Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids.

BACKGROUND: Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. METHODS: MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. RESULTS: Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short (<100 days) and long (>100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4+ T cells required longer than 6 months. CONCLUSIONS: A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

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.

Immunotoxin FN18-CRM9 induces stronger T cell signaling than unconjugated monoclonal antibody FN18.

BACKGROUND: The T-cell receptor (TCR)/CD3 complex is the target of therapeutic strategies aimed at prolonging allograft survival. The immunotoxin FN18-CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated diphtheria toxin CRM9, is useful for prolonging allograft survival in preclinical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 complex, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein tyrosine phosphorylation and ligand/receptor internalization in purified monkey peripheral blood T cells. METHODS: Purified normal rhesus monkey T cells were incubated with unconjugated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization. RESULTS: Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with similar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues and in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internalization, suggesting that tyrosine phosphorylation is involved in the internalization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-treated cells, there was a gradual decrease in cellular CD3 protein levels within 24 and 48 hr; such a decrease was not observed with the control protein Csk. CONCLUSIONS: Our findings suggest that the conjugation of the mutated diphtheria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated cross-linking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosphorylation and CD3 internalization. This may in turn contribute to the greater efficacy of the immunotoxin in prolonging allograft survival.

Tolerance and near-tolerance strategies in monkeys and their application to human renal transplantation.

Studies in non-human primates to evaluate tolerance strategies in organ transplantation have led to innovation in human transplantation. The two strategies we have studied in detail in non-human primates are T-cell depletion by anti-CD3 immunotoxin and co-stimulation blockade. Each of these strategies has been extended into early human trials in renal transplantation. The results of these human and non-human primate studies are summarized. Continued progress in better and safer immunosuppressive methods remains closely linked to research using non-human primates. However, there has not been a one-to-one correspondence between efficacy in the primate and efficacy in humans. Rather, principles can be derived from non-human primate studies that can be extended into human trials with the knowledge that regimens will likely differ in humans compared to non-human primates.

Increased glomerular deposits of von Willebrand factor in chronic, but not acute, rejection of primate renal allografts.

BACKGROUND: In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model. METHODS: Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. RESULTS: Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. CONCLUSION: Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Potentiation of CD3-induced expression of the linker for activation of T cells (LAT) by the calcineurin inhibitors cyclosporin A and FK506.

The activation of blood cells, including T cells, triggers intracellular signals that control the expression of critical molecules, including cytokines and cytokine receptors. We show that T-cell receptor (TCR) ligation increases the cellular level of the protein linker for activation of T cells (LAT), a molecule critical for T-cell development and function. T-cell activation increased LAT messenger RNA, as determined by reverse transcription-polymerase chain reaction and by Northern blotting. The TCR-induced increase in LAT expression involved the activation of the serine/threonine kinases PKC and MEK, because inhibitors of these kinases blocked the increase in LAT. Accordingly, the PKC activator phorbol myristate acetate up-regulated LAT expression. Strikingly, the calcineurin inhibitors cyclosporin A (CsA) and FK506 strongly potentiated TCR-induced LAT expression, suggesting that the activation of calcineurin following TCR ligation negatively regulates LAT expression. Accordingly, Ca(++ )ionophores, which can activate calcineurin by increasing intracellular Ca(++), blocked the TCR-induced increase in cellular LAT. CsA and FK506 blocked the Ca(++ )ionophores' inhibitory effect on LAT expression. Notably, CsA and FK506 preferentially up-regulated TCR-induced LAT expression; under the same conditions, these compounds did not increase the expression of 14 other molecules that previously had been implicated in T-cell activation. These data show that TCR-induced LAT expression involves the activation of the PKC-Erk pathway and is negatively regulated by the activation of calcineurin. Furthermore, the potentiation of TCR-induced LAT expression by CsA and FK506 suggests that the action of these agents involves up-regulating the cellular level of critical signaling molecules. These findings may have important therapeutic implications. (Blood. 2000;95:2733-2741)

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases.

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.

T cell activation up-regulates the expression of the focal adhesion kinase Pyk2: opposing roles for the activation of protein kinase C and the increase in intracellular Ca2+.

T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca2+/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca2+ ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca2+ has opposing effects on IL-2 and Pyk2 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca2+ ionophore-mediated inhibition of Pyk2 expression, implicating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells.

