Regulation Of Alloreactivity Research Articles

Type-1 Interferons impair the immunoregulatory activity of IL-10: understanding novel mechanisms of abrogation of transplant tolerance

Abstract Induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. Type-1 interferons (TI-IFNs) are recognized mediators of this effect in multiple models, but their target population and specific mechanism used remain undefined. To better understand how TI-IFN could interfere with transplant tolerance, we studied their impact on the immunomodulatory properties of IL-10. Using a murine full-mismatch skin transplant model we showed that IL-10 has a fundamental role in the protective effect of CoB. Bone marrow chimeras suggested this effect is partially dependent on IL-10 signaling in T cells. We then studied the impact of T cell exposure to TI-IFN on IL-10 signaling. Following in vitro exposure, T cells produced significantly less phospho-STAT3 in response to IL-10. This effect was very selective - IL-6 signaling remained unaltered - and resulted in the inhibition of specific IL-10 induced genes. Encouragingly, this effect was slowly reversible with removal of TI-IFN, suggesting a “druggable” pathway. This unresponsiveness was not associated with reduced IL-10-receptor expression or increased SOCS1_3 levels. Instead, microarray analysis of exposed cells suggested a novel role for STAT1 in dampening IL-10 signaling. Preliminary experiments with STAT1-KO cells supported this hypothesis. Overall, these results highlight the importance of IL-10 in the efficacy of CoB regimens and reveal a new molecular mechanism whereby TI-IFNs can interfere with IL-10 signaling, and ultimately with the regulation of alloreactivity. A strategy to selectively target this mechanism could significantly enhance the clinical efficacy of CoB-based immunomodulatory strategies.

Abstract 113: Type-1 Interferons Impair The Immunoregulatory Activity Of IL-10: Understanding The Mechanisms Of Abrogation Of Transplant Tolerance

PURPOSE: Induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. In particular, multiple studies reported that engagement of toll like receptors (TLR) abrogate the tolerogenic effect of CoB. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, their target population and specific pathway used remain undefined. To better understand how TI-IFN could interfere with the induction of transplant tolerance, we studied their impact on the immunomodulatory properties of IL-10. METHODS: Full mismatch skin transplants were performed using dorsal skin from Balb/c into C57BL/6 mice and animals received a peri-transplant regimen based on DST (107 T-depleted splenocytes; on day 0) and anti-CD154 mAb (MR-1; on day 0, 7, 14) +/- anti-IL-10R (on day 0, 7, 14). Mouse T cells were isolated by negative-selection and Tmem and Treg subsets identified by flow cytometry. IL-10R expression and phospho-STAT3 induction after IL-10 or IL-6 stimulation in T cells were measured via flow cytometry. The gene expression profile of T cell subsets exposed to TI-IFN was assessed by microarray and quantitative PCR analysis. Protein levels were measured by Western Blot. RESULTS: Our result showed that IL-10 has a fundamental role in the protective effect of CoB (transplant survival: MST 105d with CoB vs 47d with anti-IL-10R administration). We then studied the impact of T cell exposure to TI-IFN on IL-10 signaling. Following 48h of in vitro incubation with IFN-β, memory T cells (Tmem) and Tregs presented a dramatic defect in the production of phospho-STAT3 in response to IL-10. This effect was very selective, as IL-6 signaling (post IFN-β exposure) induced normal levels of phospho-STAT3. The reduced accumulation of phospho-STAT3 in conditioned cells resulted in the inhibition of the upregulation of mRNAs for LIGHT, Sphk1 and Tarm-1 - three genes we have discovered are induced by IL-10 in T cells. Encouragingly, this effect was slowly reversible with removal of TI-IFN, suggesting a “druggable” pathway. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10 receptor expression or an increase in SOCS (Suppressor of Cytokine Signaling) 1 and 3, nor with reduced STAT3 cytoplasmic availability. Instead, this analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. Using T cells from STAT1-KO mice, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-β in Treg and Tmem, supporting our new model. CONCLUSION:Overall, these results highlight the importance of IL-10 in the therapeutic effect of CoB regimens and reveal a new molecular mechanism whereby TI-IFN interfere with IL-10 signaling, and ultimately with regulation of alloreactivity. Identifying a strategy to target this mechanism could provide a valuable aid in the clinical efficacy of CoB-based immunomodulatory strategies.

