CD20-specific Monoclonal Antibody Research Articles

In vivo administration of CD4-specific monoclonal antibody: effect on provirus load in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques.

Since monoclonal antibodies (MAb) specific for CD4 are potent inhibitors of HIV and SIV replication in vitro, we explored their potential usefulness in vivo as an AIDS therapy. The anti-CD4 MAb 5A8 binds to domain 2 of the CD4 molecule and inhibits virus replication and virus-induced cell fusion at a postvirus binding step. Administration of this MAb to normal rhesus monkeys coats all circulating and lymph node CD4 cells and induces neither CD4 cell clearance nor measurable immunosuppression. In the present study, monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac) had stable levels of SIVmac provirus in PBMC prior to treatment as measured by a quantitative polymerase chain reaction technique. Six infected monkeys treated with anti-CD4 MAb demonstrated a significant decrease in SIVmac provirus level after 9 days. Of these monkeys, 3 had > 800 CD4 cells/microliter and developed strong antimouse Ig responses that prevented further treatment. The remaining 3 monkeys had < 800 CD4 cell/microliter and failed to develop antimouse Ig antibody responses. When treatment was continued for 12-21 days in these monkeys, a sustained or further decrease in SIVmac provirus load occurred over the extended treatment period. Four monkeys that received a control MAb of irrelevant specificity for 9-22 days showed either no significant change or a transient increase in SIVmac provirus. Thus, the passive administration of anti-CD4 MAb may exert a specific antiviral effect in controlling immunodeficiency virus infection in vivo.

Specific and non-specific T-cell activation in high and low antibody-producing mice (selection IV-A).

The T-cell regulatory function has been evaluated in lines of mice genetically selected according to the High (H) and Low (L) antibody responsiveness to heterologous erythrocytes. The inhibition of antibody production following CD4+ subset depletion was stronger in L than in H mice. The dose of CD4-specific monoclonal antibody (MoAb) needed for a 50% inhibition of the anti-sheep erythrocyte antibody response was much lower in L mice, even when their responsiveness was improved by repeated antigen injections or by co-injection of lipopolysaccharide as an adjuvant. Lymph-node cells from L mice responded normally or even better than those from H mice to in vitro non-specific triggering via lectins, exogenous superantigens or CD3-specific MoAb. In contrast, the antigen-specific responses of the lymph-node cells from protein-primed mice were consistently lower in the Low responder mice. Altogether the results suggest that a defective stimulation of T helper lymphocytes by the antigen contributes to the weak antibody response of L mice.

A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex-encoded class II molecules.

CD4, a cell-surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus-1 (HIV-1). Screening of microbial metabolites for CD4-binding activity using an enzyme-linked immunosorbent assay based on the binding of the CD4-specific monoclonal antibody (mAb), anti-Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti-Leu 3a and that of several other D1/D2 epitope-specific mAb with CD4, but only weakly inhibited the binding of HIV-1 gp120. Binding of a representative MHC class II molecule, HLA-DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 microM). In functional assays 411F inhibited antigen-induced CD4-dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide-induced CD4+ T cell proliferative responses but not alloantigen-induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4-dependent functions by binding over a broad area of the positively charged amino-terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.

Distinct roles for CD4 and CD8 as co‐receptors in T cell receptor signalling

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCR alpha beta) in distinct fashions. Pretreatment of CD4+ lymph node T cells with CD4-specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti-TCR alpha beta. In contrast, pretreatment of CD8+ T cells with CD8-specific mAb has no effect on DNA synthesis subsequently induced through TCR alpha beta. While inhibiting late activation signals, pretreatment with anti-CD4 does not detectably alter the pattern of anti-TCR alpha beta-induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca2+ mobilization. The distinct biological consequences of anti-CD4 and anti-CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56lck. While both T cell lineages contain similar levels of cellular p56lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti-CD4 and anti-CD8 on the distribution and activity of p56lck. Further, antibody-mediated aggregation of TCR alpha beta on CD4+ T cells induces the appearance of a p56lck species with decreased mobility in sodium dodecylsulfate-polyacrylamide gel electrophoresis. This effect is observed in CD4+ T cells exclusively and involves the fraction of p56lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger-generating systems are under distinct physical and/or functional constraints in the two T cell lineages.

