Adoptive Transfer Studies Research Articles

Sepsis erodes CD8+ memory Tcell-protective immunity against an EBV homolog in a 2B4-dependent manner.

Epstein-Barr virus (EBV) reactivation commonly occurs following sepsis, but the mechanisms underlying this are unknown. We utilized a murine EBV homolog (gHV) and the cecal ligation and puncture model of polymicrobial sepsis to study the impact of sepsis on gHV reactivation and CD8+ Tcell immune surveillance following a septic insult. We observed a significant increase in the frequency of gHV-infected germinal center B cells on day 7 following sepsis. This increase in viral load was associated with a concomitant significant decrease in the frequencies of gHV-specific CD8+ Tcells, as measured by class I MHC tetramers corresponding to the immunodominant viral epitopes. Phenotypic analysis revealed an increased frequency of gHV-specific CD8+ Tcells expressing the 2B4 coinhibitory receptor in septic animals compared with sham controls. We sought to interrogate the role of 2B4 in modulating the gHV-specific CD8+ Tcell response during sepsis. Results indicated that in the absence of 2B4, gHV-specific CD8+ Tcell populations were maintained during sepsis, and gHV viral load was unchanged in 2B4-/- septic animals relative to 2B4-/- sham controls. WT CD8+ Tcells upregulated PD-1 during sepsis, whereas 2B4-/- CD8+ Tcells did not. Finally, adoptive transfer studies revealed a Tcell-intrinsic effect of 2B4 coinhibition on virus-specific CD8+ Tcells and gHV viral load during sepsis. These data demonstrate that sepsis-induced immune dysregulation erodes antigen-specific CD8+ responses against a latent viral infection and suggest that blockade of 2B4 may better maintain protective immunity against EBV in the context of sepsis.

Cutting Edge: Local Proliferation of Uterine Tissue-Resident NK Cells during Decidualization in Mice.

NK cells accumulate in adult murine and human uteri during decidualization induced physiologically, pathologically, or experimentally. Adoptive transfer studies indicate that uterine NK (uNK) cells arise from circulating progenitors. However, virgin uteri contain few circulating NK1.1+CD49a- conventional NK cells, whereas NK1.1+CD49a+ tissue-resident NK (trNK) cells are abundant. In this study, we employed a novel, immune-competent NK cell-specific reporter mouse to track accumulation of uNK cells during unmanipulated pregnancies. We identified conventional NK and trNK cells accumulating in both decidua basalis and myometrium. Only trNK cells showed evidence of proliferation. In parabiosis studies using experimentally induced deciduomata, the accumulated uNK cells were proliferating trNK cells; migrating NK cells made no contribution. Together, these data suggest proliferating trNK cells are the source of uNK cells during endometrial decidualization.

Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

The latent viral reservoir is the critical barrier for the development of an HIV-1 cure. Previous studies have shown that potent HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) administered at the time of antiretroviral therapy (ART) discontinuation can exert direct antiviral effects, but whether bNAbs can target the viral reservoir during ART suppression remains unknown. Here we show that the V3 glycan-dependent bNAb PGT121 together with the TLR7 agonist vesatolimod (GS-9620) administered during ART suppression delayed viral rebound following ART discontinuation in SHIV-SF162P3-infected rhesus monkeys that initiated ART during early acute infection. Moreover, the subset of PGT121+GS-9620 treated monkeys that did not show viral rebound following ART discontinuation also did not reveal virus by highly sensitive adoptive transfer and CD8 depletion studies. These data demonstrate the potential of bNAb administration together with innate immune stimulation as a possible strategy to target the viral reservoir.

CD160 Stimulates CD8+ T Cell Responses and Is Required for Optimal Protective Immunity to Listeria monocytogenes.

