/c mice were infused with 2.5 x 106 CD4+ KJ1–26+ cells isolated from BALB/c DO11.10 mice, and either OVA, or beta-gal, or no Ag, and either the anti-CD4 mAb or with no mAb, as indicated in a and b. On days 46 and 56 post-Ag treatment, mice were boosted with either OVA or beta-gal as indicated in a and b. Ten days after boosting OVA- (a) and beta-gal- (b) specific Abs were measured. Data shown are mean ± SD and represent two experiments; n = 3/group.

 
Treatment with OVA and anti-CD4 mAb induces OVA-specific CD4+FOXP3+ regulatory T cells in vivo

Since the anti-CD4 mAb used in these studies, YTS 177, is known to induce Ag-specific tolerance (3, 4), the finding that treatment with OVA and anti-CD4 mAb induced nonspecific Ag unresponsiveness was unexpected. It remained a possibility that the anti-CD4 mAb treatment induced both OVA-specific tolerance, and nonspecific unresponsiveness, in the same mice. To address this issue, we determined whether treatment with OVA and anti-CD4 mAb, in the absence of anti-CD8 mAb, induced Ag-specific regulatory T cells. BALB/c mice infused with DO11.10 splenocytes were treated with either OVA and the anti-CD4 mAb/isotype control mixture, or OVA and the anti-CD4/anti-CD8 mAb mixture, or were left untreated. After 6 days, splenocytes were isolated, and the percentage of CD4+ KJ1–26+ cells that expressed FOXP3 was determined by FACS. Treatment with OVA and anti-CD4 mAb plus isotype control induced a significant increase in the percentage (Fig. 4) and total number (data not shown) of CD4+ KJ1–26+ FOXP3+ cells compared with untreated control mice. In addition, the percentage and total number of CD4+ KJ1–26+ FOXP3+ cells were not different in mice treated with OVA and anti-CD4 mAb, with or without anti-CD8 mAb, suggesting that anti-CD8 mAb was not necessary to induce Ag-specific regulatory T cells in this model (Fig. 4).


Figure 4
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  		FIGURE 4. Anti-CD4 treatment promotes Ag-specific regulatory cells in the absence of anti-CD8 blockade. BALB/c mice were infused with 2.5 x 106 CD4+ KJ1–26+ cells isolated from BALB/c DO11.10 mice. On the same day, mice were immunized with OVA and either the YTS 177/YTS 105 mixture (b) or the YTS 177/isotype control mixture (c). An additional group of mice were left untreated (d). Six days later, mice were sacrificed and spleens were removed, labeled with CD4-specific mAb and KJ1–26 (a), and either FOXP3 or the isotype control for FOXP3. a, A representative dot plot of splenocytes labeled with CD4-specific mAb and KJ1–26. Samples shown in b–d are gated on total CD4+ KJ1–26+ cells (rectangle in a) and show the expression of FOXP3 on CD4+ KJ1–26+. The marker in b–d is based on the isotype control for each cell population. Data shown on each of b–d is the mean ± SEM of the percentage of CD4+ KJ1–26+ cells that are FOXP3+ (n = 3/group). The Student t test was used to determine statistical significance. The percentage of CD4+ KJ1–26+ cells that are FOXP3+ is significantly greater in mice treated with OVA/177/105 (p = 0.03) and OVA/177/iso (p = 0.01) compared with no treatment.

 
Treatment with anti-CD4 mAb causes a significant loss of CD4+ T cells in the spleens and lymph nodes whereas treatment with the anti-CD4/anti-CD8 mAb mixture does not

