Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.

Expression of and correlational patterns among neuroinflammatory, neuropeptide, and neuroendocrine molecules from cerebrospinal fluid in cerebral palsy.

BACKGROUND: The underlying pathogenesis of cerebral palsy (CP) remains poorly understood. The possibility of an early inflammatory response after acute insult is of increasing interest. Patterns of inflammatory and related biomarkers are emerging as potential early diagnostic markers for understanding the etiologic diversity of CP. Their presence has been investigated in plasma and umbilical cord blood but not in cerebrospinal fluid (CSF). METHODS: A clinical CP sample was recruited using a single-time point cross-sectional design to collect CSF at point-of-care during a standard-of-care surgical procedure (intrathecal pump implant). Patient demographic and clinical characteristics were sourced from medical chart audit. RESULTS: Significant (p ≤ 0.001) associations were found among neuroinflammatory, neuroendocrine, and nociceptive analytes with association patterns varying by birth status (term, preterm, extremely preterm). When between birth-group correlations were compared directly, there was a significant difference between preterm and extremely preterm birth subgroups for the correlation between tumour necrosis factor alpha (TNFα) and substance P. CONCLUSION: This investigation shows that CSF can be used to study proteins in CP patients. Differences in inter-correlational patterns among analytes varying by birth status underscores the importance of considering birth status in relation to possible mechanistic differences as indicated by biomarker signatures. Future work should be oriented toward prognostic and predictive validity to continue to parse the heterogeneity of CP's presentation, pathophysiology, and response to treatment.

Engineering Functional Vasculature in Decellularized Lungs Depends on Comprehensive Endothelial Cell Tropism.

Tissue engineering using decellularized whole lungs as matrix scaffolds began as a promise for creating autologous transplantable lungs for patients with end-stage lung disease and can also be used to study strategies for lung regeneration. Vascularization remains a critical component for all solid organ bioengineering, yet there has been limited success in generating functional re-endothelialization of most pulmonary vascular segments. We evaluated recellularization of the blood vessel conduits of acellular mouse scaffolds with highly proliferating, rat pulmonary microvascular endothelial progenitor cells (RMEPCs), pulmonary arterial endothelial cells (PAECs) or microvascular endothelial cells (MVECs). After 8 days of pulsatile perfusion, histological analysis showed that PAECs and MVECs possessed selective tropism for larger vessels or microvasculature, respectively. In contrast, RMEPCs lacked site preference and repopulated all vascular segments. RMEPC-derived endothelium exhibited thrombomodulin activity, expression of junctional genes, ability to synthesize endothelial signaling molecules, and formation of a restrictive barrier. The RMEPC phenotype described here could be useful for identifying endothelial progenitors suitable for efficient vascular organ and tissue engineering, regeneration and repair.

Associations Among Diurnal Salivary Cortisol Patterns, Medication Use, and Behavioral Phenotype Features in a Community Sample of Rett Syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder resulting from mutations of the MECP2 gene. Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and abnormal stress responses have been observed in animal models of RTT, but little is known about HPA axis function among individuals with RTT. Diurnal salivary cortisol patterns from 30 females with RTT were examined in relation to mutation type, medication use, and features of the RTT behavioral phenotype. Cortisol patterns were significantly related to mutation severity, anticonvulsant medication status, and bruxism (tooth grinding). This study provides preliminary support for the hypothesis that RTT may be at risk for outcomes associated with aberrant HPA axis function, and that this risk may be mediated by mutation type.

Evidence of altered salivary cytokine concentrations in Rett syndrome and associations with clinical severity.

Background: Immune dysregulation may play a role in the development of Rett syndrome (RTT), a neurodevelopmental disorder caused by mutations of the MECP2 gene. Abnormal cytokine concentrations have been documented in the serum of individuals with RTT. Measurement of salivary cytokines has been investigated as a potential alternative approach to measurement in blood and serum, but it is unclear whether salivary cytokine concentrations can provide valid information about systemic immune function in neurodevelopmental disorders. The goal of this study was to evaluate the potential validity of salivary cytokines as biomarkers of immune dysregulation in RTT. Methods: Saliva samples from 16 individuals with RTT (all female; age range 2-40 years) and 16 healthy control females (age range 2-40 years) were analyzed for concentrations of 12 cytokines. Between-group differences in concentrations, and correlations with clinical severity in the RTT group were evaluated. Results: Concentrations of several salivary cytokines (IL-1ß, IL-6, IL-8, IL-10, GM-CSF, TNF-α, and VEGF) were increased in RTT compared to controls. The same cytokines showed significant positive correlations with clinical severity scores. There were no differences in concentrations of IL-2, IL-4, IL-5, IL-12p70, and IFN-γ. Conclusion: The results suggest that salivary cytokines may be a possible indicator of immune dysregulation in RTT. Future research should investigate whether these results can be applied to other neurodevelopmental disorders.