Type-1 Interferons Impair the Immunoregulatory Activity of IL-10

A growing body of evidence shows that induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. In particular, multiple studies reported that engagement of toll like receptors (TLR) abrogate the tolerogenic effect of CoB. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, the target population and specific pathway used by TI-IFN to induce this effect, remain unknown. CoB therapies rely on the enhancement of the frequency and suppressive activity of regulatory T cells (Tregs) that use interleukin 10 (IL-10) as a key weapon of suppression. IL-10 is a pleiotropic and immunomodulatory cytokine that in the field of transplant tolerance, despite its anticipated protective function, maintains a controversial role. To better understand how an inflammatory environment, more specifically IFN-β, could interfere with the induction of transplant tolerance, we studied its impact on the immunomodulatory properties of IL-10. Our results showed that the transient blockade of IL-10 signaling had a deleterious impact on the ability of a CoB regimen based on the infusion of donor specific transfusion (DST) and anti-CD154 (MR-1), to prolong the survival of murine full mismatch skin transplants (MST 105d vs 47d with anti-IL-10R administration). Following 48h of in vitro incubation with IFN-β, memory T cells (Tmem) and Tregs presented a dramatic defect in the production of phospho-STAT3 in response to IL-10. This effect was very selective, as IL-6 signaling (post IFN-β exposure) induced normal levels of phospho-STAT3. The reduced accumulation of phospho-STAT3 in conditioned cells resulted in the inhibition of the upregulation of mRNAs for LIGHT, Sphk1 and Tarm-1 – three genes we have discovered are induced by IL-10 in T cells. Encouragingly, this inhibition of IL-10 signaling is slowly reversible with the removal of TI-IFN. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10 receptor expression or an increase in SOCS (Suppressor of Cytokine Signaling) 1 and 3, nor with reduced STAT3 cytoplasmic availability. Instead, this analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. Using T cells from STAT1-KO mice, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-β in Treg and Tmem, supporting our new model. Overall, these results highlight the importance of IL-10 signaling in the therapeutic effect of CoB regimens in transplantation. More importantly, they reveal a new molecular mechanism whereby IFN-β interferes with IL-10 signaling, its suppressive function in T cells, and ultimately with regulation of alloreactivity. Identifying a strategy to target this mechanism will be a powerful tool to improve the efficacy of immunomodulatory strategies for transplant tolerance induction. AAI 2016-2017 Careers in Immunology Fellowship. JDRF strategic research agreement 2-SRA-2016-310-S-B.

Type-1 interferon impairs the immunoregulatory activity of IL-10: Understanding the mechanisms of abrogation of transplant tolerance

Abstract Long term transplant survival by costimulation blockade (CoB)-based regimens is impaired or eroded by inflammatory responses. Type-1 interferons (TI-IFN) are key mediators of this effect, however, their target population and mechanism of action remain unknown. To better understand how IFN-β interferes with the induction of transplant tolerance, we studied its impact on the recently discovered immunomodulatory properties of interleukin 10 (IL-10) in mouse T cells. We first demonstrate that IL-10 has an unappreciated fundamental role in transplant tolerance, as its transient blockade in recipients of full mismatch skin grafts impairs the efficacy of a peri-transplant CoB regimen based on donor specific transfusion (DST) plus anti-CD154. Moreover, when T cells are exposed to IFN-β in vitro, they develop a selective dramatic defect in their response to IL-10 (but not to IL-6) both at the signaling and gene-induction level. This IL-10-specific unresponsiveness is not associated with any reduction of IL-10R expression or an increase in SOCS1 and 3, nor with a reduced STAT3 cytoplasmic availability. Instead, our gene-expression and functional analysis suggests a novel “cross-regulatory” role for the transcription factor STAT1. Encouragingly, this inhibition is slowly reversible with the removal of TI-IFN, proving the feasibility a pharmacological intervention. Overall, these results reveal a new molecular mechanism whereby IFN-β interferes with the suppressive function of IL-10 in T cells, and potentially with regulation of alloreactivity. Identifying a strategy to target this mechanism will then be a powerful tool to improve the efficacy of CoB-based strategies for transplant tolerance induction.