CD160 promotes NK cell cytotoxicity and IFN-γ production, but the function of CD160 on CD8+ T cells remains unclear with some studies supporting a coinhibitory role and others a costimulatory role. In this study, we demonstrate that CD160 has a costimulatory role in promoting CD8+ T cell effector functions needed for optimal clearance of oral Listeria monocytogenes infection. CD160-/- mice did not clear oral L. monocytogenes as efficiently as wild type (WT) littermates. WT RAG-/- and CD160-/- RAG-/- mice similarly cleared L. monocytogenes, indicating that CD160 on NK cells does not contribute to impaired L. monocytogenes clearance. Defective L. monocytogenes clearance is due to compromised intraepithelial lymphocytes and CD8+ T cell functions. There was a reduction in the frequencies of granzyme B-expressing intraepithelial lymphocytes in L. monocytogenes-infected CD160-/- mice as compared with WT littermate controls. Similarly, the frequencies of granzyme B-expressing splenic CD8+ T cells and IFN-γ and TNF-α double-producer CD8+ T cells were significantly reduced in L. monocytogenes-infected CD160-/- mice compared with WT littermates. Adoptive transfer studies showed that RAG-/- recipients receiving CD160-/- CD8+ T cells had a higher mortality, exhibited more weight loss, and had a higher bacterial burden compared with RAG-/- recipients receiving WT CD8+ T cells. These findings demonstrate that CD160 provides costimulatory signals to CD8+ T cells needed for optimal CD8+ T cell responses and protective immunity during an acute mucosal bacterial infection.

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model. MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS). ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α+ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α− DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS. ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

Tumor necrosis factor receptor 2 promotes neuroprotection during chronic autoimmune neuroinflammation

Abstract Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS) which affects over 400,000 Americans and over 2.5 million people worldwide. Although most patients are initially diagnosed with relapsing-remitting MS, the majority of these patients later develop a chronic-progressive form of MS, for which there is no well-established mouse model. The most common genetic factor associated with MS susceptibility is the Human Leukocyte Antigen (HLA)-DR2b haplotype. Additionally, studies in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, showed that lack of TNF signaling through its receptor TNFR2 leads to disease exacerbation and severe demyelination. Here, we developed a mouse model which expresses HLA-DR2b and lacks TNFR2, designated DR2bΔR2. Strikingly, DR2bΔR2 mice develop progressive EAE with pathology and clinical features observed in progressive MS patients. Adoptive transfer studies revealed that the clinical phenotype of EAE in DR2bΔR2 mice are largely dependent on TNFR2 expression in the CNS. Subsequently, we showed that DR2bΔR2 mice have a significant increase of lesions in the cerebellum, associated with reduced of oligodendrocyte progenitor cells (OPC) recruitment or function. Moreover, we showed that DR2bΔR2 mice are presented with chronic astrogliosis in demyelinating lesions. Our studies provide key insights into CNS repair and regulatory mechanisms controlled by TNFR2 during neuroinflammation and provide novel therapeutic strategies for treating progressive MS.

Analysis of adenovirus-induced immunity to infection with Listeria monocytogenes: Fading protection coincides with declining CD8 T cell numbers and phenotypic changes

Defining correlates of T cell mediated protection is important in order to accelerate the development of efficient T cell based vaccines conferring long-term immunity.Extensive studies have provided important insight regarding the characteristics and functional properties of the effector and memory CD8 T cells induced by viral vector based vaccines. However, long-term protection has been difficult to achieve with T cell inducing vaccines, and the determinants underlying this loss in protection over time are still not fully defined.In this study we analyzed different parameters of the CD8 T cell response as a function of time after vaccination with a human serotype 5 adenovector expressing the glycoprotein (GP) of LCMV tethered to the MHC class II-associated invariant chain. Using this vector we have previously found that CD8 T cells mediate protection from challenge with GP-expressing Listeria monocytogenes at 60 days post vaccination, but only little protection after further 60 days, and we now confirm this observation. A comparison of vaccine-primed CD8 T cells early and late after vaccination revealed a minor decline in the overall numbers of antigen specific memory CD8 T cells during this interval. More importantly, we also observed phenotypic changes over time with a distinct decline in the frequency and number of KLRG1+ CD8 T cells, and, notably, adoptive transfer studies confirmed that memory CD8 T cells expressing KLRG1 are central to protection from systemic L. monocytogenes infection. Together these findings imply that multiple factors including changes in memory T cell numbers and phenotypic composition over time influence the longevity of CD8 T-cell mediated protection.

Lymphatic endothelial cells regulate B-cell homing to lymph nodes via a NIK-dependent mechanism.

