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L C Burkly

Publications and source records attributed to L C Burkly.

At least 19 recordsLinked to original sources
PubMed2001

Human platelets activate porcine endothelial cells through a CD154-dependent pathway.

BACKGROUND: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40. METHODS: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E. RESULTS: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner. CONCLUSION: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.

Animals↗
PubMed2001

Treatment with the humanized CD154-specific monoclonal antibody, hu5C8, prevents acute rejection of primary skin allografts in nonhuman primates.

BACKGROUND: Allogeneic skin transplantation remains a rigorous test of any immune intervention designed to prevent allograft rejection. To date, no single, clinically available immunosuppressant has been reported to induce long-term primary skin allograft survival in primates. We have previously shown that treatment with the humanized CD154-specific monoclonal antibody, humanized 5C8 (hu5C8), induces long-term renal allograft survival in nonhuman primates. In this study, we evaluated the efficacy of hu5C8 in preventing primary skin allograft rejection in rhesus monkeys. METHODS: Ten rhesus monkeys were transplanted with full-thickness skin allografts mismatched at both class I and class II major histocompatibility loci. Of these, two were given no treatment, five were treated with hu5C8 alone, and three received hu5C8 combined with whole blood donor-specific transfusion (DST). All recipients also received skin autografts for comparison. Animals were followed by inspection, serial biopsy, mixed lymphocyte culture, and alloantibody determination. RESULTS: Treatment with hu5C8 alone or hu5C8 plus DST greatly prolonged allograft survival. Rejection occurred in the untreated group within 7 days. Mean allograft survival in the monotherapy hu5C8 group was >236 days and in the DST group was >202 days; these differences were not significant. Rejection eventually occurred in most animals. Allograft survival was not correlated with the development of T cell hyporesponsiveness in mixed lymphocyte culture. Rejection was not predicted by the development of donor-specific alloantibody. CONCLUSION: These results show that treatment with the CD154-specific monoclonal antibody, hu5C8, greatly delays the onset of acute skin allograft rejection.

Acute Disease↗
PubMed2001

Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients.

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

Animals↗
PubMed2001

Apoptosis and altered dendritic cell homeostasis in lupus nephritis are limited by anti-CD154 treatment.

Autoimmunity results from a failure in central and/or peripheral tolerance; however, the events that initiate and maintain this dysfunction remain unclear. To better understand the mediators involved in autoimmunity, we investigated the cellular mechanisms maintaining disease in the (SWR x NZB)F(1) (SNF(1)) mouse model of systemic lupus erythematosus. Previously, we have shown that autoimmunity in this model is dependent on CD40-CD154 interactions. Herein, our studies reveal that the severity of disease in SNF(1) mice correlates with a marked increase in the frequency of apoptotic splenocytes, including a higher proportion of apoptotic dendritic cells (DC) in vivo. In addition, we demonstrate a significant disease-related increase in the absolute number of splenic CD11c(high) DC. The increased DC number appears to be attributable to DC proliferation and enhanced migration to the spleen, most likely induced by elevated splenic expression of secondary lymphoid chemokine. Importantly, these imbalances in apoptosis, secondary lymphoid chemokine expression, and DC homeostasis were reduced or normalized by anti-CD154 treatment. Thus, our data demonstrate CD154-dependent regulation of apoptosis and DC homeostasis in mice with lupus-like autoimmune disease. We suggest that these mechanisms comprise an autostimulatory loop, maintaining the cascade of autoimmunity by DC presentation of self-Ags derived from apoptotic cells and CD154-mediated costimulation.

Animals↗
PubMed2001

Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell costimulation.

Allorejection and recurrence of autoimmunity are the major barriers to transplantation of islets of Langerhans for the cure of type 1 diabetes in humans. CD40-CD154 (CD40 ligand) interaction blockade by the use of anti-CD154 monoclonal antibody (mAb) has shown efficacy in preventing allorejection in several models of organ and cell transplantation. Here we report the beneficial effect of the chronic administration of a hamster anti-murine CD154 mAb, MR1, in prolonging islet graft survival in NOD mice. We explored the transplantation of C57BL/6 islets into spontaneously diabetic NOD mice, a combination in which both allogeneic and autoimmune components are implicated in graft loss. Recipients were treated either with an irrelevant control antibody or with MR1. MR1 administration was effective in prolonging allograft survival, but did not provide permanent protection from diabetes recurrence. The autoimmune component of graft loss was studied in spontaneously diabetic NOD mice that received syngeneic islets from young male NOD mice. In this combination, a less dramatic yet substantial delay in diabetes recurrence was observed in the MR1-treated recipients when compared with the control group. Finally, the allogeneic component was explored by transplanting C57BL/6 islets into chemically induced diabetic male NOD mice. In this setting, long-term graft survival (>100 days) was achieved in MR1-treated mice, whereas control recipients rejected their grafts within 25 days. In conclusion, chronic blockade of CD154 results in permanent protection from allorejection and significantly delays recurrence of diabetes in NOD mice.

