CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1→S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1→S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

Treatment with anti-CD86 costimulatory molecule prevents the autoimmune lesions in murine Sjögren's syndrome (SS) through up-regulated Th2 response

Intraperitoneal administration with anti-CD86 (B7.2) MoAb into the murine model for primary SS in NFS/sld mutant mice resulted in dramatically inhibitory effects on the development of autoimmune lesions, while no significant effects were observed when the mice were administered with anti-CD80 (B7.1) MoAb. We found that spleen cells in the murine SS model treated with anti-CD86 MoAb showed a significant impairment of autoantigen-specific T cell proliferation. T cell activation markers (CD44high, CD45RBlow, Mel-14low) were significantly down-regulated in the spleen cells gated on CD4 in anti-CD86-treated mice. We detected a higher level of cytokine production of IL-4 from splenic T cells in anti-CD86-treated mice, but not of IL-2, and interferon-gamma (IFN-gamma), compared with those in the anti-CD80- and PBS-treated SS model. Moreover, serum autoantibody production against alpha-fodrin autoantigen was almost entirely suppressed in anti-CD86-treated mice. These data provide strong evidence that in autoimmune exocrinopathy resembling SS in NFS/sld mutant mice, the CD86 costimulatory molecule plays a crucial role in the initiation and subsequent progression of Th1-mediated autoimmunity in the salivary and lacrimal glands.     

Signalling via CD28 of human naive neonatal T lymphocytes.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.     

PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2

Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1(-/-) animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1. This study examines the functional consequences of PD-1:PD-L engagement on murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1.Fc-coated beads display dramatically decreased proliferation and IL-2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti-CD28 mAb can overcome PD-1-mediated inhibition by augmenting IL-2 production. However, PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1:PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Experiments using TCR transgenic CD4(+) or CD8(+) T cells stimulated with antigen-presenting cells expressing PD-L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8(+) T cells may be more sensitive to modulation by the PD-1:PD-L pathway because of their intrinsic inability to produce significant levels of IL-2.     

Accessory Signalling by B7-1 for T Cell Activation Induced by Anti-CD2: Evidence for IL-2-Independent CTL Generation and CsA-Rcsistant Cytokine Production

Resting T cells can be activated by selected pairs of anti-CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti-CD28, or by the combination of IL-1β and IL-6. The present study was undertaken to investigate accessory signalling by B7-1, the natural ligandof CD28, in this pathway of T cell activation. 3T6 mouse fibrobiasts were transfected with human B7-1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti-CD2 MoAb, T cell proliferation, production of cytokines (IL-2, IL-4, IL-10, GM-CSF, IFN-α and TNF-α), and generation of cytotoxic T lymphocytes were all supported by B7-l(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti-IL-2 + anti-IL-2R MoAb revealed both IL-2-dependent and IL-2-independent CTL generation after B7-1 -mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL-2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7-1 - CD28 interaction. Anti-CD2 MoAb with B7-1 costimulation could directly induce proliferation, IL-2 production and generation of CTL activity in highly purified CD8⁺ T cells without the heip of CD4⁺ T cells. We conclude that CD28 ligation with the natural ligand B7-1 provides a strong accessory signal for CD4 and CD8 cell activation through CD2.     

Persistent CD3-Crosslinking Down-Regulates Interleukin-2 Responsiveness in Interleukin-2-Competent Cloned T Cells: the Possible Involvement of Protein Kinase C

To investigate the regulation of interleukin-2 (IL-2) responsiveness of T cells, a human CD4⁺ T-cell clone with constitutive expression of IL-2 receptors was stimulated with recombinant IL-2 (rIL-2) in the presence or absence of immobilized anti-CD3 monoclonal antibodies (αCD3_imm MoAb). Incubation of T cells with αCD3_imm MoAb decreased IL-2-induced proliferation which could not be ascribed to the modulation of IL-2 receptor expression nor to cell death. Phorbol-myristate-acetate (PMA), an activator of protein kinase C (PKC), also induced down-regulation of IL-2 responsiveness. The αCD3_sol MoAb, inducing Ca²⁺-mobilization without activating PKC, did not inhibit IL-2 responsiveness whereas cyclosporine A (CsA), a drug that inhibits the Ca²⁺-dependent activation pathway, did not prevent the induction of IL-2 hyporesponsiveness induced by αCD3_imm MoAb. It is concluded that modulation of IL-2 responsiveness of T cells via the T-cell receptor/CD3 complex (TCR/CD3) may be mediated by a PKC-activating signal.     