Encephalitogenic Th1 cells are inhibited by Th2 cells with related peptide specificity: Relative roles of interleukin (IL)-4 and IL-10

Cytokines secreted by T-helper type 2 (Th2) cells inhibit the antigen-induced stimulation of type 1 (Th1) helper T cells. To study this form of regulation in an autoimmune disease model, the cytokines secreted by a Th2 clone specific for the encephalitogenic proteolipid protein (PLP) peptide 139–151 were tested for their ability to inhibit proliferation of an encephalitogenic Th1 clone specific for an epitope contained within the same peptide. Cytokines, produced by stimulation of the Th2 clone with CD3-specific monoclonal antibodies (mAbs), inhibited proliferation of the Th1 clone when stimulated by antigen and splenic antigen-presenting cells (APC). Inhibition was, however, not antigen-specific since cytokines released upon stimulation of an unrelated Th2 clone were also inhibitory. Inhibition was found to be caused by effects on either antigen presentation or co-stimulatory activity of the APC and not by direct effects on the Th1 cells. MAbs for the two major regulatory Th2 cytokines were used to identify the inhibitory component secreted by activated Th2 cells. Interleukin-10 (IL-10)-specific mAb abolished the inhibitory effect, while mAb specific for IL-4 had no effect on inhibition. The addition of recombinant IL-4 (rIL-4) and rIL-10 confirmed that inhibition of Th1 proliferation was due to secretion of Il-10 by the Th2 clone and its subsequent effects on APC. The studies described here demonstrate that PLP-specific Th2 cells which recognize peptide 139–151 inhibit encephalitogenic Th1 cells which respond to an epitope on the same peptide. This phenomenon may be important for local, antigen-specific regulation of inflammation in the central nervous system.

Cytotoxic T lymphocyte lines specific for human immunodeficiency virus type 1 Gag and reverse transcriptase derived from a vertically infected child.

Cytotoxic T lymphocytes (CTL) specific for human immunodeficiency virus type 1 (HIV-1) are thought to play an important role in controlling HIV-1 infection. HIV-1-specific CTL are readily demonstrated in unstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected adults but less frequently in PBMC from vertically infected children. HIV-1-specific CTL lines were derived from a long-term survivor of vertical HIV-1 infection using PBMC stimulated with a CD3-specific monoclonal antibody and interleukin-2; these lines had Gag- or reverse transcriptase (RT)-specific cytotoxicity. Cytotoxicity was restricted by major histocompatibility complex class I antigen and blocked by antibody to the T cell receptor complex. Fluorescence-activated cell sorting analysis demonstrated their phenotype to be CD3+CD4-CD8+. Unstimulated PBMC from this patient had no detectable HIV-1-specific cytotoxicity when tested against autologous HIV-1 envelope-, Gag-, or RT-expressing target cells. Thus, this child with vertically acquired HIV-1 infection likely has HIV-1-specific CTL precursors despite the absence of circulating, activated HIV-1-specific CTL.

In VivoAdministration to Rhesus Monkeys of a CD4-Specific Monoclonal Antibody Capable of Blocking AIDS Virus Replication

Monoclonal antibodies (mAbs) specific for CD4 are potent inhibitors of HIV replication in vitro. These agents may be useful prophylactically or in chronic HIV infection if they can be administered without inducing immunosuppression. In the present study, we explored the safety of a CD4-specific murine mAb in rhesus monkeys. The mAb 5A8, which binds to domain 2 of the CD4 molecule, inhibits AIDS virus replication noncompetitively at a postvirus binding step. This antibody, which had a similar affinity for rhesus monkey and human CD4 cells, efficiently inhibited in vitro replication of both HIV-1 and the simian immunodeficiency virus of macaques. A single 3-mg/kg injection of mAb 5A8 into normal rhesus monkeys coated all circulating and lymph node CD4 cells for 4-6 days. CD4 cells were not cleared from circulation nor was the CD4 molecule modulated from the lymphocyte surface. In fact, administration of mAb 5A8 resulted in an approximately one-to twofold increase in absolute number of circulating CD4 cells. Repeated administration in normal rhesus monkeys resulted in CD4 lymphocyte coating with mAbs for > 9 days without CD4 cell clearance or modulation. While coated with mAbs, PBLs of these monkeys retained normal in vitro proliferative responses to mitogens and these animals generated normal humoral responses in vivo to tetanus toxoid. Loss of cell coating with mAbs in normal monkeys corresponded to the appearance of anti-mouse immunoglobulin antibodies. Thus, administration of certain anti-CD4 mAbs capable of blocking HIV replication can achieve coating of the entire CD4 cell pool in rhesus monkeys without inducing significant cell loss or immunosuppression.