Mice treated with OVA and various combinations of anti-CD4, anti-CD8, or no mAb were sacrificed 3 days after boosting, and the percentage and number of CD4+ and CD8+ T cells in the spleens and lymph nodes were determined by FACS. The percentage (Fig. 5a) and total number (Fig. 5b) of CD4+ T cells in the spleen and lymph nodes are significantly less in mice treated with OVA and anti-CD4 mAb with isotype control compared with all other groups (Fig. 5a). The data suggest that anti-CD4 mAb promotes a loss of CD4+ T cells in both the spleen and lymph nodes compared with the group treated with OVA only, whereas the addition of anti-CD8 mAb in the treatment protocol inhibits that loss in the spleen and lymph nodes. The loss of CD4+ T cells in mice treated with anti-CD4 mAb in the absence of anti-CD8 mAb is specific to CD4+ T cells because CD8+ T cells are not lost in this group compared with untreated control mice (Fig. 5c). In contrast, mice treated with the anti-CD4/anti-CD8 mAb mixture have significantly fewer CD8+ T cells in the spleen and lymph nodes compared with mice in all other groups, including the group treated with anti-CD4 mAb/isotype control (Fig. 5c) suggesting that anti-CD8 mAb causes CD8+ T cell depletion. It should be noted that CD4+ and CD8+ T cell depletion caused by anti-CD4 and anti-CD8 mAbs used here is far from complete (~25% depletion in spleen and 50% in lymph nodes with anti-CD4 mAb and ~50% depletion in spleen with anti-CD8 mAb), and this is compatible with published data showing that these mAbs are "nondepleting" (4, 28, 29).


Figure 5
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  		FIGURE 5. Treatment with anti-CD4 mAb causes a significant loss of CD4+ T cells in the spleens and lymph nodes whereas treatment with the anti-CD4/anti-CD8 mAb mixture does not. BALB/c mice were treated with either OVA plus the anti-CD4/anti-CD8 mAb mixture, or OVA and anti-CD4 mAb plus isotype control, or with OVA alone, or were left untreated. Six weeks after OVA and mAb treatment, all mice were boosted with OVA and 3 days later the percentage (a) and total number (b) of CD4+ cells, and total number of CD8+ T cells (c) in the spleen and lymph nodes, was determined by FACS. Data shown are mean ± SD and are representative of four experiments. *, Statistical significance to 0.05–0.01. **, Statistical significance to 0.009–0.001. ***, Statistical significance to 0.0009–0.0001. The Student t test was used to determine statistical significance; n = 3/group.

 
Loss of CD4+ T cells by treatment with anti-CD4 mAb is not due to modulation or coating of cell surface CD4

Although we have shown that the levels of anti-CD4 and anti-CD8 mAbs at 6 wk posttreatment are insufficient to induce tolerance to OVA (Fig. 2), it was important to confirm that the loss of CD4+ T cells in spleens and lymph nodes of mice treated with anti-CD4 mAb was not due to either coating or modulation of the cell surface CD4. When measured 3 days after boosting, the percentage of CD3+ cells was equal to the sum of CD4+ cells plus CD8+ cells in all mice treated with either OVA and the anti-CD4/anti-CD8 mAb mixture, or OVA and anti-CD4 mAb plus isotype control, or OVA alone, indicating that the decrease in CD4+ and CD8+ cells in the spleens and lymph nodes of groups treated with anti-CD4 mAb/isotype control and the anti-CD4/anti-CD8 mAb mixture, respectively, reflects a decrease in the number of CD4+ and CD8+ T cells rather than modulation or coating of the cell surface proteins (Table I).


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  		Table I. Loss of CD4+ T cells is not due to modulation or coating of cell surface CD4

 
The loss of CD4+ T cells in anti-CD4 mAb-treated mice is evident as early as 3 days posttreatment

Mice treated with OVA and various combinations of anti-CD4, anti-CD8, and no mAb were sacrificed either 3 or 6 days postpriming and the number of CD4+ T cells was determined by FACS. The total number of cells that labeled positively with the anti-CD4 mAb at both 3 (Fig. 6a) and 6 (Fig. 6b) days postpriming was significantly less in both spleens and lymph nodes of mice treated with OVA and anti-CD4 mAb/isotype control than in mice treated with either, OVA alone, or with OVA plus the anti-CD4/anti-CD8 mAb mixture, or with no treatment.