Regulatory activity of activated murine peripheral CD4+CD25- T cells: a possible mechanism of feedback regulation on adaptive immunity

This study was undertaken to investigate the possibility that peripheral CD4+CD25- T cells, once fully activated, transiently obtain suppressive function against other T cells. CD4+CD25- T cells, isolated from splenocytes of BALB/c mice, were stimulated with anti-CD3 MoAb in the presence of feeder cells for 72 h. The activated CD4+ T cells (T(act)) thus obtained were able to inhibit the activation and proliferation of bystander CD4+ T cells in a non-MHC-restricted manner. T(act)-mediated suppression was cell contact dependent, reversible by exogenous IL-2 as well as anti-GITR antibody. Furthermore, adoptive transfer of T(act) cells significantly downregulated humoral response of BALB/c mice to s.c. immunization with ovalbumin. We argue that suppression mediated by activated CD4+CD25- T cells may play an important role in maintaining homeostasis of the immune system and preventing excessive T-cell responses in vivo.     

An optimized method for the functional analysis of human regulatory T cells

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.     

Anti-inflammatory action of alpha-melanocyte stimulating hormone (alpha-MSH) in anti-CD3/CD28-mediated spleen and CD4(+)CD25(-) T cells and a partial participation of IL-10

alpha-Melanocyte stimulating hormone (alpha-MSH) has been shown to inhibit the production and the effects of pro-inflammatory cytokine by inflammatory cells in innate immunity. We have determined whether alpha-MSH inhibits anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation and its mechanism of action. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells markedly were suppressed by 50-100 nM and 5-100 nM alpha-MSH, respectively. alpha-MSH (100 nM) increased the production of the anti-inflammatory cytokine IL-10 and decreased the production of the pro-inflammatory cytokine IL-2 and IFN-gamma from CD4(+)CD25(-) T cells. Moreover, anti-IL-10 blocking Ab decreased the inhibitory effects of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation by alpha-MSH, indicating a partial participation of IL-10 in its mechanism of inhibitory action. These results suggest that alpha-MSH may be useful for treatment of autoimmune diseases and transplantation involving innate and adaptive immunity.     

Modulation of cell cycle progression by CTLA4-CD80/CD86 interactions on CD4+ T cells depends on strength of the CD3 signal: critical role for IL-2

Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a well-studied T cell costimulatory receptor that is known to inhibit T cell activation. In this study, the relationship between strength of the first signal and costimulatory interactions on primary mouse CD4(+) T cells was investigated. CTLA4-CD80/CD86 interactions differentially modulate T cell cycling based on the mode of CD3 signal: Activation with plate-bound (pb) anti-CD3 generates a strong signal compared with a weak signal with soluble (sol) anti-CD3, resulting in approximately sevenfold higher amounts of interleukin (IL)-2 and an increase in cell cycling. Activation of T cells with sol anti-CD3 (weak signal) together with CTLA4-CD80/CD86 blockade lowers IL-2 production and cell cycling, demonstrating an enhancing role for these interactions. Conversely, blockade of CTLA4-CD80/CD86 interactions on T cells activated with pb anti-CD3 (strong signal) increases proliferation, which is consistent with CTLA4 as a negative regulator. Also, coculture of T cells with Chinese hamster ovary cells expressing CD80 or CD86 demonstrates that the strength of the primary signal plays an important role. It is important that modulation of IL-2 amounts leads to distinct alterations in the functional effects of CTLA4-CD80/CD86 interactions. On increasing IL-2 amounts, activation of T cells stimulated with sol anti-CD3 (weak signal) and CTLA4-CD80/CD86 blockade is greater compared with control. Concurrently, neutralization of IL-2 greatly reduces activation of T cells stimulated with pb anti-CD3 (strong signal) and CTLA4-CD80/CD86 blockade compared with control. These results underscore the importance of strength of first signal, CTLA4-CD80/CD86 interactions, and IL-2 amounts in modulating primary CD4(+) T cell responses.     

Isotype-specific cross-linking of select human Fc gamma R isoforms triggers release of IL-6.

Anti-CD3 MoAbs are widely used in T cell activation studies, and are effective in immunosuppressive therapy. We used a panel of mouse (m) anti-CD3 switch variant MoAbs of five different isotypes to study IL-6 release from accessory cells. Incubation of human (h) mononuclear cells with anti-CD3 MoAbs resulted in increased IL-6 levels with MoAbs of mIgG1 and mIgG2a isotypes, with no effect of mIgG2b or mIgA. This suggested involvement of IgG Fc receptors (Fc gamma R) in triggering IL-6 production. To evaluate the role of different Fc gamma R molecules individually we used a panel of hFc gamma R-transfected mouse fibroblasts, and Jurkat T cells as a model. IL-6 secretion by CD32 transfectants expressing the hFc gamma RIIa high-responder (HR) allelic form was triggered by mIgG1 anti-CD3 MoAb, with no effect of four other isotypes. None of the anti-CD3 MoAbs induced IL-6 secretion by CD32 transfectants expressing either a variant of this receptor, containing only a single intracellular amino acid (CT-), the hFc gamma RIIa low-responder (LR) allelic form, or hFc gamma RIIb1. hFc gamma RI (CD64) transfectants exhibited IL-6 production after incubation with mIgG2a anti-CD3 MoAb, and to a lesser extent with mIgG2b, and mIgG1 MoAb. Indirect involvement of T cells in triggering IL-6 secretion could be excluded by experiments in which transfectants were cultured with immobilized anti-CD3 MoAb. These data indicate that cross-linking of either hFc gamma RI, or hFc gamma RIIaHR by appropriate anti-CD3 MoAbs triggers IL-6 production of accessory cells, and not T cells. This may also take place in vivo during immunosuppressive therapy with anti-CD3 MoAbs, and related antibody-mediated immune responses.     

