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The Journal of Immunology, 2002, 169: 2947-2955.
Copyright © 2002 by The American Association of Immunologists

Anti-Human CD4 Induces Peripheral Tolerance in a Human CD4+, Murine CD4-, HLA-DR+ Advanced Transgenic Mouse Model1

Rüdiger Laub2,*, Rene Brecht*, Martina Dorsch{dagger}, Ulrich Valey*, Kerstin Wenk* and Frank Emmrich*

* Institute for Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany; and {dagger} Institute for Laboratory Animal Science, Medical School Hanover, Hanover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Selection in vivo of potent mAbs to human CD4 useful for immunotherapy, e.g., for the induction of immunological tolerance, is restricted for ethical reasons. We therefore used multiple transgenic mice that lack murine CD4, but express human CD4 specifically on Th cells, and HLA-DR3 as its natural counterligand (CD4/DR3 mice). The injection of CD4/DR3 mice with anti-human CD4 (mAb Max.16H5) before immunization with tetanus toxoid (TT, day 0) totally blocked the formation of specific Abs. This state of unresponsiveness persisted a subsequent boost again performed in the presence of anti-human CD4. When these mice were left untreated for at least 40 days, and were then re-exposed with TT, but in the absence of anti-human CD4, they consistently failed to induce specific Abs (long-term unresponsiveness). Exposure to second party Ags (hen egg lysozyme, human acetylcholine receptor) induced specific Abs comparable with control mice, demonstrating that the anti-CD4-induced unresponsiveness was Ag specific (immunological tolerance). Importantly, the concurrent injection of TT and anti-human CD4 at day 0, followed by another two anti-CD4 treatments, also led to tolerant animals, indicating that tolerance was inducible at the same day as the Ag exposure is provided. We finally demonstrate a limited ability of spleen cells to respond to TT in vitro, indicating that T cells are essentially involved in the maintenance of TT-specific tolerance. These data show for the first time that the human CD4 coreceptor mediates tolerance-inducing signals when triggered by an appropriate ligand in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD4 coreceptor is an attractive target for the development of therapies that allow the Ag-specific suppression of unwanted immune responses (i.e., to allo- or autoantigens) without severely affecting beneficial responses. CD4 normally initiates costimulatory signals for Th cell activation when triggered simultaneously with the TCR/CD3 complex during interaction with MHC class II (MHC II)3 molecules. The separate ligation of CD4 with mAbs can initiate signaling events independently of those induced by triggering the TCR/CD3 complex. Such signals have been shown to induce aberrant Th cell differentiation in many systems.

In rodents, selected mAbs to CD4 deplete Th cells from peripheral circulation, leading to immunosuppression (1, 2, 3). It has been demonstrated later that depletion of Th cells is not an absolute requirement for the suppression of immune responses to soluble proteins (4, 5). Similarly, following complex application protocols, selected mAbs to mouse and rat CD4 have been shown to suppress acute rejection of allogeneic solid organ grafts, such as heart and kidney, allow complete vascularization, and thus ensure long-term graft survival (6, 7, 8, 9).

Functional studies of the human CD4 coreceptor are restricted to Th cells in vitro and have shown that mAbs to human CD4 can initiate stimulatory as well as inhibitory cellular signals. Some mAbs raise both intracellular calcium and IL-2 production in the absence of TCR/CD3 stimulation (10), while others can prime Th cells to activation-dependent cell death that can be triggered by subsequent TCR/CD3-mediated signals (11, 12). Using mAbs recognizing different CD4 epitopes, Baldari and coworkers (13, 14, 15) ascertained that gene-activating and proapoptotic potential are associated with different CD4 epitopes, and that the CD4-associated phosphotyrosine kinase p56lck delivers signals inherent to apoptosis commitment.

Although such in vitro studies indicate isolated properties of potent functional relevance, the benefit to human therapy, such as tolerance induction, can be proven only in the context of an entire immune system.

To this end, we have developed a mouse model with transgenic expression of both human CD4 and the MHC II counterligand HLA-DR3, in a mouse CD4-deficient background (CD4/DR3 mice) (16). The expression of human CD4 on mouse T cells is restricted to the helper subset, i.e., the alternative functional T cell subsets are fully preserved. The majority of mouse MHC II-positive cells in spleen, lymph node, and peripheral blood were shown to coexpress HLA-DR3, with no significant bias to particular leukocytes.

Via its cytoplasmic tail, CD4 interacts functionally with a phosphotyrosine kinase, p56lck. This interaction is crucially involved in both the maintenance of posttranscriptional CD4 expression levels and the transduction of coreceptor signals for Th cell activation. We could show that both functions are enabled in Th cells isolated from CD4/DR3 mice (16). Treatment with anti-human CD4 led to a decrease of CD4 surface expression, a process referred to as modulation. Modulation seen with CD4/DR3 mice Th cells was quite similar to human Th cells, both in magnitude and kinetics.