Proopiomelanocortin (POMC) sequencing and developmental delay: Preliminary evidence for a SNP in the 3' UTR region of the POMC gene-Possible relevance for biological risk and self-injurious behavior.

The proopiomelanocortin (POMC) molecule has been implicated in models of self-injurious behavior (SIB) in neurodevelopmental disorders, but it has never been specifically sequenced in search of base specific polymorphisms. The empirical focus of this preliminary study was to sequence the POMC gene in 11 children (mean age = 41.8 months, range = 12-60 months; 73% male) with clinical concerns regarding global developmental delay, 5 with reported self-injury. Genomic DNA was extracted from blood samples, and the POMC gene was amplified by specific oligonucleotide primers via polymerase chain reaction. The amplified gene products were sequenced by the University of Minnesota Genomic Center, and the results were analyzed using Sequencher software. A single nucleotide polymorphism (SNP), 1130 C>T, was found in the 3' untranslated region (UTR) of two samples (one of whom had SIB). The program TargetScanHuman was used to predict the function of this mutation. Variant c.1130 C<T was predicted to be located in the target site of two microRNAs (miRNAs; hsa-mir-3715 and hsa-mir-1909), and the variant allele T may result in an increased minimum free energy for the two miRNAs. Further work with much larger samples is needed to continue the investigation of POMC's possible function as a risk factor for the development of SIB in children with developmental delay/disability. The findings presented in this study show that the SNP found in the 3' UTR could alter the binding of miRNAs to POMC 3'UTR, thus, increasing POMC expression and affecting several biological systems with high relevance to the biology of self-injury. There was a significant difference in ß-endorphin levels between SIB (M = 169.25 pg/mL) and no SIB (M = 273.5 pg/mL, SD = 15.2) cases (p < .01). Intervention implications are tied to prior observations of individual differences among SIB responders and nonresponders to treatment with the opioid antagonist naltrexone. Stratifying individuals with SIB by POMC mutation status may provide a potential tailoring-like variable to guide the selection of who is more (or less) likely to respond to opiate antagonist treatment. Currently, opioid antagonistic treatment for SIB is empiric (trial and error).

The FDA approved PI3K inhibitor GDC-0941 enhances in vitro the anti-neoplastic efficacy of Axitinib against c-myc-amplified high-risk medulloblastoma.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/ß in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi-kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti-neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c-myc-amplified Non-WNT/Non-SHH and SHH-TP53-mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/ß and c-kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi-modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC-0941 enhances the anti-neoplastic efficacy of Axitinib against c-myc-amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.

Early assessment of dosimetric and biological differences of total marrow irradiation versus total body irradiation in rodents.

PURPOSE: To develop a murine total marrow irradiation (TMI) model in comparison with the total body irradiation (TBI) model. MATERIALS AND METHODS: Myeloablative TMI and TBI were administered in mice using a custom jig, and the dosimetric differences between TBI and TMI were evaluated. The early effects of TBI/TMI on bone marrow (BM) and organs were evaluated using histology, FDG-PET, and cytokine production. TMI and TBI with and without cyclophosphamide (Cy) were evaluated for donor cell engraftment and tissue damage early after allogeneic hematopoietic cell transplantation (HCT). Stromal derived factor-1 (SDF-1) expression was evaluated. RESULTS: TMI resulted in similar dose exposure to bone and 50% reduction in dose to bystander organs. BM histology was similar between the groups. In the non-HCT model, TMI mice had significantly less acute intestinal and lung injury compared to TBI. In the HCT model, recipients of TMI had significantly less acute intestinal injury and spleen GVHD, but increased early donor cell engraftment and BM:organ SDF-1 ratio compared to TBI recipients. CONCLUSIONS: The expected BM damage was similar in both models, but the damage to other normal tissues was reduced by TMI. However, BM engraftment was improved in the TMI group compared to TBI, which may be due to enhanced production of SDF-1 in BM relative to other organs after TMI.

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition.

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants.

A generic viral dynamic model to systematically characterize the interaction between oncolytic virus kinetics and tumor growth.

Oncolytic viruses (OV) represent an encouraging new therapeutic concept for treatment of human cancers. OVs specifically replicate in tumor cells and initiate cell lysis whilst tumor cells act as endogenous bioreactors for virus amplification. This complex bidirectional interaction between tumor and oncolytic virus hampers the establishment of a straight dose-concentration-effect relation. We aimed to develop a generic mathematical pharmacokinetic/pharmacodynamics (PK/PD) model to characterize the relationship between tumor cell growth and kinetics of different OVs. U87 glioblastoma cell growth and titer of Newcastle disease virus (NDV), reovirus (RV) and parvovirus (PV) were systematically determined in vitro. PK/PD analyses were performed using non-linear mixed effects modeling. A viral dynamic model (VDM) with a common structure for the three different OVs was developed which simultaneously described tumor growth and virus replication. Virus specific parameters enabled a comparison of the kinetics and tumor killing efficacy of each OV. The long-term interactions of tumor cells with NDV and RV were simulated to predict tumor reoccurrence. Various treatment scenarios (single and multiple dosing with same OV, co-infection with different OVs and combination with hypothetical cytotoxic compounds) were simulated and ranked for efficacy using a newly developed treatment rating score. The developed VDM serves as flexible tool for the systematic cross-characterization of tumor-virus relationships and supports preselection of the most promising treatment regimens for follow-up in vivo analyses.