CD25 Blockade protects T Cells from Activation-induced Cell Death (AICD) via Maintenance of TOSO Expression

CD25 monoclonal antibody binding to the α -chain of the Interleukin-2 (IL-2) receptor, blocks high-affinity IL-2 binding, thereby preventing complete T-cell activation and being of ample importance in transplantation medicine and potentially the treatment of autoimmune disease. However, CD25 antibodies do not only block T-cell activation but also prevent activation-induced cell death (AICD) attributing a dual function to IL-2. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of humanized anti-CD25 mAb was investigated. PBMC were stimulated with CD3 antibody OKT-3 together with recombinant IL-2 in the absence or presence of anti-CD25 mAb. RNA was extracted and subjected to microarray analysis on U133A microarrays (Affymetrix). Anti-CD25 treatment inhibited several genes typically expressed during T-cell activation including granzyme B, signalling lymphocyte activation molecule, family member 1 (SLAMF1), CD40-Ligand (CD40-L), IL-9 and interferon (IFN)- γ . Interestingly, anti-CD25 mAb also blocked the expression of several genes important for susceptibility to apoptosis, such as death receptor 6 (DR6) or reversed IL-2-mediated repression of anti-apoptotic genes, such as Fas apoptotic inhibitory molecule 3 (FAIM3)/TOSO. Functional significance of DR6 and TOSO expression in IL-2-dependent T-cell activation was subsequently evaluated by RNA interference in AICD: While siRNA specifically directed against DR6 did not modulate FAS-L-mediated apoptosis induction in primary T cells, down-regulation of TOSO significantly increased susceptibility to apoptosis, emphasizing an important role for TOSO in IL-2-mediated AICD.     

Immobilized anti-CD3 mAb induces anergy in murine naive and memory CD4+ T cells in vitro

The induction of non-responsiveness in resting murine CD4+ T cells was investigated using immobilized anti-CD3 mAb. Incubation of freshly isolated CD4+ T cells with immobilized anti-CD3 mAb led to apoptosis in 40-60% cells. The surviving cells were profoundly non-responsive to subsequent mitogenic stimulation. The non-responsive state was characterized by a lack of IL-2 production and hyper-responsiveness to added IL-2, but was not explained by further activation-induced cell death. The induction of non-responsiveness was not due to modulation of the TCR-CD3 complex, and required partial activation of the T cells in that it was accompanied by an increase in cell size and was inhibited by addition of cyclosporin A. Finally, analysis of anti-CD3-mediated responses in naive and memory CD4+ T cells, separated on the basis of CD44 expression, showed that both naive and memory T cells have similar sensitivity to immobilized anti-CD3 mAb-induced activation, apoptosis and anergy. These results demonstrate that TCR-CD3 engagement on freshly isolated resting CD4+ naive and memory T cells, In the absence of co-stimulation, as achieved by plastic-immobilized anti-CD3 mAb, induces both anergy and cell death.      

Mouse B cell activation is inhibited by CD44 cross-linking

Lymphocyte activation and trafficking are indispensable to the immune system. CD44 is an adhesion molecule with known importance in T cell activation, lymphocyte trafficking, and tumor metastasis. Although CD44 has been shown to participate in the activation, rolling and adhesion, and homing of T cells, the role of CD44 on B cells is relatively unknown. The effects of CD44 cross-linking on murine B cell activation via CD40L was explored using the anti-CD44 mAbs RK3G9 and IM7. When immobilized on a plate, both RK3G9 and IM7 were found to strongly inhibit B cell proliferation and Ig production, especially at lower cell input concentrations. IgE inhibition was especially prominent. In contrast, soluble RK3G9 added to the B cell cultures had no effect. The inhibitory effect of anti-CD44 on B cell activation was not influenced by the addition of the anti-FcγRII, indicating that Fc cross-linking did not play a role in this inhibition. As Ig production requires several days for both B cell proliferation and differentiation to occur, the effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. Finally, B cells were activated by either LPS or anti-IgM F(ab')₂. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. These results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.     