Furthermore, recall responses to tetanus toxoid (TT) in vitro were clearly dependent on the interaction between the human transgenic surface molecules, CD4 and HLA-DR. mAbs to either molecule inhibited proliferation in spleen cell suspensions of CD4/DR3 mice previously immunized with TT (16).

Based on these in vitro data, we sought to determine whether the administration of anti-human CD4 would have functional significance to immune responses in vivo. We show in this study that the treatment of CD4/DR3 mice with anti-human CD4 (clone Max.16H5) (17) blocks primary immune responses, and may induce Ag-specific tolerance toward a complex protein Ag, TT. These results demonstrate for the first time that the human CD4 coreceptor can mediate immunological tolerance when appropriately triggered in vivo.


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

C57BL/6 (H2b) mice were obtained from the animal facilities of the Medical Faculty, University of Leipzig. CD4/DR3 mice express human CD4 and HLA-DR17, a split Ag of HLA-DR3, in a murine CD4-deficient background. These mice were bred at Institute for Laboratory Animal Science, Medical School Hanover, as previously reported (18). Briefly, TgN(HLA-DR17a/b)1Dkfz (19) and TgN(hCD4)1Lit-cd4tm1Lit mice (20) served as founders to establish a strain with transgenic CD4 and HLA-DR3 and inactivated mouse CD4. Animals bearing the desired mutations were identified by PCR or FACS, and were used for further inbreeding (21). CD4/DR3 mice used in the present investigations were of the 5. and 6. inbred generation, respectively. Siblings with the genotype human CD4+/+/mouse CD4-/- which segregated during early inbreeding, were used for control purposes. Animal housing and experiments were performed in accordance with institutional guidelines.

Flow cytometry

T lymphocytes were monitored using peripheral blood obtained by puncture of the retro-orbital venous plexus. Whole blood samples were treated with Fc block, and aliquots were incubated with anti-human CD4 (clone Max.16H5). After 20 min, samples were depleted of erythrocytes using lysing solution, and incubated for another 10 min. After rinsing, the cells were incubated with biotinylated rat anti-mouse IgG1, washed again, and finally stained with SA-PeCy5, FITC anti-CD3, or PE anti-CD8. Abs were from BD Biosciences (Heidelberg, Germany), except clone Max.16H5, which was produced in our laboratory (see below). To test the coating of Th cells with anti-human CD4 in vivo, PBMC of mAb-treated animals were stained with biotinylated rat anti-mouse IgG1, followed by SA-PeCy5.

Anti-human CD4 reagents and treatment protocols

The anti-human CD4 mAb Max.16H5 (17) was purified from hybridoma supernatants produced in an Acusyst-Maximizer 500 bioreactor (Endotronics, Minneapolis, MN). Chromatography-guided digestion with papain (Sigma-Aldrich, Deisenhoven, Germany), followed by protein A isolation and gel filtration, was used to produce F(ab')2 and Fab, respectively. The purity was >98% for F(ab')2, and >91% for the Fab fraction. The latter contained ~7% L chains, and less than 2% F(ab')2. Preparations were adjusted to 1 mg/ml with PBS, and were i.p. injected at the time points indicated. Monoclonal mouse IgG1 (MOPC21; Sigma-Aldrich) was used as isotype control.

Treatment protocols

TT, which was a kind gift of Dr. K. Enzle (Behring/Chiron, Marburg, Germany), was used as a model Ag. TT was delivered by i.p. injection of 50 µg dosages at days 0 and 13. The individual capabilities to produce TT-specific Abs were measured in CD4/DR3 or control mice that had been treated either with anti-human CD4 or the respective isotype control mAb. If not stated otherwise, animals were i.p. injected with 15 µg/g body weight (BW) anti-human CD4 at days -2, -1, 0, +3, and +12. Alternative protocols were run to test the effects of either different mAb dosages (3 and 0.5 µg/g BW), or aberrant injection times. For instance, in some experiments, animals were not pretreated at days -2 and -1; instead, they received mAbs at days 0, +1, and +2, respectively. Finally, fragments of anti-human CD4 were tested at dosages equimolar to 15 µg/g entire IgG.

TT-specific Abs were individually monitored. Therefore, sera were prepared from peripheral blood obtained by retro-orbital bleeding. To test for recall responses or unresponsiveness, respective animals were again injected with TT at later time points, as indicated in the experiments. These injections were done in the absence of anti-human CD4. TT-specific Abs were determined in sera taken 10 days later.

To test for second party responses, CD4/DR3 mice were injected with the respective Ags (recombinant human acetylcholine receptor-{epsilon} (AcHR), hen egg lysozyme), and serum Abs were determined 10 days later using Ag-specific ELISAs.