B cells home to the lymph nodes (LNs) via high endothelial venules (HEVs) under the guidance of chemokines, particularly CXCL13. However, as CXCL13 is not directly made in HEVs, the molecular mechanism mediating B-cell homing to LNs has remained unclear. We show here that nuclear factor (NF)-κB-inducing kinase (NIK), a kinase mediating activation of the noncanonical NF-κB pathway, functions in lymphatic endothelial cells (LECs) to regulate B-cell homing to LNs. LEC-conditional deletion of NIK in mice did not affect the integrity or global function of lymphatic vessels but caused a severe reduction in the frequency of B cells in LNs. The LEC-specific NIK deficiency did not affect the survival of B cells or the frequency of B cells in the spleen. B-cell adoptive transfer studies revealed that the LEC-specific NIK deletion impairs the ability of LNs to recruit B cells. We further show that NIK mediates expression of the chemokines CXCL13 and CCL19 in LECs. Although CCL19 is also expressed in blood endothelial cells (BECs), CXCL13 is not produced in BECs. These results suggest that NIK regulates naive B-cell homing to LNs via mediating production of the B-cell homing chemokine CXCL13 in LECs.

Neonatal adoptive transfer of lymphocytes rescues social behaviour during adolescence in immune-deficient mice.

Accumulating evidence has shown that lymphocytes modulate behaviour and cognition by direct interactions with the central nervous system. Studies have shown that reconstitution by adoptive transfer of lymphocytes from wild type into immune-deficient mice restores a number of neurobehavioural deficits observed in these models. Moreover, it has been shown that these effects are mostly mediated by T lymphocytes. Studies of adoptive transfer thus far have employed adult mice, but whether lymphocytes may also modulate behaviour during development remains unknown. In this study, neonate lymphocyte-deficient Rag2-/- mice were reconstituted within 48hours after birth with lymphoid cells from transgenic donors expressing green fluorescent protein, allowing for their identification in various tissues in recipient mice while retaining all functional aspects. Adolescent Rag2-/- and reconstituted Rag2-/- along with C57BL/6J wild-type mice underwent a series of behavioural tests, including open field, social interaction and sucrose preference tests. At 12weeks, they were evaluated in the Morris water maze (MWM). Reconstituted mice showed changes in almost all aspects of behaviour that were assessed, with a remarkable complete rescue of impaired social behaviour displayed by adolescent Rag2-/- mice. Consistent with previous reports in adult mice, neonatal reconstitution in Rag2-/- mice restored spatial memory in the MWM. The presence of donor lymphocytes in the brain of neonatally reconstituted Rag2-/- mice was confirmed at various developmental points. These findings provide evidence that lymphocytes colonize the brain during post-natal development and modulate behaviour across the lifespan supporting a role for adaptive immunity during brain maturation.

Abstract IA24: Identification of therapies for plexiform neurofibromas, a precursor of malignant peripheral nerve sheath tumors

Abstract Plexiform neurofibromas (PNs) are complex nerve and soft tissue tumors that affect 25-50% of individuals with neurofibromatosis type 1 (NF1), the most common genetic disease in man with a predisposition to cancer. PNs cause lifelong morbidity ranging from deformity to paralysis and mortality due to organ compression, and progress to MPNST in ~8-13% of patients. The generation of genetically engineered mouse models that closely recapitulate the development of human plexiform neurofibromas has allowed our laboratory and others to pursue genetic and pharmacologic studies to identify novel therapies. Genetic and adoptive transfer studies conducted by our lab in collaboration with Dr. Luis Parada identified a key role for c-kit/kit-ligand in tumor initiation and progression. These data led to a clinical trial administering imatinib mesylate to target aberrant stem cell factor (SCF)/c-kit signaling in the tumor microenvironment, which induced the only objective clinical responses ever obtained in a therapeutic clinical trial in pNF. Based on the observation that young children with head, neck, and airway tumors were most likely to have partial tumor regression, a registration trial in this patient population is currently being pursued. Further, additional phase 2 trials using other receptor tyrosine kinases that include c-kit are under way as well. More recently, studies in GEM models informed clinical trials of MEK inhibitors led by Dr. Brigitte Widemann (Pediatric Branch of NCI) to directly inhibit aberrant Ras signaling in PN cells. Studies are ongoing to extend these promising initial clinical findings to investigate the developmental, molecular, and pharmacokinetic effects of c-kit and MEK inhibition alone and in combination in preclinical models. Specifically, studies to explore adaptive responses to MEK inhibition monotherapy in mice and patients treated with selumetinib via integrated exome, RNAseq, and kinome studies in pre- and post-treatment tumor tissue are under way. Citation Format: D. Wade Clapp. Identification of therapies for plexiform neurofibromas, a precursor of malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA24.