Figure 6
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  		FIGURE 6. The loss of CD4+ T cells in anti-CD4 mAb-treated mice is evident as early as 3 days posttreatment. BALB/c mice were infused with CD4+ KJ1–26+ cells and treated with either OVA plus the anti-CD4/anti-CD8 mAb mixture, OVA and anti-CD4 mAb plus isotype control, OVA alone, or were left untreated. On days 3 (a) and 6 (b) after OVA and mAb treatment, the total number of CD4+ cells in the spleen and lymph nodes was determined by FACS. Data shown are mean ± SD and are representative of two experiments. **, Statistical significance to 0.009–0.001. ***, Statistical significance to 0.0009–0.0001. The Student t test was used to determine statistical significance; n = 3/group.

 
To determine whether the loss of CD4+ T cells was due to modulation of the cell surface CD4, or was due to a loss of the CD4+ T cells, the percentage of CD4+, CD8+, and CD3+ was calculated. At 6 days postpriming, we found that the percentage of CD3+ T cells was equal to the sum of CD4+ and CD8+ cells in both spleens and lymph nodes, indicating that the loss of CD4+ T cells in anti-CD4 mAb/isotype control-treated mice at this time point was due to a loss of the CD4+ T cells and not modulation or coating of the cell surface CD4 (Table II). CD8+ T cell loss is also significantly depleted by day 6 postpriming in the spleens and lymph nodes of mice treated with OVA and the anti-CD4/anti-CD8 mAb mixture. By day 3 postpriming, the percentage of CD3+ T cells in the spleen of mice treated with OVA and anti-CD4 mAb/isotype control was equal to the sum of the percentage of CD4+ cells and CD8+ cells indicating that CD4+ cells were lost in the spleens of these mice as early as 3 days postpriming (Table II). However, this is not true in the lymph nodes of these mice. Thus, data show that the percentage of CD3+ T cells in the lymph nodes of mice treated 3 days previously with OVA and anti-CD4 mAb/isotype control is 71 ± 3.6 whereas the sum of CD4+ and CD8+ cells is 60 ± 7.9. These data indicate that there are more CD3+ T cells than there are CD4+ plus CD8+ cells suggesting that 10% of the CD3+ cells have undetectable CD4/CD8 cell surface proteins (probably due to modulation and/or coating). Because these mice were only treated with anti-CD4 mAb it is reasonable to suggest that it is cell surface CD4 that is modulated/coated due to excess CD4-specific mAb in the recipient. At this same time point, the mice that received the anti-CD4/anti-CD8 mAb mixture also show an increase in CD3+ cells compared with the sum of CD4 plus CD8 cells in both spleen and lymph nodes suggesting modulation by one or both cell surface CD4 and/or CD8.


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  		Table II. Loss of CD4+ T cells is evident by day 3 post-mAb treatment

 
These data suggest that treatment with anti-CD4 mAb alone induces CD4+ T cell depletion as early as 3 days posttreatment and this is inhibited by cotreatment with anti-CD8 mAb. Again, and as expected, the depletion seen at these early time points with anti-CD4 mAb is incomplete and this is consistent with published data.

The loss of CD4+ T cells from the spleens of mice treated with anti-CD4 mAb/isotype control is due to apoptosis of CD4+ T cells

Mice treated with either OVA plus anti-CD4 mAb/isotype control, OVA plus the anti-CD4/anti-CD8 mAb mixture, OVA alone, or left untreated were sacrificed at 3 and 6 days postpriming and the number of CD4+ T cells that were undergoing apoptosis and proliferation was determined by FACS. Fig. 7 shows the strategy used to analyze the percentage of CD4+ splenocytes that are BrdU+ and annexin V+. Fig. 7a is a dot plot of splenocytes from OVA-treated mice labeled with anti-CD4 mAb. The histograms in plots Fig. 7, b–e, are gated on CD4+ T cells and show the percentage of CD4+ cells that are BrdU+ (Fig. 7b) and annexin V+ (Fig. 7d). Fig. 7, c and e, show CD4+ cells stained with isotype control for BrdU-specific mAb (Fig. 7c) and control for annexin V (Fig. 7e).