Assessment of anti-human CD4 serum levels

Serum was prepared from peripheral blood obtained by retro-orbital bleeding. Anti-CD4 titers were assessed using an ELISA with human rCD4, which was a kind gift of Biogen (Cambridge, MA), coated on Maxisorb 96-well microtiter plates (Nunc, Roskilde, Denmark). Retrieved anti-CD4 was detected by sequential processing with polyclonal biotinylated goat anti-mouse (Caltec, Burlingham, CA), ExtrAvidin/AP (Sigma-Aldrich), and p-nitrophenylphosphate (Sigma-Aldrich). Anti-human CD4 serum concentrations were determined based on a standard curve made with different concentrations of Max.16H5.

TT immunization and assessment of specific Abs

Anti-TT serum titers were recorded using an ELISA with TT coated on Maxisorb plates. TT-specific Abs were detected by sequential processing with polyclonal biotinylated goat anti-mouse (Caltec), ExtrAvidin/AP (Sigma-Aldrich), and p-nitrophenylphosphate (Sigma-Aldrich). Anti-TT titers were compared with gradual dilutions of a high-titer mixed reference serum. The IgG content of the reference serum was determined by ELISA detection, whereby ChromPure mouse IgG (Dianova, Hamburg, Germany) served as standards. TT-injected CD4/DR3 mice were considered unresponsive if their sera provided ELISA signals not exceeding those obtained with control sera of mice responding to various protein Ags different from TT (response threshold, 1.4 ± 0.8 ng/ml IgG). Note: Although a titer of 2 ng/ml TT Abs is extremely low, the animal cannot be considered unresponsive.

Serum Abs to second party Ags were assessed similarly.

T cell proliferation assay

Spleen cell suspensions of TT-responsive and TT-tolerant CD4/DR3 mice were cultured at 4e5 cells/200 µl Xvivo15 (BioWhittaker, Mannheim, Germany). Cultures were restimulated with TT or left untreated, and control cultures were polyclonally stimulated with 1 µg/ml PHA (Wellcome, Dartford, U.K.). Abs for blocking studies, and the corresponding isotype controls, were added 30 min before the Ag. Cells were cultured in triplicate in 96-well round-bottom tissue plates (Greiner, Frickenhausen, Germany) at 37°C in 5% CO2 atmosphere. Cultures were pulsed for the last 12 h with 0.5 µCi/well tritiated thymidine (Amersham, Little Chalfont, U.K.). Cells were harvested onto glass filter mats and air dried, and incorporated radioactivity was measured using a Matrix 96 ionization chamber beta counter (Packard Instruments, Dreieich, Germany). Proliferation data are expressed as stimulation indices (SI), referring to untreated cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4/DR3 mice use the human transgenic molecules and develop primary Ab responses comparable with wild-type mice

CD4/DR3 mice were investigated with respect to the formation of specific Abs to TT, which was used as model Ag for the present study. As shown in Fig. 1GoA, the kinetics of Ab formation in CD4/DR3 mice was very similar to that in wild-type mice (C57BL/6). Significant serum titers for TT-specific Abs developed by day 5 following immunization and reached a plateau after day 10.



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  		FIGURE 1. Formation of TT-specific Abs. A, Kinetics of Ab formation in CD4/DR3 and BL/6 mice. Four mice each were injected i.p. with 50 µg TT at day 0 and bled at the indicated time points, and TT-specific Abs were assessed by ELISA. B, Dependence of Ab responses on human CD4-HLA-DR interaction. CD4/DR3 mice and transgenic mice bearing human CD4, but lacking its counterreceptor HLA-DR (DR-deficient, filled column), were i.p. immunized with 50 µg TT, and specific Abs were measured at day 7. CD4/DR3 mice were treated with anti-HLA-DR (hatched columns) or the respective isotype control Ab (open column) at day 0 and every second day thereafter. Columns represent mean and SD of three animals each; differences were significant for *, p < 0.05; **, p < 0.01 (Student’s t test).

 
The human transgenes were necessary for the effective induction of TT Abs, because mice with disrupted interaction between HLA-DR and the CD4 coreceptor showed significantly decreased TT-Ab titers (Fig. 1GoB). Anti-human HLA-DR (clone L243) injected in CD4/DR3 mice at the day of immunization, and every second day thereafter, inhibited specific Abs. Dosages of 30 µg/g BW per single shot were more effective at inhibiting specific Abs than dosages of 15 µg/g. Likewise, mice expressing transgenic human CD4, but lacking HLA-DR3 (segregants of the inbreeding process), failed to produce specific Abs upon injection with TT.