Activation of T lymphocytes for adhesion and cytokine expression by toxin-conjugated anti-CD3 monoclonal antibodies.

BACKGROUND: Immunosuppressive drugs that target T cells are useful for prolonging allograft survival. The anti-CD3 immunotoxin FN18-CRM9 has been shown to effectively prolong renal allograft survival in a rhesus monkey model of transplantation. However, immunotoxin-treated monkeys showed increased levels of inflammatory cytokines and produced antibodies to donor proteins. To better understand the role of FN18-CRM9 in the production of cytokines and anti-donor antibodies in the monkey model, we examined whether this immunotoxin elicits functional responses in T cells. METHODS: Purified normal rhesus monkey T cells (>98% purity) were incubated with immunotoxin FN18-CRM9 or the unconjugated anti-CD3 monoclonal antibodies and then examined for changes in protein tyrosine phosphorylation, adhesion to fibronectin, gene expression, and proliferation in the presence or absence of anti-CD28 monoclonal antibodies (mAb) and interleukin-2. RESULTS: Immunotoxin treatment of T cells in vitro increased protein tyrosine phosphorylation, cell adhesion to the extracellular matrix, and expression of the inflammatory cytokines interferon-gamma and tumor necrosis factor-alpha. These immunotoxin effects were similar in magnitude to those induced by the unconjugated mAb. In contrast, immunotoxin-induced T cell proliferation was markedly less than that induced by the unconjugated mAb. Interestingly, the mitogenic molecules IL-2 and anti-CD28 mAb did not prevent immunotoxin-induced inhibition of cell proliferation. CONCLUSIONS: The activation of T cells for protein phosphorylation, adhesion, and cytokine expression strongly suggests that the actions of FN18-CRM9 in vivo are not limited to the inhibition of protein synthesis.

CD28 ligation induces rapid tyrosine phosphorylation of the linker molecule LAT in the absence of Syk and ZAP-70 tyrosine phosphorylation.

CD28 is a T cell surface molecule that is important for T cell activation. CD28-triggered T cell stimulation involves protein tyrosine phosphorylation, a process that is critical for CD28 function. Recently, a linker molecule has been identified as LAT (Linker for Activation of T cells). Studies involving LAT mutants and reconstitution experiments strongly implicate LAT in playing a critical role in T cell activation. We show in the present report that CD28 ligation induces tyrosine phosphorylation of LAT. CD28-induced tyrosine phosphorylation of LAT was rapid, as it was apparent within 1 min of CD28 ligation, reached a peak by 5 min, and declined thereafter. Previous studies implicated the protein tyrosine kinases ZAP-70 and Syk in the TCR-induced tyrosine phosphorylation of LAT. Here, tyrosine phosphorylation of Syk and ZAP-70 was detected after TCR but not after CD28 ligation. Thus, CD28 ligation appears to induce tyrosine phosphorylation of LAT by mechanisms that are independent of ZAP-70 and Syk. The concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of LAT. These results implicate LAT in CD28 signal transduction pathways and in the co-stimulatory process in T cells.

CD28 ligation induces tyrosine phosphorylation of Pyk2 but not Fak in Jurkat T cells.

Protein tyrosine kinases are critical for the function of CD28 in T cells. We examined whether the tyrosine kinases Pyk2 and Fak (members of the focal adhesion kinase family) are involved in CD28 signaling. We found that ligating CD28 in Jurkat T cells rapidly increases the tyrosine phosphorylation of Pyk2 but not of Fak. Paxillin, a substrate for Pyk2 and Fak, was not tyrosine-phosphorylated after CD28 ligation. CD28-induced tyrosine phosphorylation of Pyk2 was markedly reduced in the absence of external Ca2+. Previous studies have shown that the T cell antigen receptor (TCR) induces tyrosine phosphorylation of Pyk2. In this report, the concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of Pyk2; however, the extent of phosphorylation by both receptors was equivalent to the sum of that induced by each receptor alone. The Syk/Zap inhibitor piceatannol blocked CD28, and TCR induced tyrosine phosphorylation of Pyk2, suggesting that Syk/Zap is involved in Pyk2 phosphorylation. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin blocked TCR- but not CD28-induced phosphorylation of Pyk2, suggesting that CD28 and TCR activate distinct pathways to induce tyrosine phosphorylation of Pyk2. Notably, depleting phorbol 12-myristate 13-acetate-sensitive protein kinase C did not block CD28- and CD3-induced tyrosine phosphorylation of Pyk2. These data provide evidence for the involvement of Pyk2 in the CD28 signaling cascade and suggest that neither Fak nor paxillin is involved in the signaling pathways of CD28.