Exacerbation of Acute Traumatic Brain Injury by Circulating Extracellular Vesicles.

Inflammatory lesions in the brain activate a systemic acute-phase response (APR), which is dependent on the release of extracellular vesicles (EVs) into the circulation. The resulting APR is responsible for regulating leukocyte mobilization and subsequent recruitment to the brain. Factors that either exacerbate or inhibit the APR will also exacerbate or inhibit central nervous system (CNS) inflammation as a consequence and have the potential to influence ongoing secondary damage. Here, we were interested to discover how the circulating EV population changes after traumatic brain injury (TBI) and how manipulation of the circulating EV pool impacts on the outcome of TBI. We found the number of circulating EVs increased rapidly post-TBI, and this was accompanied by an increase in CNS and hepatic leukocyte recruitment. In an adoptive transfer study, we then evaluated the outcomes of TBI after administering EVs derived from either in vitro macrophage or endothelial cell lines stimulated with lipopolysaccharide (LPS), or from murine plasma from an LPS challenge using the air-pouch model. By manipulating the circulating EV population, we were able to demonstrate that each population of transferred EVs increased the APR. However, the characteristics of the response were dependent on the nature of the EVs; specifically, it was significantly increased when animals were challenged with macrophage-derived EVs, suggesting that the cellular origins of EVs may determine their function. Selectively targeting EVs from macrophage/monocyte populations is likely to be of value in reducing the impact of the systemic inflammatory response on the outcome of traumatic CNS injury.

Induction of immunity following vaccination with a chemically attenuated malaria vaccine correlates with persistent antigenic stimulation.

ObjectivesBlood stage malaria parasites attenuated with seco‐cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction.MethodsMice were vaccinated with chemically attenuated Plasmodium chabaudi parasites. PCR and adoptive transfer studies were used to determine the presence of parasites and antigen in vivo. In other experiments, Plasmodium falciparum parasitised red blood cells were attenuated in vitro and RNA and antigen expression studied.ResultsWe show that blood transferred from vaccinated mice into naïve mice activates T cells and induces complete protective immunity in the recipient mice strongly suggesting that there is persistence of parasite antigen postvaccination. This is supported by the presence of parasite RNA in vaccinated mice and both RNA and antigen expression in P. falciparum cultures treated with CPI drugs in vitro. In addition, drugs that block parasite growth also prevent the induction of immunity in vaccinated mice, indicating that some growth of attenuated parasites is required for immune induction.ConclusionsAttenuated parasites persist at submicroscopic levels in the blood of mice postvaccination with the ability to activate T cells and induce ongoing protective immune responses.

The Spleen Promotes the Secretion of CCL2 and Supports an M1 Dominant Phenotype in Hepatic Macrophages During Liver Fibrosis

Background/Aims: Liver fibrosis is a complex process of tissue remodeling in response to injury. Hepatic macrophages have been identified as a key player in this process. As the largest lymphoid organ in the body, the spleen exerts both local and systemic effects on immune cell response. Splenectomy can improve hepatic function during the treatment of liver cirrhosis. However, whether the spleen influences disease progression through the modulation of hepatic macrophages remains unclear. Methods: We examined ex vivo hepatic macrophage responses from splenectomized or sham operated rats and performed splenocyte adoptive transfer studies, in combination with in vivo CCL2 blockade, in splenectomized or sham operated rats. Results: We found that splenectomy reduced fibrosis severity and monocyte/ macrophage infiltration within the injured liver. Splenectomy also reduced secretion of the monocyte chemokine CCL2 by hepatic macrophages. Ex vivo, splenocytes, especially splenic macrophages, promoted CCL2 secretion via upregulation of SOCS3 signaling in hepatic macrophages. Migration of splenic monocytes in response to conditioned medium from hepatic macrophages was inhibited by the blockade of SOCS3–CCL2–CCR2 signaling. Splenectomy also attenuated the establishment of an M1-dominant hepatic macrophage phenotype whilst the adoptive transfer of splenocytes could partly reverse this effect and exacerbate fibrosis. However, CCL2 blockade following adoptive splenocyte transfer restored the protective effects of splenectomy. Conclusion: Our study demonstrates that splenic macrophages can promote hepatic macrophage secretion of CCL2, which in turn facilitates monocyte recruitment and the establishment of an M1-dominant hepatic macrophage phenotype, and thus increase the severity of liver fibrosis.