Animals↗
PubMed2001

Characterization of IL-4 receptor components expressed on monocytes and monocyte-derived macrophages: variation associated with differential signaling by IL-4.

The anti-inflammatory effects of IL-4 on activated monocytes differ from those on monocyte-derived macrophages (MDMac). While IL-4 suppresses LPS-induced IL-1beta , IL-12, IL-10 and TNFalpha production by monocytes, IL-4 suppresses only IL-1beta and IL-12 production by MDMac. The U937 and Mono Mac 6 cell lines have similar cytokine responses to IL-4 as monocytes and MDMac, respectively. The IL-4Ralpha and IL-2Rgamma (gammac) chains are well-characterized components of the IL-4 receptor. Cross-linking studies with 125I-IL-4 revealed that for monocytes and U937 cells, the binding of IL-4 to the receptor components was approximately 1:1 for IL-4Ralpha:gammac. In contrast, for MDMac and Mono Mac 6 cells that have a relative reduction in gammac surface expression, the binding of IL-4 to IL-4Ralpha:gammac was approximately 3:1. Furthermore, IL-4 induced IL-4Ralpha chain phosphorylation more rapidly in MDMac and Mono Mac 6 cells than in monocytes and U937 cells. This study identifies a correlation between altered 125I-IL-4 cross-linking to IL-4Ralpha:gammac, IL-4-induced signaling and regulation of pro-inflammatory cytokine production by IL-4.

Blotting, Western↗
PubMed2000

Costimulatory molecules are active in the human xenoreactive T-cell response but not in natural killer-mediated cytotoxicity.

BACKGROUND: T-cell costimulatory blocking agents inhibit allospecific T-cell responses in vitro and prevent allograft rejection in vivo. Costimulatory requirements for discordant xenospecific cellular responses remain undefined. We have evaluated costimulatory molecule expression by porcine endothelial cells (PEC) after interaction with human cells and tested agents known to inhibit allospecific responses for their ability to inhibit xenospecific responses in vitro. METHODS: Human-specific agents were screened for their ability to bind porcine costimulatory molecules by FACS. Up-regulation of B7 molecules on PEC was evaluated by FACS after exposure to human cells or supernatants. The effect of human and/or porcine costimulatory blockade was tested in xeno-mixed lymphocyte reactions (XMLRs) and in natural killer (NK) cell cytotoxicity assays. RESULTS: B7 expression was induced on PEC after exposure to human T and NK cells or T cell-conditioned medium. The human XMLR was attenuated by human CTLA4-Ig and anti-human CD154 (hu5C8), and the combination was synergistic. Anti-human CD80 and CD86 antibodies alone had minor effects in the XMLR, but in combination with hu5C8 were as effective as human CTLA4-Ig plus hu5C8. Anti-hCD80 and hCD86 antibodies that did not cross-react with porcine CD80 or CD86 were as effective in blocking the MLR as those that did cross-react, indicating that the predominant costimulation in vitro was derived from the responding cells. None of the agents affected the xeno-NK response. CONCLUSIONS: We conclude that the costimulation-modulating agents block human anti-porcine T-cell responses in vitro predominantly through interruption of costimulation derived from responding cells. They have no effect on NK cell-mediated cytotoxicity.

Abatacept↗
PubMed2000

Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype.

In the present study, we investigated the role of the CD40L-CD40 pathway in a model of progressive atherosclerosis. ApoE-/- mice were treated with an anti-CD40L antibody or a control antibody for 12 wk. Antibody treatment started early (age 5 wk) or was delayed until after the establishment of atherosclerosis (age 17 wk). In both the early and delayed treatment groups, anti-CD40L antibody did not decrease plaque area or inhibit lesion initiation or age-related increase in lesion area. The morphology of initial lesions was not affected, except for a decrease in T-lymphocyte content. Effects of anti-CD40L antibody treatment on the morphology of advanced lesions were pronounced. In both the early and delayed treatment groups, T-lymphocyte content was significantly decreased. Furthermore, a pronounced increase in collagen content, vascular smooth muscle cell/myofibroblast content, and fibrous cap thickness was observed. In the delayed treatment group, a decrease in lipid core and macrophage content occurred. Interestingly, advanced lesions of anti-CD40L antibody-treated mice exhibited an increased transforming growth factor beta1 immunoreactivity, especially in macrophages. In conclusion, both early and delayed treatment with an anti-CD40L antibody do not affect atherosclerotic lesion initiation but do result in the development of a lipid-poor collagen-rich stable plaque phenotype. Furthermore, delayed treatment with anti-CD40L antibody can transform the lesion profile from a lipid-rich to a lipid-poor collagen-rich phenotype. Postulated mechanisms of this effect on plaque phenotype are the down-regulation of proinflammatory pathways and up-regulation of collagen-promoting factors like transforming growth factor beta.

Age Factors↗
PubMed2000

Role of CD154 in the secondary immune response: the reduction of pre-existing splenic germinal centers and anti-factor VIII inhibitor titer.