Recognition of a highly conserved region of human immunodeficiency virus type 1 gp120 by an HLA-Cw4-restricted cytotoxic T-lymphocyte clone

Human immunodeficiency virus type 1 (HIV-1) isolates exhibit extensive sequence variation, particularly in the gp120 subunit of the envelope glycoprotein, and the degree of this variation has raised questions as to whether conserved regions of the HIV-1 envelope can be recognized by the host immune response. A CD8+ cytotoxic T-lymphocyte (CTL) clone specific for the HIV-1 envelope was derived by culturing peripheral blood mononuclear cells from an HIV-1 seropositive subject in the presence of a CD3-specific monoclonal antibody, interleukin-2, and irradiated allogeneic peripheral blood mononuclear cells. Lysis of target cells was restricted by an HLA-C molecule, Cw4, which has not been previously shown to present viral antigen to CTL. Mapping of the specificity of this CTL clone by using synthetic HIV-1 peptides localized the epitope to an 8-amino-acid region of gp120 (amino acids 376 to 383) which is conserved among approximately 90% of sequenced viral isolates. Examination of the recognition of variant peptides by this CTL clone demonstrated that a single, nonconservative amino acid substitution within the 8-amino-acid minimal epitope could abrogate lysis of targets incubated with the variant peptide. The identification of a CTL epitope in a highly conserved region of gp120 documents the ability of cellular immune responses of infected persons to respond to relatively invariant portions of this highly variable envelope glycoprotein. However, the ability of even a single-amino-acid change in gp120 to abolish lysis by CTL supports the hypothesis that sequence variation in HIV-1 may serve as a mechanism of immune escape. In addition, the identification of an HLA-C molecule presenting viral antigen to CTL supports a functional role for these molecules.

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.

Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and CD8+ T cells.

The role of CD4+ and CD8+ T cells in mediating pulmonary clearance of a cryptococcal infection was investigated. Intratracheal inoculation of BALB/c and C.B-17 mice with a moderately virulent strain of Cryptococcus neoformans (52D) resulted in a pulmonary infection, which was cleared by a T cell-dependent mechanism. During this clearance, there was a significant influx of both CD4+ and CD8+ T cells into the lungs. Depletion of CD4+ T cells by injections of CD4-specific monoclonal antibody (mAb) prevented pulmonary clearance and also resulted in significant colonization of the brain and spleen of infected mice. CD4 depletion did not prevent the influx of CD8+ T cells into the lungs. Surprisingly, depletion of CD8+ T cells by mAb also ablated pulmonary clearance. CD8-depleted mice also had a small but significant increase in brain and spleen colony-forming unit compared to control mice by the end of the study. CD4+ T cell pulmonary influx was independent of the presence of CD8+ T cells. The lungs of T cell-depleted mice were examined histologically. CD4+ and CD8+ T cells each mediated a degree of inflammatory influx seen in the lungs of infected mice and raised the possibility that CD4+ and CD8+ T cells may synergize to generate the inflammatory response in the lungs. Numerous phagocytized but intact cryptococci were seen in the inflammatory foci of CD8-depleted mice but not in control or CD4-depleted mice. We propose that CD4+ T cells may recruit and activate effector phagocytes while CD8+ T cells predominantly function to lyse cryptococcus-laden unactivated phagocytes similar to the function of CD8+ T cells during listeria and mycobacteria infections.

Identification of cross-reactive T cell restriction epitopes located on the DR7β1 and DRβ4 molecules

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DRα:DR7β1 and DRα:DRβ4 (DRw53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cel clones. One clone is restricted by the DR7β1 molecule, another clone by the DRβ4(DRw53) molecule, and a third clone by a cross-reactive T cell epitope on DR7β1 and DRβ4(DRw53) molecules. The two DRβ4(DRw53)-restricted clones react, however, differently with a panel of HLA-DR DRw53-positive human peripheral blood lympocytes used as APC. Therefore the data presented here clearly document that the DRβ4 (DRw53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DRβ loci and in addition emphasize the high complexity of the supertypic HLA-DRw53 specificity.

Long-term culture and fine specificity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1.

The definition of human immunodeficiency virus type 1 (HIV-1) immunogenic epitopes is central to the rational design of AIDS vaccine strategies. In this study, we have generated seven HIV-1 reverse transcriptase-specific cytotoxic T-lymphocyte (CTL) clones from the peripheral blood of two seropositive subjects. Epitopes recognized by these CTL clones were identified by using target cells infected with recombinant HIV-1-vaccinia virus vectors expressing truncated reverse transcriptase proteins and further defined by using target cells incubated with overlapping 25-amino acid synthetic reverse transcriptase peptides. Five different CTL epitopes were identified, and in each case recognition was restricted by class I human leukocyte antigens (HLA). Clones maintained specific cytolytic function in continuous culture for up to 11 months, requiring only periodic restimulation with a CD3-specific monoclonal antibody. These results indicate that HIV-1-specific, major histocompatibility class I-restricted CTL recognize multiple epitopes of a single viral gene product in conjunction with different host HLA antigens. In addition, they demonstrate that human virus-specific CTL can be grown in long-term culture without the need for reexposure to viral antigen.

