Gary S. Gray

B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4

The functional necessity for two CD28 counterreceptors (B7-1 and B7-2) is presently unknown. B7-1 and B7-2 equivalently costimulate IL-2 and interferon-gamma (IFN gamma) production and IL-2 receptor alpha and gamma chain expression. B7-2 induces significantly more IL-4 production than B7-1, with the greatest difference seen in naive T cells. Repetitive costimulation of CD4+ CD45RA+ T cells with B7-2 results in moderate levels of both IL-4 and IL-2, whereas repetitive costimulation with B7-1 results in high levels of IL-2 and low levels of IL-4. Therefore, B7-1 and B7-2 costimulation mediate distinct outcomes, since B7-2 provides an initial signal to induce naive T cells to become IL-4 producers, thereby directing the immune response more towards Th0/Th2, whereas B7-1 is a more neutral differentiative signal.

Absence of B7-dependent responses in CD28-deficient mice

Costimulation of T cell proliferation can occur through the CD28 signal transduction pathway. In addition, other cell surface receptors, including the CD28 homolog CTLA-4, have been proposed to be capable of providing costimulatory signals. We have examined the response of CD28-deficient T cells to activation by a variety of agonists. We demonstrate that proliferation of CD28-deficient T cells in the presence of antigen-presenting cells or B7-1 transfectants is markedly reduced. Although CTLA-4 can be expressed on CD28-deficient T cells, we observed no B7-dependent costimulation in the absence of CD28. This data demonstrates that CD28 is the major B7-binding costimulatory ligand on T cells. Furthermore, our data suggest that CD28 is the primary, and perhaps exclusive, costimulatory receptor used by traditional antigen-presenting cells to augment the proliferation of antigen-activated T cells.

Inhibition Of Transplant Rejection Following Treatment With Anti-b7-2 And Anti-b7-1 Antibodies

Antigen-specific T cell activation depends initially on the interaction of the T cell receptor (TCR) with peptide/MHC. In addition, a costimulatory signal, mediated by distinct cell surface accessory molecules, is required for complete T cell activation leading to lymphokine production and proliferation. CD28 has been implicated as the major receptor on T cells responsible for delivering the costimulatory signal. Although two distinct ligands for CD28, B7-1 and B7-2, have been identified on antigen-presenting cells (APC), the co-stimulatory role of each molecule during a physiological immune response remains unresolved. In the present study, the relative roles of B7-1 and B7-2 interactions were evaluated in an allogeneic pancreatic islet transplant setting. In isolation, anti-B7-2 mAbs and, to a much lesser degree, anti-B7-1 mAbs suppressed T cell proliferative responses to allogeneic islets or splenic APC in vitro. Maximal inhibition of the allogeneic response was observed using a combination of the anti-B7-1 and anti-B7-2 mAbs. Administration of anti-B7-2 but not anti-B7-1 mAbs prolonged C3H allograft survival in B6 recipients, with a combination of both mAbs significantly prolonging rejection beyond either mAb alone. The immunosuppressive effects of the in vivo mAb treatment were not manifested in in vitro analyses as T cells isolated from suppressed mice responded normally to allogeneic stimuli in terms of both proliferation and lymphokine production. However, combined mAb therapy in vivo selectively delayed CD4+ T lymphocyte infiltration into the graft. These data suggest that both B7-1 and B7-2 costimulatory molecules are active in vivo, although B7-2 plays a clearly dominant role in this allograft model. The mechanism of immune suppression in vivo remains unresolved but may occur at sites distinct from the allograft.

Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis.

The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.

Induction Therapy With Monoclonal Antibodies Specific For Cd80 And Cd86 Delays The Onset Of Acute Renal Allograft Rejection In Non-human Primates1

CD80 and CD86 (also known as B7–1 and B7–2, respectively) are both ligands for the T cell costimulatory receptors CD28 and CD152. Both CD80 and CD86 mediate T cell costimulation, and as such, have been studied for their role in promoting allograft rejection. In this study we demonstrate that administering monoclonal antibodies specific for these B7 ligands can delay the onset of acute renal allograft rejection in rhesus monkeys. The most durable effect results from simultaneous administration of both anti-B7 antibodies. The mechanism of action does not involve global depletion of T or B cells. Despite in vitro and in vivo evidence demonstrating the effectiveness of the anti-B7 antibodies in suppressing T cell responsiveness to alloantigen, their use does not result in durable tolerance. Prolonged therapy with murine anti-B7 antibodies is limited by the development of neutralizing antibodies, but that problem was avoided when humanized anti-B7 reagents are used. Most animals develop rejection and an alloantibody response although still on antibody therapy and before the development of a neutralizing antibody response. Anti-B7 antibody therapy may have use as an adjunctive agent for clinical allotransplantation, but using the dosing regimens we used, is not a tolerizing therapy in this non-human primate model.

Envelope gene sequence of HTLV-1 isolate MT-2 and its comparison with other HTLV-1 isolates

The DNA sequence of the envelope gene of the HTLV-1 proviral clone MT-2 was determined and compared with envelope sequences of other HTLV-1 isolates. The comparison of this sequence with another of Japanese origin and two of Caribbean origin does not support the proposal by K.T.A. Malik, J. Even, and A. Karpas (J. Gen. Virol. 69, 1695-1710, 1988) that isolates of similar geographic origins are more homologous than isolates of different origins. No significant differences in the degree of homology could be found among the HTLV-1 envelope gene sequences from Japanese and Caribbean isolates. The comparison confirms the high degree of homology previously found between various HTLV-1 isolates (greater than 97%).