The CD44 variant isoforms CD44v6 and CD44v7 are expressed by distinct leukocyte subpopulations and exert non-overlapping functional activities

We have described recently that anti-CD44s, anti-CD44v6 and anti-CD44v7 interfere with delayed-type hypersensitivity (DTH) reactions. Yet, TNBS-induced colitis can be cured only by anti-CD44v7. To clarify the mechanisms underlying the divergent functional activities of CD44v6 and CD44v7 we explored their contribution to lymphocyte activation in vivo and in vitro. CD44v6 and CD44v7 are distinctly expressed on subpopulations of activated lymphocytes. Expression of CD44v6 is mainly restricted to T cell blasts. CD44v7 has been detected on CD4(+) cells, B cells and monocytes. Mitogenic and antigenic stimulation of lymphocytes in vitro was impaired in the presence of anti-CD44v6 and anti-CD44v7. Accordingly, anti-CD44v6 and anti-CD44v7 mitigated the DTH reaction in 2,4-dinitro-1-fluorobenzene-sensitized and challenged mice. However, the seemingly similar effects of CD44v6- and CD44v7-specific antibodies resulted from different activities. Anti-CD44v6 treatment led to a down-regulation of IL-2 and IFN-gamma production predominantly by CD8(+) cells. In anti-CD44v7-treated mice expression of IL-12 was decreased. Elevated levels of IL-10 accompanied this reduction. The latter resulted from an anti-CD44v7-mediated blockade of interactions between CD4(+) cells and monocytes as well as an active triggering of B cells. Thus, anti-CD44v6 and anti-CD44v7 interfere with lymphocyte activation at very specific points. CD44v6 functions predominantly at the T cell level. CD44v7 influences production of proinflammatory cytokines by B cells as well as an interaction between CD4(+) cells and antigen-presenting cells. As CD44 isoforms do not differ in their intracytoplasmatic tail, the distinct activities must result from expression on different leukocyte subsets and interactions with distinct ligands.     

Mouse B Cell Activation is Inhibited by CD44 Cross-Linking

Lymphocyte activation and trafficking are indispensable to the immune system. CD44 is an adhesion molecule with known importance in T cell activation, lymphocyte trafficking, and tumor metastasis. Although CD44 has been shown to participate in the activation, rolling and adhesion, and homing of T cells, the role of CD44 on B cells is relatively unknown. The effects of CD44 cross-linking on murine B cell activation via CD40L was explored using the anti-CD44 mAbs RK3G9 and IM7. When immobilized on a plate, both RK3G9 and IM7 were found to strongly inhibit B cell proliferation and Ig production, especially at lower cell input concentrations. IgE inhibition was especially prominent. In contrast, soluble RK3G9 added to the B cell cultures had no effect. The inhibitory effect of anti-CD44 on B cell activation was not influenced by the addition of the anti-FcγRII, indicating that Fc cross-linking did not play a role in this inhibition. As Ig production requires several days for both B cell proliferation and differentiation to occur, the effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. Finally, B cells were activated by either LPS or anti-IgM F(ab′)₂. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. These results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.     

Role of monocytes in anti-CD3-induced T-cell DNA synthesis: Effect of chloroquine and monensin on anti-CD3-induced human T-cell activation

Anti-CD3 monoclonal antibody (MoAb) induces proliferation of freshly isolated peripheral blood T cells only in the presence of monocytes/macrophages and requires binding of the Fc portion of antibody to monocytes/macrophages. In this investigation, we examined whether monocytes process anti-CD3 similar to any soluble antigen and present to T cells in context with HLA-DR to induce maximal DNA synthesis. Adherent monocytes were pulsed with anti-CD3 MoAb in the presence or absence of the lysozomotropic agents chloroquine and monensin, which are known to inhibit processing of soluble antigens, washed extensively, and then incubated with autologous T cells in the absence of soluble anti-CD3, and³H-thymidine incorporation and CD25 expression were measured. Both monensin and chloroquine inhibited anti-CD3-pulsed monocyte-induced T-cell DNA synthesis and CD25 expression in a dose-dependent manner. This inhibitory effect was not due to any loss in cell viability or the effect on the expression of HLA-DR on monocytes. Paraformaldehyde-fixed monocytes pulsed with anti-CD3 MoAb induced significantly less DNA synthesis, HLA-DR expression, and CD25 antigen expression on autologous T cells as compared to responses induced by unfixed anti-CD3-pulsed monocytes. The treatment of anti-CD3-pulsed monocytes with frame-work-specific anti-HLA-DR MoAb inhibited their capacity to induce T-cell DNA synthesis. These data suggest that monocytes, in addition to serving as the matrix for cross-linking, also process anti-CD3 MoAb and present to the T cells in the context of HLA-DR antigens to induce optimal DNA synthesis.