Anti-human CD4 modulates CD4 expression in vivo, but does not deplete Th cells from circulation

We next analyzed the anti-human CD4 serum levels after i.p. injection of the mAb at different dosages. Whereas single i.p. injection of 3 or 0.5 µg/g BW was insufficient to establish significant anti-CD4 serum titers, a dose of 15 µg/g BW led to a peak of ~80 µg/ml between 3 and 6 h postinjection, and a rapid decline afterward (Fig. 2GoA). Stainings of PBMC with anti-mouse IgG1 plus SA-PeCy5 led to strong signals of a subpopulation of CD3-expressing T cells, indicating that Th cells were coated soon after the mAb was distributed in the circulation (not shown). If 15 µg/g BW were injected at 3 consecutive days, excessive anti-CD4 serum levels of ~80 µg/ml were established, and were maintained at least for another 3 days (data not shown). Under conditions of repeated mAb injections, cellular CD4 expression levels declined within 24 h to less then 30% as compared with those of isotype-treated control CD4/DR3 mice (in vivo modulation, Fig. 2GoB). This state of modulated CD4 expression was maintained for several days. The experiment shown in Table IGo demonstrates that total recovery of normal CD4 expression levels may require several weeks; at day 8, mean CD4 expression levels were 48% of the isotype-treated CD4/DR3 mice.



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  		FIGURE 2. Influence of anti-human CD4 treatment on circulating T cells. A, Effect of anti-CD4 dosage on serum levels. Groups of three CD4/DR3 mice each were i.p. injected with 15, 3, or 0.5 µg/g Max.16H5. Mice were bled at the indicated time points, and anti-CD4 serum levels were assessed by ELISA. B, Effect of anti-human CD4 on CD4 expression of peripheral Th cells. Three CD4/DR3 mice each were i.p. injected with 15 µg/g BW anti-human CD4, or irrelevant mouse IgG1, on 3 consecutive days. Animals were bled at days 0, 1, and 3, and PBMC were triple stained with anti-CD3 FITC, anti-CD8 Pe, and anti-CD4 (Max.16H5), followed by biotinylated rat anti-mouse IgG1 and SA-PeCy5. CD4-derived signals of the CD3+/CD8- fractions are displayed as mean fluorescence intensities (MFI). C, Composition of peripheral T cells at days 0, 1, and 3. Display of CD8+ and CD8- peripheral T cells of anti-CD4-treated mice shown in B (SD for each group <20%).

 

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  		Table I. CD4 expression levels after administration of anti-human CD4 in vivoa

 
Th cells were not depleted, because the number of peripheral T lymphocytes did not change significantly. Rather, they appear as T cells without any coreceptor expression (CD3+/CD8-/CD4-), or varying CD4 expression. Thus, a comparison of the ratios between CD3+/CD8+ and CD3+/CD8- cells would reflect the changes in the peripheral Th cell population. As shown in Fig. 2GoC, significant changes were not detected after anti-human CD4 administration, showing that Th cells were not depleted from circulation.

Anti-human CD4 prevents the primary humoral response to the protein Ag, TT

We investigated the potential of anti-human CD4 to prevent humoral responses to TT. CD4/DR3 mice were therefore immunized with TT (day 0), in the presence of either anti-human CD4 (Max.16H5), or the respective isotype control mAb (MOPC 21). The development of TT-specific Abs in the sera of immunized mice was detected by ELISA.

The treatment with 15 µg/g BW of anti-human CD4 at days -2, -1, 0, and +3 clearly prevented the formation of TT-specific Abs (Fig. 3Go). This inhibitory effect was specific to anti-human CD4, because the control mAb did not impair Ab production.



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  		FIGURE 3. Influence of anti-human CD4 on primary immune response in CD4/DR3 mice. A, Effect of different anti-CD4 fragments. Groups of three CD4/DR3 mice each were injected with 15 µg/g BW of the entire Ig, or equimolar amounts of F(ab')2, and Fab at days -2, -1, and 0. Control animals received irrelevant IgG1, or were left untreated. Animals were immunized i.p. with 50 µg TT at day 0, and TT-specific Abs were determined by ELISA at days 0 and 7. Decreases in Ab titers vs isotype-treated mice were significant for **, p < 0.01, or *, p < 0.05 (Student’s t test). B, Dosage analysis of anti-CD4-induced unresponsiveness. Groups of four CD4/DR3 mice each were injected i.p. with 15, 3, or 0.5 µg/g BW anti-CD4, or 15 µg/g irrelevant mIgG1 (iso) at days -2, -1, 0, and +3. Animals were immunized i.p. with 50 µg TT at day 0, and TT-specific Abs were determined by ELISA at days 0 and 7. *, Differences between the 15 and 3 µg group and the 15 and 0.6 µg group were significant (p < 0.1; Student’s t test).

