NK Cell and Monocyte Dysfunction in Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multiorgan involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong Ab production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 wk postinfection. Therefore, we hypothesized that dysfunctional cell-mediated Ab responses downstream of Ab production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, whereas 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. Taken together, our results reveal dysregulation in Ab-mediated cellular responses of myeloid and NK cells that likely contribute to the immune pathology of this disease.

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.

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.

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.

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.

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.

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.

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.

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.