Figure 7
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  		FIGURE 7. BrdU+ and annexin V+CD4+ cells can be detected in the splenocytes of mice primed with OVA as early as 3 days postpriming. BALB/c mice were infused with CD4+ KJ1–26+ cells and primed with OVA. Mice were fed BrdU for 3 days and then the spleens were removed. Splenocytes from all mice were stained with either CD4- and BrdU-specific Ab (b), CD4-specific Ab and annexin V (d), CD4-specific Ab and the isotype control for BrdU-specific Ab (c), or CD4-specific Ab and annexin V control reagent (e). a, The gate used to identify CD4+ cells for analysis in b–e. The percentage of BrdU+CD4+ cells (b) and the percentage of CD4+ cells that were annexin V+ (d) was determined by FACS. Data shown are mean ± SEM and are representative of two experiments; n = 3/group.

 
Treatment with OVA induces significant increase in the number of proliferating CD4+ T splenocytes at both 3 and 6 days posttreatment compared with CD4+ T cells from untreated mice (Fig. 8a). The addition of anti-CD4 mAb significantly inhibits the total number of CD4+ T cells undergoing proliferation and this is not affected by cotreatment with anti-CD8 mAb, suggesting that promoting proliferation at these early time points is not a major mechanism used by anti-CD8 mAb to inhibit CD4+ cell loss from the spleen.


Figure 8
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  		FIGURE 8. The loss of CD4+ T cells from the spleens of mice treated with anti-CD4 mAb/isotype control is due to apoptosis of CD4+ T cells. BALB/c mice were treated with either OVA plus the YTS 177/YTS 105 mixture, OVA and YTS 177 plus isotype control, OVA alone, or were left untreated. On days 0–3, half of the mice in each group were fed BrdU and splenocytes were removed on day 3. The remaining mice were fed BrdU on days 3–6 and splenocytes were removed from those mice on day 6. The total number of CD4+BrdU+ cells in the spleen (a), the percentage of CD4+ cells that were annexin V+ (b), and the total number of CD4+annexin V+ cells (c) was determined by FACS. Data shown in each case are mean ± SEM and are representative of two experiments. *, Statistical significance to 0.05–0.01. **, Statistical significance to 0.009–0.001. The Student t test was used to determine statistical significance; n = 3/group.

 
The number of CD4+ cells that were annexin V+ in the spleens of mice treated with the anti-CD4/anti-CD8 mAb mixture was significantly less than the percentage in the group that received the anti-CD4 mAb/isotype control mixture at both 3 and 6 days posttreatment. These data suggest that cotreatment with anti-CD8 mAb protects CD4+ T cells from apoptosis (Fig. 8c). The effect of anti-CD8 mAb on CD4+ T cell apoptosis is also reflected in the percentage of CD4+ T cells undergoing apoptosis at day 3, but not day 6, postpriming further suggesting a role for anti-CD8 mAb in protecting CD4+ T cells from apoptosis during the first 3 days posttreatment, but not afterward. (Fig. 8b).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A beneficial effect of CD8 blockade in promoting immune tolerance has been reported in a variety of tolerance models, but the mechanism for this effect is not entirely understood. Using a well-established model for the study of Ag responsiveness and tolerance, we show that while CD8 blockade is not necessary to induce Ag-specific regulatory CD4+ FOXP3+ T cells, it is essential for the maintenance of Ag responsiveness to irrelevant Ag in tolerant mice. In this model, the effect of CD8 blockade is to prevent the loss of nontransgenic CD4+ T cells in mice treated with OVA and anti-CD4 mAb and this is consistent with previous findings indicating that CD8 blockade also prevents the loss of TCR-transgenic CD4+ T cells in mice treated with anti-CD4 mAb (12). However, the mechanism for this effect on transgenic and nontransgenic CD4+ T cells is not the same. Thus, anti-CD4 mAb treatment causes significant apoptosis of the nontransgenic CD4+ T cell population whereas apoptosis was not detected in the CD4+ TCR-transgenic population. In addition, whereas cotreatment with anti-CD8 mAb inhibits apoptosis in the nontransgenic CD4+ T cell population, it promoted proliferation of the CD4+ TCR-transgenic cells (12). There are at least two explanations that might explain this paradox. The first is that the transgenic expression of the OVA-specific TCR causes the transgenic CD4+ T cells respond differently to both anti-CD4 mAb and CD8 blockade than nontransgenic CD4+ T cells. Alternatively, Ag-specific cells, including the OVA-specific TCR-transgenic cells, might be protected from anti-CD4 mAb-induced apoptosis by stimulation with Ag leading to survival of TCR-transgenic cells. We suggest that the latter is the more likely explanation because engagement of TCR is reported to inhibit anti-CD4 mAb-induced apoptosis (30). This would also apply to OVA-specific CD4+ T cells that are not transgenic but protection of these nontransgenic CD4+ T cells is not detected because the frequency of OVA-specific cells in the nontransgenic CD4+ T cell population is very low. Similarly, we find that the cotreatment with anti-CD8 does not promote proliferation of nontransgenic CD4+ cells. But again, significant proliferation might not be detected in the nontransgenic CD4+ T cell population because the frequency of cells likely to be OVA-specific is too low.