Using the murine model of hemophilia A, we have examined the role of CD154 in the secondary immune response to factor VIII (FVIII). We previously reported that repeated i.v. injection of FVIII in hemophilia A mice induces a T cell-dependent anti-FVIII antibody formation. Herein, blocking of CD154 by a monoclonal antibody in FVIII-primed hemophilia A mice resulted in the disappearance of pre-existing spleen germinal centers (GC) in the white pulp within 24 h of treatment. Moreover, further expansion of GC in response to FVIII challenge was completely inhibited. In parallel, anti-FVIII antibody titers were markedly reduced and T cell responses to FVIII were abolished. The rapid disappearance of the GC after anti-CD154 treatment was not accompanied by increased B cell apoptosis; instead B cells accumulated in the peripheral zone of the splenic white pulp. Interestingly, repeated exposure to FVIII with anti-CD154 antibody administration blocked anti-FVIII antibody formation but failed to induce long-lasting unresponsiveness. Our data demonstrate that the CD40-CD154 interaction is critical for B cell homeostasis and the secondary immune response to FVIII. For potential clinical application, the data also suggest that therapies targeting the CD154 molecule may be useful for the treatment of high titer FVIII inhibitors in hemophilia A.

Animals↗
PubMed2000

Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration.

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Animals↗
PubMed1999

Prolongation of primate cardiac allograft survival by treatment with ANTI-CD40 ligand (CD154) antibody.

BACKGROUND: We evaluated whether a humanized anti-CD154 antibody (hu5c8) prolongs primate cardiac allograft survival. METHODS: Heterotopic cardiac allografts were performed between MHC class II-mismatched cynomolgus monkeys. Survival was compared between groups treated with a perioperative dosing of hu5c8 (group 1; n=6), sustained dosing with hu5c8 (group 2; n=3), and control regimens (n=4). All recipients received fresh donor-specific transfusions during surgery. RESULTS: Median graft survival was 49 days (range 14 to 56) in group 1 and 106 days (range 56 to 245) in group 2, compared with 5 days (range 5 to 6) for controls (P<0.05 for all comparisons). Lymphocytic infiltrates were often present in hu5c8-treated grafts with stable contractility. Donor-specific mixed lymphocyte reaction was generally preserved. Vasculitis and cellular intimal proliferation were prevalent in rejected grafts but occurred later and were less prevalent in group 2. CONCLUSIONS: Anti-CD154 antibody markedly prolongs the survival of cardiac allografts in primates and is well tolerated. Sustained dosing with hu5c8 yielded improved survival and may be associated with a lower incidence of vascular pathology. We conclude that hu5c8 therapy is an effective approach for inhibiting acute cardiac allograft rejection in primates.

Animals↗
PubMed1999

Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154.

Reported effects of anti-CD154 treatment on autoimmunity, alloreactivity, and inflammatory events mediated by macrophages and endothelial cells indicated that it might be an ideal agent for the prevention of intrahepatic islet allograft failure. This hypothesis was tested in MHC-mismatched rhesus monkeys. Transplantation of an adequate number of viable islets resulted in engraftment and insulin independence in six of six recipients treated with anti-CD154 (hu5c8) induction plus monthly maintenance therapy (post-operative day >125, >246, >266, >405, >419, >476). Anti-CD154 (hu5c8) displayed no inhibitory effect on islet cell function. For monkeys followed for >100 days, continued improvement in graft function, as determined by first phase insulin release in response to intravenous glucose, was observed after the first 100 days post-transplant. No evidence of toxicity or infectious complications has been observed. All recipients treated with anti-CD154 became specifically nonresponsive to donor cells in mixed lymphocyte reactions. Furthermore, three monkeys are now off therapy (>113, >67, and >54 days off anti-CD154), with continued insulin independence and donor-specific mixed lymphocyte reaction hyporeactivity. In striking contrast to all previously tested strategies, transplantation of an adequate number of functional islets under the cover of anti-CD154 (hu5c8) monotherapy consistently allows for allogeneic islet engraftment and long-term insulin independence in this highly relevant preclinical model.

Animals↗
PubMed1999

Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.

Animals↗
PubMed1999

Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154.

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.

Animals↗
PubMed1998

Interaction affinity between cytokine receptor components on the cell surface.

The anti-common gamma chain (gammac) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Ralpha and gammac receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Ralpha, and to PHA blasts expressing both IL-4Ralpha and gammac, were used to estimate the affinity of the key interaction between gammac and the binary IL-4Ralpha.IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Ralpha.IL-4.gammac]/[IL-4Ralpha.IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR approximately 9, implying that approximately 10% of the limiting IL-4Ralpha chain remains free of gammac even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.

Animals↗
PubMed1998

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.

Amino Acid Sequence↗
PubMed1998

Molecular mapping with functional antibodies localizes critical sites on the human IL receptor common gamma (gamma c) chain.

The IL receptor common gamma (gamma c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how gamma c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human gamma c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-gamma c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the gamma c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on gamma c chain required for its association with multiple ligands.

Alanine↗