Anti-CD20 Antibody Prevents Red Blood Cell Alloimmunization in a Mouse Model.

Alloimmunization against RBCs can cause life-threatening delayed hemolytic transfusion reactions. Anti-CD20 Ab has recently been used to prevent alloimmunization. However, its effects remain unclear, particularly in lymphoid organs. We investigated the impact of murine anti-CD20 Ab in the blood and spleen. We assessed protocols for preventing primary alloimmunization and for abolishing established alloimmunization. Prophylactic protocols prevented alloimmunization. However, anti-CD20 treatment could only limit the further amplification of established alloimmunization. Residual B cell subtype distribution was disrupted in the spleen, but adoptive transfer studies indicated that these cells were neither plasma nor memory cells. Anti-CD20 Ab had a major effect on alloreactive CD4+ T cells, increasing the expansion of this population and its CD40 expression, while lowering its CD134 expression, thereby confirming its role in alloimmunization. In conclusion, this study shows that anti-CD20 immunotherapy can prevent RBC Ab development. However, this immunotherapy is limited by the increase in alloreactive CD4+ T lymphocytes. Nevertheless, treatment with anti-CD20 Abs should be considered for patients requiring transfusion with a very high risk of alloimmunization and life-threatening complications.

Dengue virus-reactive CD8+ T cells mediate cross-protection against subsequent Zika virus challenge

Zika virus (ZIKV) and dengue virus (DENV) are antigenically related flaviviruses that share cross-reactivity in antibody and T cell responses, and co-circulate in increasing numbers of countries. Whether pre-existing DENV immunity can cross-protect or enhance ZIKV infection during sequential infection of the same host is unknown. Here, we show that DENV-immune Ifnar1−/− or wild-type C57BL/6 mice infected with ZIKV have cross-reactive immunity to subsequent ZIKV infection and pathogenesis. Adoptive transfer and cell depletion studies demonstrate that DENV-immune CD8+ T cells predominantly mediate cross-protective responses to ZIKV. In contrast, passive transfer studies suggest that DENV-immune serum does not protect against ZIKV infection. Thus, CD8+ T cell immunity generated during primary DENV infection can confer protection against secondary ZIKV infection in mice. Further optimization of current DENV vaccines for T cell responses might confer cross-protection and prevent antibody-mediated enhancement of ZIKV infection.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

PurposeWe investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)–derived cells to the neurosensory retina (NSR) and RPE layer.MethodsGFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 μm) with 30 applications were placed 50 to 100 μm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed.ResultsWithin 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle–dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid.ConclusionsSRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle–dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Distinctive Roles for α7*- and α9*-Nicotinic Acetylcholine Receptors in Inflammatory and Autoimmune Responses in the Murine Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

Previous studies have demonstrated immunosuppressive and anti-inflammatory effects of nicotine, including in the experimental autoimmune encephalomyelitis (EAE) model in mice of some forms of multiple sclerosis (MS). Other studies using knock-out (KO) mice have implicated nicotinic acetylcholine (ACh) receptors containing α7, α9, or β2 subunits (α7*-, α9*- or β2*-nAChR) in different, disease-exacerbating or disease-ameliorating processes. These outcomes are in harmony with gene expression analyses showing nAChR subunit mRNA in many classes of immune system cell types. Consistent with influences on disease status, predictable effects of nAChR subunit (and subtype) KO, or of nicotine exposure, are seen on immune cell numbers and distribution and on cytokine levels or other markers of immunity, inflammation, demyelination, and axonal degradation. Providing support for our hypotheses about distinctive roles for nAChR subtypes in EAE, here we have used direct and adoptive EAE induction and a nAChR subunit gene double knock-out (DKO) strategy. Immune cell expression of nAChR α9 subunits as protein is demonstrated by immunostaining of isolated CD4+, CD8+, CD11b+ and CD11c+ cells from wild-type (WT) mice, but not in cells from nAChR α9 subunit KO animals. Nicotine exposure is protective against directly-induced EAE in WT or α7/α9 DKO animals relative to effects seen in WT/vehicle-treated mice, but, remarkably, EAE is exacerbated in vehicle-treated α7/α9 DKO mice. Brain lesion volume and intra-cranial inflammatory activity similarly are higher in DKO/vehicle than in WT/vehicle-treated animals, although nicotine’s protective effects are seen in each instance. By contrast, in adoptive transfer studies, disease severity is attenuated and disease onset is delayed in recipients of splenocytes from WT animals treated with nicotine rather than with vehicle. Moreover, protection as seen in nicotine-treated WT animals is the same in recipients of splenocytes from nAChR α7/α9 DKO mice irrespective of their exposure to nicotine or vehicle. When combined with previous observations, these findings are consistent with disease exacerbation (or even induction) being mediated at least in part via α9*-nAChR in peripheral immune cells. They also suggest protective roles of central nervous system (CNS) α7*-nAChR. The results suggest that both α7*- and α9*-nAChR are potential targets of therapeutic ligands to modulate inflammation and autoimmunity.