 
We next tested whether the entire Ig is required, or whether fragments of the entire mAb could eventually mediate this inhibitory effect. F(ab')2, which represent a bivalent ligand for CD4, as the entire mAb does, and Fab, representing monovalent CD4 ligands, were used. We found that F(ab')2 at a dosage equimolar to the entire IgG also prevented TT-specific Abs (Fig. 3GoA). In contrast to the bivalent CD4 ligands, monovalent Fab of Max.16H5 were less efficient at inhibiting primary Ab responses.

The inhibitory effect of anti-human CD4 was dose dependent. As shown in Fig. 3GoB, CD4/DR3 mice receiving 15 µg/g anti-human CD4 showed lowest titers for TT Abs, and all mice were unresponsive. Despite reduced titers in all groups, only two of four mice treated with 3 µg/g BW, and one of three receiving 0.6 µg/g, respectively, remained unresponsive in terms of not exceeding the response threshold (Table IIGo, settings 4 and 5).


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  		Table II. Unresponsive CD4/DR3 mice obtained by different anti-human CD4 treatment protocolsa

 
Of interest, protocols with the first anti-CD4 injection at day 0 (4 h before TT) also blocked primary Ab responses to TT. None of eight CD4/DR3 mice either receiving three single doses at days 0, +1, and +2, or at days 0, +3, and +12, respectively, had initiated TT-specific Abs at day 7 (Table IIGo, settings 1 and 2).

Anti-human CD4 induces long-term unresponsiveness

Having demonstrated that anti-human CD4 prevents formation of TT-specific Abs in the primary immune response, we investigated the possibility that anti-human CD4 could induce a long-term state of unresponsiveness that is maintained without the requirement of further anti-human CD4 treatment.

Therefore, CD4/DR3 mice injected with TT under the cover of anti-human CD4 (day 0) were re-exposed to the Ag at different time points. Initial experiments with a second TT exposure at day 13 following primary injecion with TT always resulted in the formation of specific Abs (data not shown). However, a single injection of 15 µg/g anti-human CD4 1 day before this re-exposure, i.e., at day 12, was efficient enough to prevent Ab formation as measured 10 days later (day 22). In contrast, this ínjection boosted TT Ab titers in the isotype-treated control groups; therefore, differential serum TT Ab levels of anti-human CD4-treated vs control animals became much more significant. We consequentially used this treatment protocol to further investigate Ab responses to TT injected at later time points without anti-human CD4.

CD4/DR3 mice, which were made unresponsive by anti-human CD4 given at the time of immunization and day 12, responded again with the formation of specific Abs when injected within the next 30 days, i.e., as early as day 32 or 42, respectively. In none of seven CD4/DR3 mice, an already induced unresponsiveness was maintained when animals were injected in that time window (not shown). However, when animals were not re-exposed to TT until day 52 (the Ag-free interval was 40 days), their ability to generate TT-specific Abs was abrogated, or dramatically reduced, even when injected in the absence of anti-human CD4. Animals with TT Ab titers not exceeding the threshold of 1.4 ng/ml were regarded unresponsive. In three independent experiments, 70% of CD4/DR3 mice remained unresponsive when the first two injections with TT were performed under the cover of anti-human CD4 (15 µg/g per single dose) or its F(ab')2 at equimolar dosages (Table IIGo, settings 1–3 and 6). Moreover, unresponsiveness in such animals resisted multiple expositions with TT performed later. Fig. 4Go shows the mean TT Ab titers of an experiment with all animals remaining unresponsive. The remaining 30% of animals that were responsive by definition showed varying Ab titers ranging from a few to nearly 100 ng (not shown).



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  		FIGURE 4. Long-term maintenance of anti-CD4-induced unresponsiveness in CD4/DR3 mice. Groups of CD4/DR3 mice were injected i.p with 15 µg/g anti-CD4 (filled symbols), or isotype-matched control Ab (open symbols) at days -2, -1, 0, 3, and 12. At days 0, 13, 52, 62, 72, 146, and 156, mice were injected with TT. Injections with Ab (open arrows) and TT (filled arrows) are indicated on the x (time)-axis. Ab serum levels were determined at the times indicated by line symbols.

 
In contrast to anti-human CD4, the isotype control Ab did not impair TT-specific Abs. Animals of the control groups responded to repeated TT exposures by Ab formation, which was apparent in that Ab titers did not decline over time. TT Ab titers in these animals varied between 30 and several 100 ng/ml.

Anti-human CD4-induced unresponsiveness is Ag specific (tolerance)

To ascertain whether unresponsiveness was specific for TT, or eventually resulted from a general immunosuppression of anti-human CD4-treated CD4/DR3 mice, unresponsive animals were exposed to second party Ags. Fig. 5GoA compares Ab responses to the human AChR, which were induced in TT-tolerant animals and in the respective control animals treated with irrelevant IgG1 at primary immunization and at boost. Both treatment groups developed significant titers for AChR-specific Abs. Of interest, the induction of an immune response to second party Ags did not basically break TT-specific tolerance. Two of three CD4/DR3 mice maintained unresponsiveness toward TT after the induction of Ab to AChR. Similar results were obtained with TT-tolerant animals injected with hen egg lysozyme as second party Ag (Fig. 5GoB).