mAbs can cause depletion of their target cell by a number of different mechanisms depending, to a large extent, on their isotype. These mechanisms include cell opsonization followed by uptake by FcR-bearing phagocytes (31, 32), activation of the complement cascade (33), and apoptosis via Ab-dependent cellular cytotoxicity (Refs. 33, 34, 35, 36). The isotype of both YTS 177 and YTS 105 mAbs is rat IgG2a, an isotype known to be less efficient in inducing depletion in mice compared with other rat Ig isotypes, including rat IgG2b (37, 38, 39) and rat IgG1 (39). However, depletion and partial depletion with rat IgG2a mAbs has been described (38, 40), but this has not been general (7, 9), and might depend on the specificity of the mAb (41). The anti-CD4 mAb, YTS 177, and the anti-CD8 mAb, YTS 105, used in these studies are not depleting when used to induce tolerance in transplant models (4, 28, 29). In contrast, in the model described here, both YTS 177 and YTS 105 induce partial depletion of the cells that they target. This is not due to aggregation of the mAbs used because centrifuging at 25,000 x g for 10 min before injection did not alter the effect of these mAb on CD4+ and CD8+ cell depletion (data not sown). This might again be explained by difference in the frequency of cells responding to Ag in the two systems. The frequency of CD4+ and CD8+ T cells responding to OVA is significantly lower than the frequency of cells responding to either multiple major transplantation Ags (MHC) or multiple minor transplantation Ags (42, 43). If TCR engagement inhibits anti-CD4 mAb-induced apoptosis in this model, as is described for other models (30), the CD4+ and CD8+ T cells in mice that received a transplant would be less susceptible to depletion by the anti-CD4 and anti-CD8 mAbs than CD4+ and CD8+ in mice that were primed with OVA.

The induction of CD4+ T cell apoptosis with anti-CD4 mAb has been reported previously (44, 45). In those published models the mechanism for the induction of apoptosis by anti-CD4 mAb involves CD4 cross-linking (46), an up-regulation of cell surface Fas on CD4+ T cells (47, 48), and expression of Fas ligand on monocytes (46), which results in CD4+ T cell apoptosis via the Fas-FasL pathway. However, the effect of CD8 blockade on inhibiting CD4+ T cell apoptosis is novel. It is possible that simultaneous apoptosis of CD8+ and CD4+ T cells might result in competition for effector factors necessary for the induction of apoptosis, including Fas ligand-expressing monocytes. In addition, removal of CD8+ T cells might alleviate the competition for survival factors that inhibit apoptosis including IL-2 (49, 50), IL-7 (51, 52, 53), and APC (30). A beneficial effect of CD8+ cell depletion in CD4+ T cell survival has been reported in other systems but the mechanism, and the effect on Ag responsiveness, was not investigated. In a clinical study the incidence and severity of graft-vs-host disease was significantly reduced by depletion of donor CD8+ T cells (54, 55) and CD8+ T cell depletion was associated with an increase in CD4+ T cell recovery (54). CD8 blockade has also been shown to promote chimerism in bone marrow transplantation in the NOD mouse (56) and in nonautoimmune susceptible mice (57). It is important to note that, in the model described here, we cannot rule out the possibility that the effect of CD8 blockade on CD4+ T cell survival does not require CD8+ T cell depletion. It is also possible that CD8+ T cells are not the relevant target for the CD8 blockade effect.