The PI3K inhibitor GDC-0941 displays promising in vitro and in vivo efficacy for targeted medulloblastoma therapy.

Deregulation of the Phosphoinositide 3-kinase (PI3K)/AKT signalling network is a hallmark of oncogenesis. Also medulloblastoma, the most common malignant brain tumor in children, is characterized by high levels of AKT phosphorylation and activated PI3K signalling in medulloblastoma is associated with enhanced cellular motility, survival and chemoresistency underscoring its role of as a potential therapeutic target. Here we demonstrate that GDC-0941, a highly specific PI3K inhibitor with good clinical tolerability and promising anti-neoplastic activity in adult cancer, also displays anti-proliferative and pro-apoptotic effects in pediatric human medulloblastoma cell lines. Loss in cell viability is accompanied by reduced phosphorylation of AKT, a downstream target of PI3K. Furthermore, we show that GDC-0941 attenuates the migratory capacity of medulloblastoma cells and targets subpopulations expressing the stem cell marker CD133. GDC-0941 also synergizes with the standard medulloblastoma chemotherapeutic etoposide. In an orthotopic xenograft model of the most aggressive human medulloblastoma variant we document that oral adminstration of GDC-0941 impairs tumor growth and significantly prolongs survival. These findings provide a rational to further investigate GDC-0941 alone and in combination with standard chemotherapeutics for medulloblastoma treatment.

Newcastle disease virotherapy induces long-term survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death.

The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-γ(+) T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2(-/-) mice and in CD8(+) T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity.

MSC therapy attenuates obliterative bronchiolitis after murine bone marrow transplant.

RATIONALE: Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease. OBJECTIVE: Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model. METHODS: Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT. MEASUREMENTS AND MAIN RESULTS: No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206. CONCLUSIONS: These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.

In comparative analysis of multi-kinase inhibitors for targeted medulloblastoma therapy pazopanib exhibits promising in vitro and in vivo efficacy.

Regardless of the recent advances in cytotoxic therapies, 30% of children diagnosed with medulloblastoma. succumb to the disease. Therefore, novel therapeutic approaches are warranted. Here we demonstrate that Pazopanib a clinically approved multi-kinase angiogenesis inhibitor (MKI) inhibits proliferation and apoptosis in medulloblastoma cell lines. Moreover, Pazopanib profoundly attenuates medulloblastoma cell migration, a prerequisite for tumor invasion and metastasis. In keeping with the observed anti-neoplastic activity of Pazopanib, we also delineate reduced phosphorylation of the STAT3 protein, a key regulator of medulloblastoma proliferation and cell survival. Finally, we document profound in vivo activity of Pazopanib in an orthotopic mouse model of the most aggressive c-myc amplified human medulloblastoma variant. Pazopanib reduced the growth rate of intracranial growing medulloblastoma and significantly prolonged the survival. Furthermore, to put these results into a broader perspective we analysed Pazopanib side by side with the MKI Sorafenib. Both compounds share a similar target profile but display different pharmacodynamics and pharmacokinetics with distinct cytotoxic activity in different tumor entities. Thus, we identified Pazopanib as a new promising candidate for a rational clinical assessment for targeted paediatric medulloblastoma therapy.

TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection.

Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) are synthetic ODNs with unmethylated DNA sequences that mimic viral and bacterial DNA and protect against infectious agents and tumor challenge. We show that CpG ODNs markedly accelerated graft-versus-host disease (GVHD) lethality by Toll-like receptor 9 (TLR9) ligation of host antigen-presenting cells (APCs), dependent upon host IFNgamma but independent of host IL-12, IL-6, or natural killer (NK) cells. Imaging studies showed significantly more green fluorescent protein-positive (GFP(+)) effector T cells in lymphoid and nonlymphoid organs. In engraftment studies, CpG ODNs promoted allogeneic donor bone marrow (BM) rejection independent of host IFNgamma, IL-12, or IL-6. During the course of these studies, we uncovered a previously unknown and critical role of donor BM APCs in modulating the rejection response. CpG ODNs promoted BM rejection by ligation of donor BM, but not host, TLR9. CpG ODNs did not impair engraftment of TLR9(-/-) BM unless wild-type myeloid (CD11b(+)) but not B-lineage (CD19(+)) BM cells were added to the donor inoculum. The importance of donor BM APCs in modulating the strength of the host antidonor rejection response was underscored by the finding that B7-1/B7-2(-/-) BM was less likely than wild-type BM to be rejected. Collectively, these data offer new insight into the mechanism of alloresponses regulating GVHD and BM rejection.