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  		FIGURE 5. Specificity of unresponsiveness to TT as determined by second party Ags. A, CD4/DR3 mice (n = 3) that were either responsive (filled columns) or were made tolerant to TT by anti-CD4 treatment (open columns) were i.p. injected with rAChR. Animals were injected with TT at days 0 and +13; tolerance was induced with 15 µg/g BW anti-human CD4 injected at days -2, -1, 0, +3, and +12. At the time points indicated, AChR-specific (left bar chart) and TT-specific Abs (right bar chart) were assessed. Time axis (middle) indicates the following treatments: day 74, assessment of AChR Abs (blank value) and TT-specific Abs; day 95, immunization with rAChR; day 103, assessment of AChR-specific Abs; day 122, immunization with TT; day 127, determination of TT-specific Abs (day 0, start of the tolerance induction experiment, i.e., TT injection in the presence or absence of anti-human CD4). B, Humoral responses of TT-tolerant vs TT-responsive mice to immunization with HEL. Mean and SD of three mice each.

 
In vitro responses to TT of immunocompetent and tolerant mice

Spleen cell suspensions of TT-responsive and TT-tolerant CD4/DR3 mice were investigated with regard to their ability to respond to TT by proliferation in vitro. By analyzing [3H]thymidine uptake at different time points (2, 3, and 5 days, respectively), we first investigated basic proliferative responses induced by a polyclonal stimulator, PHA. PHA stimulated proliferative responses at similar extent in cells of both tolerant and responsive mice (Fig. 6GoA). Although there were no differences in polyclonal stimulation, we observed a markedly different proliferative behavior in cultures stimulated with Ag specifically (Fig. 6GoB): TT stimulated some proliferation in spleen cell cultures of tolerant mice, whereas cells of responsive mice showed a more progressive increase in [3H]thymidine incorporation over time. Stimulation of spleen cell cultures of both responsive and tolerant mice was dependent on CD4 and HLA-DR3, as mAbs to these human transgenic molecules blocked proliferation to nearly background levels (not shown). As shown in Fig. 6GoC, there was an apparent correlation between proliferation obtained upon restimulation with TT in vitro and TT-specific Abs produced in anti-CD4-treated and control mice.



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  		FIGURE 6. Proliferative responses in spleen cells of tolerant vs responsive CD4/DR3 mice. Triplicates of 4e5 spleen cells of tolerant (filled columns) or responsive CD4/DR3 mice (open columns) were stimulated with 1 µg/ml PHA (A) or 10 µg/ml TT (B). SI were calculated from nonstimulated vs stimulated cultures for the time points indicated. Differences in TT-specific proliferation were significant for p < 0.1 (*) or p < 0.05 (**), respectively. C, Coherence between TT-specific serum Abs and SI in TT-restimulated spleen cell cultures (84 h) of TT-responsive and TT-tolerant (filled symbols) CD4/DR3 mice (coefficient of correlation p = 0.68).

 
The tolerance-inducing anti-human CD4 treatment failed to establish immunological tolerance in responsive animals

Finally, we sought to determine whether the exposure of TT-responsive CD4/DR3 mice to Ag under the cover of anti-CD4 would be able to establish tolerance. In two independent experiments, the same administration that led to TT-specific tolerance in naive mice failed to abrogate or to diminish TT-Ab titers (data not shown). On the contrary, titers further increased regardless of anti-human CD4. Tolerance could also not be established when animals were subjected to a second treatment course with Max.16H5 6 wk later (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments presented in this work show for the first time that triggering the human CD4 coreceptor in vivo with a specific mAb may: 1) prevent the establishment of specific immune responses toward Ags delivered at the same time as anti-human CD4, 2) implement a long-term state of Ag-specific unresponsiveness that is sustained without further anti-human CD4 treatment, and 3) affect both the humoral and cellular limb of an immune response.

CD4/DR3 mice represent an animal model with a partially humanized pathway for the stimulation of Th cells. These mice exhibit normal T lymphocyte subsets, which strongly suggests appropriate signaling via the transgenic coreceptor in thymic selection (16).

As a model Ag for tolerance induction studies, we have used the xenogeneic protein TT, which consists of >1300 aa residues. In humans, thymus-independent antigenic epitopes are not known for TT, hence the induction of TT-specific Abs depends on appropriate T cell help. Indeed, several HLA-DR3-restricted T cell epitopes have been found (22, 23). Furthermore, TT contains promiscuous epitopes recognized by >80% of immunized human subjects (24, 25).