The CD8-specific mAb, YTS 105, used in these studies is specific for the {alpha}-chain of CD8 (4) which is expressed on CD8{alpha}+ dendritic cells (DC) as well as CD8+ T cells (58). A role for CD8{alpha}+ DC in limiting T cell responsiveness is also well-documented. Examples include failure to support proliferation of T cells (59), inhibition of alloreactive T cell expansion resulting in prolonged cardiac allograft survival (60), and, more relevant to this study, the induction of T cell apoptosis (61) including apoptosis of self-reactive T cells (62). However, CD8{alpha}+ DC also play a role in inducing primary Th1-type rather than Th2-type responses (63, 64). These findings suggest the possibility that anti-CD8 mAb might promote CD4+ regulatory T cell expansion, including Th2-type cells, by blocking CD8{alpha}+ DC function and thus removing CD8{alpha}+ DC-mediated suppression of T cell responses.

Our finding that treatment with Ag and anti-CD4 mAb, in the absence of CD8 blockade, can induce Ag-specific regulatory CD4+FOXP3+ T cells, is consistent with data from the Waldmann group (15) who showed an increase in CD4+FOXP3+ cells in mice made tolerant to skin allografts with anti-CD4 mAb. However, the unresponsiveness to nontolerizing Ags that accompanies tolerance in our model was not seen in previously published studies (15). This is not likely to be due to a qualitative difference between models of skin transplantation and the OVA system because Ag-specific tolerance, in the absence of nonspecific unresponsiveness, has also been induced by anti-CD4 mAb to rat IgG (4). Moreover, we do not believe that our findings are due to an idiosyncrasy of the model that we are using because the DO11.10 model is a well-established model for the study of tolerance and immune regulation. In addition, the anti-CD4 mAb that we use, YTS 177, and the protocols for priming and inducing tolerance to OVA, including the dose of anti-CD4 mAb used, are the same as those used in published reports (4, 15). However, there is a notable difference between these studies in the way tolerance was tested. Whereas we test tolerance by challenging mice with Ag given i.p., the published studies test for tolerance using protocols that are potentially more immunogenic, specifically, by challenging mice with either a skin allograft (15), or, with Ag in CFA (4). It is possible that nonspecific unresponsiveness might be reversed in situations of increased immunogenicity, while tolerance is maintained. In contrast, in the experiments described here, the less immunogenic challenge might be less able to break unresponsiveness.

To our knowledge, this is the first report on an effect of CD8 blockade on promoting responsiveness to nontolerizing Ag in tolerant mice. Together with previously published data showing that CD8 blockade also promotes the responsiveness of Ag-specific CD4+ regulatory T cells in tolerant mice (12), we suggest that CD8 blockade exerts a beneficial effect on the recipient by promoting responsiveness to both nontolerizing and tolerizing Ag, resulting in enhanced protection from pathogens, and long-term tolerance by expansion of Ag-specific regulatory CD4+ T cells.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Diabetes National Research Group (DNRG0603), the Alzheimer and Aging Research Center (AARC5104), and the National Institutes of Health (DK61334) to J.D.D. Back

2 Address correspondence and reprint requests to Dr. Joanna D. Davies, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. E-mail address: jdavies{at}tpims.org Back

3 Abbreviations used in this paper: beta-gal, beta-galactosidase; DC, dendritic cell. Back

Received for publication November 13, 2006. Accepted for publication February 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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