Impaired Generation of Anti‐Viral Cytotoxicity against Lymphocytic Choriomeningitis and Vaccinia Virus in Mice Treated with CD4‐Specific Monoclonal Antibody

The role of CD4+ helper T cells in induction of anti-viral cytotoxic T-cell response was investigated by treating normal and thymectomized C57B1/6 mice with CD4-specific monoclonal antibodies (MoAb). In CD4-specific MoAb-treated mice infected with Vaccinia or lymphocytic choriomeningitis virus (LCMV), cytotoxic T-cell activity was 5-15 times lower than in normal controls when measured in a 51Cr release assay and computed as lytic units 6 and 8 days respectively after virus inoculation. This difference in the levels of effector T-cell activities did not reflect slower kinetics of cytotoxic T-cell induction in antibody-treated versus control mice, since it was also obvious at 8 days after infection for Vaccinia virus and 10 and 12 days after inoculation with LCMV. CD4-specific MoAb-induced inhibition of cytotoxic T-cell responses in vivo was seen up to 150 days after treatment in thymectomized mice. However, no significant suppressive effect of the same antibody treatment on T-cell cytotoxicity could be observed in animals treated on day 3 or later after infection with Vaccinia virus. Injection of CD4-depleted mice with recombinant interleukin 2 (rIL-2) partially corrected the impaired virus-specific cytotoxic T-cell response, suggesting that IL-2 supply may be limiting in mice lacking T helper cells.

HIV-Related Lentiviruses of Nonhuman Primates

The purpose of this review is to summarize the current status of known natural infections with SIV, the pathogenic potential of SIV in natural and unnaturual hosts, the genetic diversity of SIV, and possible monkey origins for HIV. In addition, we discuss the importance of SIV for studying the pathogenesis of AIDS and strategies for therapeutic and vaccine intervention

In vitro effect of dynorphin on hcg production and release by trophoblast tissue explants

Some CD28-specific monoclonal antibodies (mAbs) have the surprising property to activate T-cells without the need for TCR ligation. This review summarizes the differences between these “superagonistic” and conventional CD28-specific mAbs with regard to binding specificity and signalling properties. Futhermore, the dramatic effects of in vivo application of CD28 superagonists with regard to the induction of regulatory T-cells and polyclonal T-cell expansion in lymphopenic settings are discussed under the aspect of potential therapeutic applications.

Increased expression of CD4 molecules on Jurkat cells mediated by human immunodeficiency virus tat protein.

The tat gene of the human immunodeficiency virus, tat-III, when introduced into T-lymphoblastoid Jurkat cells by a Moloney retroviral recombinant DNA vector expressed high levels of the functional tat protein as measured by the chloramphenicol acetyltransferase assay. Immunofluorescence analysis with CD4-specific monoclonal antibodies demonstrated that the cell surface levels of the CD4 antigen were increased by 5- to 10-fold in the tat-III-infected Jurkat cells. Cellular cytoplasmic RNA analysis indicated that the enhanced CD4 expression was mediated at the mRNA level. Our findings suggest that the single expression of the human immunodeficiency virus tat protein in the absence of the other viral proteins causes an upregulation of CD4 gene expression on helper T cells, although infection of these cells by the virus, thus expressing all the viral gene products including tat, is known to downregulate CD4 antigen expression.

Location and Chemical Synthesis of a Binding Site for HIV-1 on the CD4 Protein

The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus.

HIV-specific cytotoxic T lymphocytes in seropositive individuals.

Virus-specific cytotoxic T lymphocytes (CTL) which kill virus-infected cells are thought to be a major host defence against viral infections. Here we report the existence of human immunodeficiency virus (HIV)-specific CTL in persons infected with this virus, the aetiological agent of AIDS (acquired immunodeficiency syndrome). Recombinant HIV-vaccinia viruses were used to express HIV antigens in B-cell lines established from subjects seropositive for HIV and seronegative controls. Circulating lymphocytes capable of killing HIV env-expressing autologous B cells were detected in eight of eight seropositive subjects; in addition, at least three seropositive subjects demonstrated gag-specific cytotoxic responses. No HIV-specific cytotoxicity was observed in seronegative subjects. Selective inhibition of the env-specific cytotoxicity by a CD3-specific monoclonal antibody indicates that the effectors are T cells. This demonstration of a cytotoxic T-cell immune response to HIV in infected individuals should prove useful in investigating the immunopathogenesis of HIV infection further and in evaluating AIDS vaccine strategies.