Similar to the situation in humans, appropriate Th cells were required for the generation of TT-specific Abs in CD4/DR3 mice. A single injection of TT without adjuvant was sufficient to induce appropriate help, leading to significant Ab titers in the primary immune response. However, the in vivo blockade of HLA-DR with mAb L243 decreased primary Ab responses. This finding underlines the requirement for a productive interaction of human CD4 with HLA-DR to generate effective B cell help. Furthermore, cross species interaction between human CD4 and mouse MHC II was ineligible to support primary Ab responses to TT. A single injection of TT did not induce a primary Ab response in control mice expressing human CD4, but lacking HLA-DR as its pristine ligand. Obviously, the interaction of human CD4 with mouse MHC II is less efficient at inducing genuine helper cells for humoral responses, i.e., Th2 cells.

Requirement for CD4 coreceptor stimulation apparently differs among various Ags. For example, T cell responses to myelin basic protein in HLA-DR1 transgenic mice were independent of human CD4 because littermates either expressing or lacking human CD4 showed similar T cell responses (26).

The CD4 coreceptor interacts with a nonpolymorphic region on the {beta}-chain of MHC II. Murine CD4 is apparently incapable of productively interacting with HLA molecules (20). This suggests an interaction of mouse CD4 with mouse MHC II {beta}-chains, which can form interspecies heterodimers with human MHC II {alpha}-chains in HLA-DR-transgenic mice (19). The inactivation of mouse CD4 in the CD4/DR3 mice used in this study precludes such exceptional activation pathways, and is therefore an important prerequisite for the realistic assessment of tolerance induction by anti-human CD4.

The anti-human CD4 mAb Max.16H5 blocked the induction of primary Ab responses to TT in CD4/DR3 mice. One possibility is that the failure to produce TT-specific Abs results from the lack of help by Th cells that cannot be stimulated productively after occupancy of their coreceptor with anti-CD4. In fact, this could be true for the primary Ab response to TT. However, Th cell block would not explain the persistent failure to produce Abs later on, when CD4/DR3 mice are re-exposed to TT in the absence of anti-human CD4. The ability to respond to second party Ags at the same time strongly suggests that a regulatory mechanism specifically suppresses immune responses to TT (tolerance).

For the implementation of peripheral tolerance in CD4/DR3 mice, TT must be delivered within a critical time of anti-human CD4 administration. This period is characterized by excessive anti-CD4 serum levels, resulting in the complete saturation of transgenic surface CD4 and its subsequent disappearance (modulation). FACS analyses show that Th cells were not depleted from the circulation, but rather redistributed in the CD8-negative T cell subset. This is demonstrated by unperturbed ratios of CD3+/CD8- T cells (Fig. 2GoC). Although previous experiments in rodents have suggested that Th cell depletion is important to tolerance induction (27, 28), nondepleting Abs were later found to induce tolerance in rodent models. Modulation, however, was a prominent feature during the administration period, affecting the induction of transplantation tolerance (29). Because it is an epiphenomenon of coating, the occupancy and prolonged blockade of the CD4 coreceptor are likely to cause the tolerance induction (30).

We have tested the ability of several alternative treatment protocols to induce peripheral tolerance. Pretreatment with 15 µg/g anti-CD4 at days -2, -1, and 0 (immunization), followed by a single shot at day +3, most effectively induced tolerance in CD4/DR3 mice (Table IIGo). Importantly, pretreatment with anti-CD4 was not essentially required, because a protocol with the first Ab injection at the same time as the immunization was done, and further injections on the following 2 days also established tolerance to TT (Table IIGo). This would be of particular importance to a potential human application, i.e., in organ transplantation, because anti-CD4 treatment cannot be initiated until the graft is available.

Dose reduction to 3 and 0.5 µg/g BW resulted in unproportional diminished anti-human CD4 serum levels, indicating that saturating Ab concentrations were not achieved (Fig. 2GoA). Although TT Abs were also dramatically reduced in low-dose trials (Fig. 3GoB), mice could not be considered tolerant because their Ab levels did not fall below the response threshold.

TT-specific tolerance required a certain time to become established. It was not apparent when CD4/DR3 mice were again challanged within ~40 days following the last TT injection in the presence of anti-human CD4. A critical period of 4 wk was also shown in a rodent heart transplantation model in which tolerance to alloantigens is induced by donor-specific blood transfusion under the cover of anti-mouse CD4 (31). Apparently, during that critical period, regulatory cells can develop that counterbalance normal immune reactivity. Although regulatory Th cells have not been identified yet, experimental data from anti-CD4-treated mice do suggest their existence: anti-CD4-induced tolerance can be transferred to naive animals by Th cells (28). The tolerance-maintaining principle is self spreading among CD4 T cells, a phenomenon known as infectious tolerance (32).