Insights into the mechanism of FTY720 and compatibility with regulatory T cells for the inhibition of graft-versus-host disease (GVHD).

The immunomodulator FTY720 (FTY) has been shown to be beneficial in experimental models of organ transplantation and autoimmunity. We show that FTY significantly inhibited but did not prevent graft-versus-host disease (GVHD) in lethally irradiated or nonirradiated allogeneic recipients. Although most studies implicate prevention of lymphocyte egress from lymphoid organs as the primary mechanism of action, our data indicate that FTY effects on the host are more likely to be responsible for GVHD inhibition. FTY reduced splenic CD11c+ cells by 50%, and similarly reduced CD4+ and CD8+ T-cell responder frequencies in the spleen early after transplantation. Imaging of GFP+ effectors indicated that FTY modified donor effector T-cell migration to secondary lymphoid organs, but did not uniformly trap T cells in lymph nodes or prevent early effector migration to GVHD parenchymal target organs. Administration of FTY only prior to transplantation inhibited GVHD, indicating that the primary function of FTY may be targeted to host cells. FTY was additive with regulatory T cells for GVHD inhibition. FTY slightly impaired but did not abrogate a graft-versus-leukemia (GVL) effect against C1498, a myeloid leukemia. Our data further define the mechanisms of action and provide insight as to the potential clinical uses of FTY in allogeneic bone marrow transplant recipients.

Preformed antibody, not primed T cells, is the initial and major barrier to bone marrow engraftment in allosensitized recipients.

Multiply-transfused individuals are at higher risk for BM rejection. We show that whereas allosensitization resulted in the priming of both cellular and humoral immunity, preformed antibody was the major barrier to engraftment. The generation of cross-reactive alloantibody led to rejection of BM of a different MHC-disparate strain. Imaging studies indicated that antibody-mediated rejection was very rapid (<3 hours) in primed recipients, while T-cell-mediated rejection in nonprimed mice took more than 6 days. Antibody-mediated BM rejection was not due to a defect in BM homing as rejection occurred despite direct intra-BM infusion of donor BM. Rejection was dependent upon host FcR+ cells. BM cells incubated with serum from primed mice were eliminated in nonprimed recipients, indicating that persistent exposure to high-titer antibody was not essential for rejection. High donor engraftment was achieved in a proportion of primed mice by mega-BM cell dose, in vivo T-cell depletion, and high-dose immunoglobulin infusion. The addition of splenectomy to this protocol only modestly added to the efficacy of this combination strategy. These data demonstrate both rapid alloantibody-mediated elimination of BM by host FcR+ cells and priming of host antidonor T cells and suggest a practical strategy to overcome engraftment barriers in primed individuals.

Combined effects of calcineurin inhibitors or sirolimus with anti-CD40L mAb on alloengraftment under nonmyeloablative conditions.

The immunosuppressive drugs, cyclosporine A (CsA), tacrolimus, or sirolimus, were analyzed as single agents and in combination with anti-CD40L monoclonal antibody (mAb) for their effects on alloengraftment in mice conditioned with minimal total body irradiation (TBI). Whereas anti-CD40L mAb facilitated chimerism, neither sirolimus nor CsA resulted in substantial alloengraftment. However, sirolimus was synergistic with anti-CD40L mAb for inducing donor chimerism. Contrary to expectations, CsA, a T-cell receptor (TCR) signaling inhibitor, did not abrogate anti-CD40L mAb-facilitated engraftment but rather increased engraftment in anti-CD40L mAb-treated mice. Although tacrolimus alone or with anti-CD40L mAb resulted in similar levels of donor chimerism, donor T-cell reconstitution was very low in tacrolimus-treated mice. At 1 week after transplantation, CsA decreased thymic numbers more profoundly than sirolimus or tacrolimus in anti-CD40L mAb-treated recipients. In contrast, only sirolimus resulted in a decrease in host splenic T-cell numbers in anti-CD40L mAb-treated recipients. Importantly, sirolimus and anti-CD40L mAb induced profound donor tolerance with 100% acceptance of donor skin grafts placed early after bone marrow transplantation (BMT). In contrast, anti-CD40L mAb alone or in combination with CsA resulted in 12% or less donor skin graft acceptance early (1 month) and 60% or less later (3 months) after BMT. These data have clinical relevance and indicate that immunosuppressive pharmacologic agents enhance anti-CD40L mAb-facilitated alloengraftment and tolerance induction under nonmyeloablative conditioning.