Opposing effects of CTLA4-Ig and anti-CD80 (B7-1) plus anti-CD86 (B7-2) on experimental allergic encephalomyelitis.

The roles of the B7 receptors, CD80 and CD86, during actively induced experimental allergic encephalomyelitis were examined with specific monoclonal antibodies and CTLA4-Ig. Injection of CTLA4-Ig on day 2 post-immunization resulted in decreased incidence and severity of resultant disease. Anti-CD80 injection on day 2 blocked development of the first disease episode. Subsequent relapses were unaffected. In contrast, injection of anti-CD86 alone had no effect. Surprisingly, combined anti-CD80 + anti-CD86 monoclonal antibody injection on day 2 resulted in marked exacerbation of disease. Examination of cytokine production in the draining lymph node cells demonstrated a reduction in both interferon (IFN)-gamma and interleukin (IL)-2 producing cells, but a dramatic increase in tumor necrosis factor (TNF)-alpha secretion in animals receiving both monoclonal antibodies. These results suggest distinct roles for CD80 and CD86 in the initiation of EAE, resulting in the diverse clinical outcomes observed in this model of EAE.

Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD154 in nonhuman primate renal transplantation.

Background. Antibodies and fusion proteins specific for CD80, CD86, and CD154 have shown promise as agents capable of inducing donor-specific tolerance in rodents. These agents have also been shown to be synergistic with one another in many settings of counter-adaptive immunity. In the nonhuman primate, monoclonal antibodies specific for CD80 and CD86 have prolonged the time to rejection of renal allografts but have not resulted in tolerance. A monoclonal antibody specific for CD154 has resulted in markedly prolonged survival of kidney, islet, cardiac, and skin allografts, but again most animals have eventually developed rejection after prolonged periods of rejection-free survival off therapy. Methods. A combination of monoclonal antibodies specific for CD80, CD86, and CD154 were used in a mismatched nonhuman primate renal-allograft model. Doses used were based on optimized treatment protocols for each agent individually. Results. Treatment of four rhesus macaques with this combination yielded a mean rejection-free survival of 565 days (311–911 days), significantly greater than untreated controls (mean survival=7.0 days, P =0.001) and animals treated with only a combination of anti-CD80 and CD86 (mean survival=191 days, P =0.01). The survival of animals treated with this combination of monoclonal antibodies was not significantly greater than those treated with anti-CD154 alone, but the production of alloantibody was delayed compared with monotherapy anti-CD154. Conclusion. These data suggest that a synergy exists between these agents, particularly with regard to T-dependent B-cell responses, but that they fail to induce durable tolerance in nonhuman primates.

Induction, maintenance, and reversal of streptozotocin-induced insulin-dependent diabetes mellitus in the juvenile cynomolgus monkey (Macaca fascilularis)

BACKGROUND Insulin-dependent diabetes mellitus (IDDM) is the second most prevalent chronic illness of children. Investigation of the treatment of IDDM is hindered by the lack of a reproducible and easily maintained non-human primate model of this disorder. METHODS We induced IDDM in 11 juvenile cynomolgus monkeys after a single (150 mg/kg) intravenous injection of streptozotocin (STZ). All diabetic monkeys were treated with insulin twice daily, based on a sliding scale. Subcutaneous vascular access ports were surgically placed in each monkey to facilitate serial blood sampling and drug administration. Allogeneic pancreatic islet cells from unrelated donors were subsequently transplanted into the mesenteric circulation of all STZ-treated monkeys. RESULTS Mild, transient nausea and vomiting occurred in all animals after STZ injection; however, no additional signs of toxicity occurred. Within 36 hr, all monkeys required twice daily administration of exogenous insulin to maintain a non-ketotic state. Serum C-peptide levels decreased from >1.2 ng/ml before STZ, to between 0.0 and 0.9 ng/ml after STZ, confirming islet cell destruction. Animals were maintained in an insulin-dependent state for up to 147 days without any observable clinical complications. Subcutaneous vascular access port patency was maintained up to 136 days with a single incidence of local infection. Islet cell transplantation resulted in normoglycemia within 24 hr. Serum C-peptide levels increased (range: 2-8 ng/ml) for 6 - 8 days in immune competent animals, and for 39-98 days after transplant in immunosuppressed monkeys. CONCLUSIONS IDDM can be consistently induced and safely treated in juvenile cynomolgus monkeys. Chronic vascular access can be maintained with minimal supervision and complications. This model is appropriate for studies investigating potential treatments for IDDM including islet cell transplantation.

Defining critical residues m the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies

A peptide display library [Scott and Smith, Science 249 (1990) 386-390] was constructed that expressed 1.5 x 10(8) unique 20-amino-acid (aa) peptides fused to the N-terminus of the pIII coat protein of filamentous phage fd. This phage display library (PDL-20) was prepared using a degenerate oligodeoxyribonucleotide designed to minimize bias towards most aa. Characterization of the PDL-20 showed that all aa were present at the expected frequency and that there was no positional bias. Screening of this library with a HIV-1 isotype MN envelope reactive monoclonal antibody (mAb 58.2) using two different panning procedures showed that the biopanning technique was sensitive to one phage in 10(8). Analysis of peptide sequences from panning the mAb identified a core antibody recognition sequence of four aa residues (GPGR) and two preferred flanking residues on either side. This epitope occurred at various locations within the random aa segment demonstrating an absence of positional or nearest neighbor effects. Parallel panning experiments using an array of 266 synthetic peptides identified an epitope similar to that defined by the phage display library.