Several alternative mechanisms for anti-CD4-induced tolerance to allografts have been proposed in rodent models. The interference of anti-CD4 mAbs with CD4 signaling in the thymus suggests a central mechanism. The intrathymic presence of Ag would then predispose developing Ag-specific Th cells to selective deletion, or anergy (33). Furthermore, a functional switch of Th cells in vivo toward Th2, associated with the production of IL-4 and IL-10, was proposed to prevent the rejection of allografts due to impairment of Th1-dependent, cellular and cytotoxic mechanisms (34). This concept is challenged by the findings that allotolerance could be maintained by IL-4-independent Th cells, and that IL-2 production was not changed (35). Furthermore, no differences in mRNA for IL-2, IL-4, IL-10, and IL-13 were found in grafts either rejected or maintained due to the treatment with the rat anti-CD4 mAb OX38 (36). Our data also argue against immune deviation to Th2 as causative mechanism. If the administration of Max.16H5 would only induce a shift toward Th2, the mAb should facilitate, but not prevent humoral responses. Rather, Ag-specific suppressive mechanisms independent of Th1 and Th2 must have developed.

In the rat, treatment with anti-CD4 (OX38) depleted mature, but left out naive Th cells. Pretransplant thymectomia abrogated tolerance induction by anti-CD4, suggesting a role for naive, but not memory cells (36). Indeed, this finding seemingly contradicts earlier data showing that anti-CD4 depletes resting naive and spared memory Th cells (37). An important clue to explain the conflicting data was given by in vitro experiments with human Th cells, demonstrating that each individual CD4 epitope may induce a unique signal upon encounter with a particular mAb. Anti-CD4 mAbs B66.6, BMA031, 63G4, OKT4, and 3F11 differentially affected p56lck, p59fyn, Shc, and NF-AT activation, and calcium influx (13, 15). CD4 ligation also down-regulates Bcl-2 expression and predisposes Th cells to CD95 (Fas)-mediated apoptosis (38).

The anti-human CD4 mAb Max.16H5 recognizes the CDR2-like region of CD4 V1 (17, 39); Max.16H5 was shown to interfere with anti-CD3-driven T cell activation in vitro via a mechanism involving the cross-linking of CD4 with CD3 in a time-dependent fashion. Although preincubation times with Max.16H5 <15 min before anti->CD3 and subsequent cross-linking enhance DNA synthesis, longer times lead to a decrease (40). This finding indicates that anti-CD4-coated cells in vivo, i.e., in Max.16H5-treated CD4/DR3 mice, would not clonally expand upon Ag presentation. Indeed, Max.16H5 was shown to inhibit IL-2-dependent proliferation of transformed human Th cells (41). Finally, Max.16H5 induces a Th1>Th2 cytokine shift in human PBMC (42). A uniform finding in this respect is that CD4 needs to be oligomerized. The present in vivo experiments also confirm the need for at least dimerized CD4, as treatment with F(ab')2, but not Fab, of Max.16H5 was able to induce tolerance. Importantly, oligomerization of CD4 is also required for physiological Th cell activation, and is attained by formation of multimeric complexes, including several MHC II and CD4 molecules (43).

In summary, we have shown that triggering the human CD4 coreceptor can induce immunological tolerance in vivo. Based on the heterogeneity of mAbs to human CD4, and the various functional changes they induce, it seems likely that identification of those mAbs most appropriate for tolerance induction in humans will require detailed studies in vivo. Our data show that new preclinical models, such as the transgenic model used in this study, should facilitate that search.


   Acknowledgments
 
We gratefully acknowledge the provision of CD4-/CD4+ transgenic mice by Drs. Dan Littman and Nigel Killeen (University of California, San Francisco, CA), and of the HLA-DR3 transgenic mice by Dr. Günter J. Hämmerling (German Cancer Research Center, Heidelberg, Germany). We thank Matthias Meyer, Medical School Hanover, for screening of cross breedings. We particularly thank Dr. Michael Cross (University of Leipzig) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the German Bundesministerium für Bildung, Forschung und Technologie (BMB+F), and the Interdisziplinaeres Zentrum für Klinische Forschung (IZKF) at the University of Leipzig (01KS9504/1, Project A2). Back

2 Address correspondence and reprint requests to Dr. Rüdiger Laub, Institute for Clinical Immunology and Transfusion Medicine, University of Leipzig, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. E-mail address: laur{at}server3.medizin.uni-leipzig.de Back

3 Abbreviations used in this paper: MHC II, MHC class II; TT, tetanus toxoid; AChR, acetylcholine receptor; BW, body weight; SI, stimulation index. Back

Received for publication March 22, 2002. Accepted for publication July 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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