Strategies for tolerance induction in nonhuman primates.

Organ transplants in nonhuman primates provide a model which closely simulates the biological conditions of human organ transplantation, due to similarities between human and primate MHC (class I and II) structure and expression. Several strategies for tolerance induction have been developed in nonhuman primate models. These include targeting the T cell receptor or costimulatory molecules and the generation of mixed chimerism. Tolerance can be reliably induced in several such models, although none with 100% success.

Primate renal transplants using immunotoxin.

BACKGROUND: T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey and allograft survival, on antibody production, and on T-cell recovery. METHODS: Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. RESULTS: Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. CONCLUSIONS: Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. T-cell-dependent B-cell responses remain intact after treatment.

Reconstitution of high affinity IgE receptor-mediated secretion by transfecting protein tyrosine kinase pp125FAK.

To study the role of the focal adhesion tyrosine kinase (FAK) in receptor-mediated secretion, we transfected FAK cDNA into a variant (3B6) of the RBL-2H3 mast cell line. This 3B6 cell line expressed low levels of FAK and was defective in high affinity IgE receptor (FcepsilonRI) but not Ca2+ ionophore-mediated secretion. FcepsilonRI-mediated secretion was reconstituted after transfection of wild-type FAK. Histamine release was also enhanced by the stable expression of two mutants of FAK: a kinase-inactive form in which the ATP binding site Lys-454 was replaced by Arg or a mutant in which the autophosphorylation site Tyr-397 was replaced by Phe. Therefore, the catalytic activity and the autophosphorylation site of FAK are not essential for secretion. FcepsilonRI aggregation increased the tyrosine phosphorylation of both mutants of FAK to the same extent as wild-type FAK. Therefore, tyrosine kinases activated by FcepsilonRI aggregation are phosphorylating FAK and some of these phosphorylation sites are other than Tyr-397. These results strongly suggest that FAK plays a role in FcepsilonRI-induced secretion by functioning as an adapter or linker molecule.

Fc epsilon RI aggregation induces tyrosine phosphorylation of a novel 72 kDa protein downstream of Syk.

Tyrosine phosphorylation of proteins is critical for the Fc epsilon RI-induced signal transduction that leads to the release of inflammatory mediators from mast cells. Here we report the isolation of a monoclonal antibody, mAb BD2, to a 72 kDa protein that becomes rapidly tyrosine phosphorylated after Fc epsilon RI aggregation. By immunoprecipitation, immunoblotting and/or protease digestion this 72 kDa protein was different from the previously identified 68-76 kDa tyrosine phosphorylated proteins Btk, paxillin, SLP-76 or Syk. The phosphorylation of this 72 kDa protein was detectable within 15 sec after receptor aggregation and was independent of Ca2+ influx or the activation of protein kinase C. By in vitro kinase reaction, the 72 kDa protein did not autophosphorylate, which suggests that it is not a kinase, but is associated with a 140 kDa protein that was strongly phosphorylated. Studies in Syk deficient and Syk transfected variants of the RBL-2H3 cells demonstrated that the tyrosine phosphorylation of this 72 kDa protein was downstream of Syk. These data indicate that the 72 kDa protein precipitated by mAb BD2 is a novel phosphoprotein involved in Fc epsilon RI signaling.

Large scale isolation of intact rat basophilic leukemia (RBL-2H3) cells.

The rat basophilic leukemia RBL-2H3 mast cell line is widely used for studies of the structure and function of the high affinity IgE receptor (Fc epsilonRI). Here we report on a simple method to isolate large numbers of intact RBL-2H3 cells from tumors produced by injection of the cells into newborn rats. Collagenase treatment of rat tumors yields approximately 3.5 x 10(8) viable cells/animal. Aggregating Fc epsilonRI on these cells induced tyrosine phosphorylation of proteins including the protein tyrosine kinase Syk. This procedure should prove useful for the isolation and characterization of cellular molecules important for mast cell and basophil function.