Abstract 001: Sympathetic Innervation Promotes Bone Marrow Homing of Hypertension-specific CD8 + Effector Memory T Cells

We have recently identified a critical role of hypertension-specific effector memory T lymphocytes (T EM cells) in the blood pressure elevation and renal dysfunction caused by repeated hypertensive stimuli. Formed during an initial immune challenge, T EM cells reside in the bone marrow (BM) in a quiescent state for prolonged periods, and can be reactivated upon re-exposure to the hypertensive stimulus. Hypertension is associated with increased sympathetic outflow. We therefore hypothesized that sympathetic nerves regulate accumulation and reactivation of hypertension-specific T EM cells in the BM. We performed unilateral superior cervical ganglionectomy (SCGx) in wild-type C57BL/6 mice, causing selectively sympathectomy of the forelimb on the surgical side. After recovery, mice received Ang II infusion for two weeks. To determine the changes of T cells in the BM that were specific to hypertension, 5х10 6 BM cells were isolated from either the SCGx or control limbs, loaded with proliferation marker CFSE, and co-cultured with 0.5х10 6 splenic dendritic cells isolated from another Ang II-infused mouse. We found 30% less CD8 + T cell proliferation in the SCGx BM compared to control side (1.8±0.1 vs. 2.6±0.3х10 4 ), but no difference in CD4 + T cells. To further study the effect of sympathetic nerves on BM T cell homing, 1х10 7 pan T cells were isolated from CD45.2 + wild-type mice after Ang II infusion and adoptively transferred to CD45.1 mice that had previously undergone unilateral SCGx. Flow cytometry indicated that 7 days after transfer, 25% fewer CD8 + T EM cells from the hypertensive donors homed to the SCGx BM compared to the innervated BM (27.8±2.6 vs. 20.8±2.5 per 10 3 total BM cells). This effect was specific for hypertension as homing of OT-I T EM cells, which are responsive to ovalbumin, was not influenced by sympathectomy. Further adoptive transfer studies using mice lacking beta 2 adrenergic receptors (β2AR) indicate that β2AR in the bone marrow niche, rather than T cell β2AR is critical for T EM cell homing. These data define a novel role of sympathetic nerves in regulation of memory T cell trafficking, and this likely contributes to the predisposition to hypertension and end-organ damage for prolonged periods following an initial episode of hypertension.

Dynamics of Recent Thymic Emigrants in Young Adult Mice.

The peripheral naive T-cell pool is generally thought to consist of a subpopulation of recent thymic emigrants (RTEs) and a subpopulation of mature naive (MN) T cells with different dynamics. Thymus transplantation and adoptive transfer studies in mice have provided contradicting results, with some studies suggesting that RTEs are relatively short-lived cells, while another study suggested that RTEs have a survival advantage. We here estimate the death rates of RTE and MN T cells by performing both thymus transplantations and deuterium labeling experiments in mice of at least 12 weeks old, an age at which the size of the T-cell pool has stabilized. For CD4+ T cells, we found the total loss rate from the RTE compartment (by death and maturation) to be fourfold faster than that of MN T cells. We estimate the death rate of CD4+ RTE to be 0.046 per day, which is threefold faster than the total loss rate from the MN T-cell compartment. For CD8+ T cells, we found no evidence for kinetic differences between RTE and MN T cells. Thus, our data support the notion that in young adult mice, CD4+ RTE are relatively short-lived cells within the naive CD4+ T-cell pool.