Neuroimmunomodulation and Aging: A Role for Transferrin and the Hypothalamus/Thymus Axis

Advanced age is associated with an increased incidence of immune and degenerative disorders, mediated by metabolic changes, dysregulation of proinflammatory signals, and apoptosis. Concurrently, there is a progressive decline in self-recognition. Investigations on biologic functions of transferrin (Tf) other than iron transport showed that Tf has a profound cytoprotective (anti-apoptotic) effect on lympho-hematopoietic cells and the thymus, and interferes with stress-induced signals. Tf protects hepatocytes against Fas-induced cell death by reducing BID cleavage, inhibiting caspase-3 and -9 activation and up-regulating survival signals such as Bcl-xL. The involvement in the regulation of alloreactivity and apoptosis suggests that Tf participates in the maintenance of "self-identity" mechanisms, which are tightly linked to the capacity of the immune system to recognize and react against any noxious agent. Some of the disorders associated with aging are thought to be related to thymic involution, reflecting alterations in the interplay of neural, endocrine and immune factors. We established a murine model of thymic involution induced by stereotactically placed electrolytic lesions in the anterior hypothalamic area. The events observed in this model mimic those observed during senescence including thymus involution, i.e. enhanced glucocorticoid reaction to distress, and obesity. The described properties of Tf can be exploited to modify immune responses and provide cytoprotection against pro-apoptotic and cytotoxic signals when neuroimmunomodulatory mechanisms are impaired, as is the case with aging.

GvHD Prophylaxis with Everolimus and Mycophenolate in Allogeneic Hematopoietic Cell Transplantation - A Phase I/II Study.

Abstract 4642Next to the control of malignant disease, tolerance induction of the grafted cells remain to be a critical issue for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are believed to be involved in the process of tolerance induction to solid organ grafts and in the regulation of alloreactivity, e.g. graft versus host disease (GvHD) and graft versus leukemia effect (GvL), after allogeneic HCT. Since GvHD causes substantial morbidity, medication with calcineurin inhibitors (CI) like cyclosporine are established prophylactic measures for the prevention of GvHD after HCT. Next to the substantial renal toxicity of CI, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. In contrast, data from in vitro and animal experiments suggest that inhibition of the mammalian target of rapamycin (mTOR) has not only an antiproliferative effect on many malignant cell lines but also results in an inhibition of proliferation of alloreactive T cells with sustained Treg function in a murine HCT model. Therefore we initiated a phase I/II, monocenter trial using everolimus and mycophenolate sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after conditioning with fludarabine, melphalan, and BCNU (FBM). No additional T cell depleting agents were used for conditioning/GvHD prophylaxis. Enrolment was started in april 2008, and up to august 2009 10 patients were included (median age: 50.7 years, range: 26-64). The diagnoses included de novo AML (n=3), sAML (n=4), RAEB II (n=1), CML (n=1), T-PLL (n=1). 6/10 patients were regarded as high risk (not in CR1) for early relapse. PBSC grafts were obtained from unrelated (n=5) and related (n=5) HLA-matched donors. With no graft failures, engraftment kinetics for myeloid cells were normal, and reconstitution of the T cell compartment reached median cell counts of 251 CD4+ cells/μl and 163 CD8+ cells/μl at day +30. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow-up of 6 month two patients have died. The causes were acute GvHD, refractory to several lines of treatment, in a patient with CML, and severe pulmonary toxicity/BOOP in a patient with sAML. Out of 9 patients reaching CR after HCT, only one high risk patient relapsed after 6 month. In total 6 patients are alive and show complete donor chimerism for time periods of 1-14 months post transplant. The observed early recovery of T cell immunity correlated in 8/10 patients with an early brief period of acute GvHD, with 4 patients experiencing grade III/IV severities. Most of the cases could be controlled with steroids alone. Chronic GvHD could be observed in 6/7 patients, with mild to moderate forms in 5 cases, mainly involving skin, mucosa and liver. Interestingly, while early tapering of MMF-Na did not cause any problems, reduction of everolimus earlier as 6 month after HCT resulted in an induction of GvHD symptoms. Although viral reactivation (CMV, HHV6) did occur in patients receiving additional immunosuppression with steroids, no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of mild to moderate chronic GvHD. Since this sustained mild alloreactivity might reduce the risk of relapse, this GvHD prophylaxis could well be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures:Marks:Novartis: Research Funding. Off Label Use: Everolimus for prophylaxis of GvHD. Finke:Novartis: Research Funding.

Decreases in circulating CD4+CD25hiFOXP3+ cells and increases in intragraft FOXP3+ cells accompany allograft rejection in pediatric liver allograft recipients

We examined CD4(+)CD25(hi)FOXP3(+) cells Treg in children following liver transplantation and determined the relationship between Treg cell levels in the blood and in the graft. Peripheral blood was obtained from pediatric liver transplant patients at sequential time points: pre-transplant, one month, 3-4 months, 6-7 months, and 11-12 months post-transplant. PBMC were isolated, labeled for CD4, CD25 and FOXP3 expression and analyzed by flow cytometry for CD4(+)CD25(hi)FOXP3(+) cells. Sorted CD4(+)CD25(hi) cells were assessed for functional activity. Pretransplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were not significantly different from post-transplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells. However, the blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were significantly decreased during acute rejection compared with levels when graft function was stable. Immunohistochemistry revealed that FOXP3(+) cells were increased in the portal region of livers with histopathologic evidence of acute graft rejection compared with livers without evidence of rejection and were localized primarily within the inflammatory infiltrate. These data indicate that Treg cells are found at the site of allograft rejection and may play a role in regulation of alloreactivity. Moreover, monitoring peripheral CD4(+)CD25(hi)FOXP3(+) Treg cell levels may be useful in improving the post-transplant management of pediatric liver allograft recipients.

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

AbstractAlloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2b), BALB/c (H2d), CBA (H2k), and DBA/1 (H2q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.

Human CD4 Negative NKT Cells Potently Suppress Alloreactive Responses in a TGF-β and IL-10-Independent Manner.

Acute graft versus host disease (aGVHD) remains a major source of morbidity and mortality in allogeneic stem cell transplantation. There is a need for therapeutic interventions to ameliorate its severity. NKT cells, a novel class of regulatory T cells, are restricted by the glycolipid presenting MHC-like molecule CD1d. They constitute <0.1% of T cells and modulate responses of the innate and adaptive immune system in autoreactivity and against pathogens and tumours through interactions with antigen presenting cells (APC). The regulation of alloreactivity and aGVHD by NKT cells has not been studied. We tested the effect of expanded human NKT cells on the mixed lymphocyte reaction (MLR). First, NKT cells were expanded ex vivo from peripheral blood mononuclear cells (PBMC) of normal donors in the presence of the CD1d-presented glycolipid α-galactosylceramide. Under these conditions NKT cells (n=7), identified by staining with mAbs specific to their TCR α and β chains, expanded by 238-fold±102 by day 10. Total, CD4+ (69%±17 of total) and CD4- (30%±17 of total) NKT were purified to >95% and tested for their effect on the MLR using 3H incorporation assays. Total and NKT subsets from a panel of donors were added to the MLR at a ratio of NKT:Responder (consisting of autologous T cells) of 1:10 and a panel of allogeneic irradiated PBMC were used as stimulators. Co-culture with total or CD4+ NKT had a variable effect on the MLR. By contrast, in all MLR tested using 7 different responders against a panel of 3–5 stimulators, the presence of CD4- NKT had a profound suppressive effect (mean suppression from baseline MLR: 67%±18%). Titration of the NKT numbers revealed that the magnitude of the suppressive effect was inversely proportional to the NKT:R ratio, suggesting that NKT can exert opposing effects of differential magnitude and the maximum suppression depends on an optimal NKT:R ratio. In addition we demonstrated that CD4- NKT cell lines exert linked suppression, i.e., that they have suppressive effect (mean 52%±16.5, n=2) in MLR when the responding T cells are allogeneic to the NKT themselves. In transwell experiments we determined that the suppressive effect is at least partially contact-dependent and it is not mediated by either IL-10 or TGFβ as these anti-proliferative cytokines were not detected in the supernatants of the MLR and the presence of the corresponding neutralising mAbs did not reverse the suppressive effect. These findings indicate that the suppressive effect of NKT is not due to induction of anergy on the alloresponsive T cells. Instead, it is likely that it is mediated by a modulatory effect on the APC. The mechanisms of this modulation are the subject of current investigation. Finally, all CD4- NKT cells lines tested (n=4) had markers of activation (>90% were CD25+ CD69+), demonstrated potential for homing to peripheral tissues (mean expression 52%±23.9 were CD62L−CCR7−), especially to mucosal tissues (mean expression of integrin β7 84%±8) but not to the skin (mean expression of CLA <1%). In summary, we have identified a subset of NKT cells that when expanded ex vivo are very efficient in suppressing the alloreactive response. These findings form the basis for the use of such cells at a clinical level for the treatment of aGVHD, especially of mucosal localisation, through their adoptive transfer. The procedure of expanding suppressive NKT can be easily adapted for clinical purposes as using autologous PBMC as APC can result in a >200-fold expansion after just one round of stimulation.

Regulation of alloreactivity in the popliteal lymph node assay by the new immunosuppressants: malononitrilamides.

Malononitrilamides (MNAs) represent a new class of low molecular weight immunosuppressants and have been shown to prevent and reverse ongoing acute allograft rejection and effectively prolong xenograft survival in rodents. MNAs were also found to be potent inhibitors of B and T cell-mediated autoimmune processes and mediate their effects by binding specifically to dihydro-orotate dehydrogenase (DHODH), inhibiting de novo pyrimidine biosynthesis, thereby blocking T and B cell proliferation and strongly suppressing the IgM and IgG antibody production. Here we evaluated the effects of the MNAs (HMR 1279 and HMR 1715) on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local graft-versus-host reaction in Lewis x Brown Norway F1 hybrid rats by measuring the enlargement of the popliteal lymph nodes (PLN) draining the site of allogeneic cell injection. Oral administration of the MNAs dose-dependently prevented the localized lymphoproliferative response in the PLN assay and suppressed the lymph node hyperplasia. The MNAs even acted therapeutically when they were given during an ongoing alloreactivity as late as days 4 or 5 after challenge. Consistent with the mode of action, a complete reversal of the immunosuppression on the lymphoproliferation in vivo was attempted in this protocol by the addition of exogenous uridine during days 0-5. These data suggest the HMR 1279 and HMR 1715 mediate their antiproliferative and immunosuppressive effects in the PLN assay in vivo by decreasing the activity of DHODH in the lymph node cells and thereby inhibiting pyrimidine biosynthesis.

Regulation of alloreactivity in the popliteal lymph node assay by the new immunosuppressants: malononitrilamides

Abstract Malononitrilamides (MNAs) represent a new class of low molecular weight immunosuppressants and have been shown to prevent and reverse ongoing acute allograft rejection and effectively prolong xenograft survival in rodents. MNAs were also found to be potent inhibitors of B and T cell-mediated autoimmune processes and mediate their effects by binding specifically to dihydr-orotate dehydrogenase (DHODH), inhibiting de novo pyrimidine biosynthesis, thereby blocking T and B cell proliferation and strongly suppressing the IgM and IgG antibody production. Here we evaluated the effects of the MNAs (HMR 1279 and HMR 1715) on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local graft-versushost reaction in Lewis × Brown Norway F1 hybrid rats by measuring the enlargement of the popliteal lymph nodes (PLN) draining the site of allogeneic cell injection. Oral administration of the MNAs dose-dependently prevented the localized lymphoproliferative response in the PLN assay and suppressed the lymph node hyperplasia. The MNAs even acted therapeutically when they were given during an ongoing alloreactivity as late as days 4 or 5 after challenge. Consistent with the mode of action, a complete reversal of the immunosuppression on the lymphoproliferation in vivo was attempted in this protocol by the addition of exogenous uridine during days 0–5. These data suggest the HMR 1279 and HMR 1715 mediate their antiproliferative and immunosuppressive effects in the PLN assay in vivo by decreasing the activity of DHODH in the lymph node cells and thereby inhibiting pyrimidinebiosynthesis.

Regulation of alloreactivity in vivo by IL-4 and the soluble IL-4 receptor.

Although numerous in vitro studies have demonstrated that cytokines are involved in the generation of alloreactive effector cells, the role of these regulatory substances in vivo is less well defined. We have recently cloned and expressed cDNAs encoding both membrane bound and soluble forms of the murine IL-4R. The effects of murine rIL-4 and a recombinant, soluble, extracellular portion of the murine IL-4R soluble(s) IL-4R on the generation of alloresponsiveness in vivo were evaluated by measuring the lymphoproliferative response to a localized injection of allogeneic cells and the survival of cardiac allografts. Administration of IL-4 to BALB/c mice resulted in a slight augmentation of the anti-C57BL/6 lymphoproliferative response compared to that occurring in control, mouse serum albumin-(MSA) treated mice. In contrast, the sIL-4R suppressed this response to allogeneic cells in a dose-dependent manner, with a dose of 50 micrograms/kg/day causing nearly complete inhibition of the response as compared to the response observed in controls. The inhibitory effect of sIL-4R was reversed by simultaneous administration of IL-4. A neutralizing antibody against IL-4 (11B11) and another against the IL-4R (M1) were also effective inhibitors of the response when given at 100- to 1000-fold higher concentrations than the amount of sIL-4R required to inhibit the response. In cardiac allograft experiments, BALB/c mice were engrafted with newborn C57BL/6 hearts in the ear pinnae and treated with sIL-4R (50 micrograms/kg) or MSA. Such allografts survived an average of 4 days longer in sIL-4R-treated mice than in MSA-treated controls. In conclusion, neutralization of IL-4 inhibits alloresponsiveness in vivo. These results confirm a regulatory role for this pleiotropic cytokine in allograft rejection and suggest a therapeutic value for IL-4 antagonists alone or in combination with other immunosuppressive regimens.

Regulation of Alloreactivity in Vivo by a Soluble Form of the Interleukin-1 Receptor

In vitro studies have shown that cytokines are involved in the regulation of the immune response, but their role in vivo is less well defined. Specific cytokine antagonists enable the identification of particular cytokines involved in the response and offer a means for modifying it. Systemic administration of a soluble, extracellular portion of the receptor for interleukin-1 (sIL-1R) had profound inhibitory effects on the development of in vivo alloreactivity. Survival of heterotopic heart allografts was prolonged from 12 days in controls to 17 days in mice treated with sIL-1R. Lymph node hyperplasia in response to a localized injection of allogeneic cells was completely blocked by sIL-1R treatment. The inhibition was overcome by simultaneous administration of interleukin-1 (IL-1); thus, sIL-1R acts by neutralizing IL-1. These results implicate IL-1 as a regulator of allograft rejection and demonstrate the in vivo biological efficacy of a soluble cytokine receptor.

Perturbation of the idiotypic network: I. Induction with multiple alloantigen stimulation

The effect of hyperimmunization on the immune network with allostimulated syngeneic lymphocytes responding to different haplotypes was analyzed. Ten different haplotypes were used to stimulate syngeneic donor mice. Control mice were multiply immunized with incomplete Freund's adjuvant alone or with syngeneic mixed lymphocyte culture-generated lymphocytes. BALB/c mice were immunized consecutively with alloreactive blasts or allogeneically stimulated spleen cells at 10-day intervals. After a rest period of 2 months, the ratio of T helpers to T suppressors was determined by immunofluorescent staining. The functional network was probed by immunizing the mice with phosphorylcholine (PC) coupled to hemocyanin. The sera were analyzed for anti-PC antibodies and TEPC15 (T15) idiotypic expression. The results demonstrated (i) a decrease in the level of anti-PC antibody titer and T15 idiotypic expression; (ii) a decrease in the number of T helper cells and an increase in the number of T suppressor cells; (iii) a loss of PC epitope specificity; (iv) an increase of IgM antibodies expressing T15 without anti-PC specificity; and (v) an elevated level of preimmune lymphocyte proliferation and Ig secretion. These results reveal a functional network linkage in the regulation of alloreactivity and antigen response and show how repeated exposure to alloantigens can induce a perturbation of the idiotypic network controlling the response of a non-alloantigen-related BALB/c strain dominant idiotype (T15).