Peripheral Lymphoid Tissues Research Articles

In Vivo Generation of Gut-Homing Regulatory T Cells for the Suppression of Colitis.

Current therapies for gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that direct in vivo augmentation of the induction of gut-homing regulatory T (Treg) cells is a strategy of expected specificity for the treatment of chronic intestinal inflammation (e.g., inflammatory bowel disease). We showed that dendritic cells (DCs), engineered to de novo produce high concentrations of both 1,25-dihydroxyvitamin D, the active vitamin D metabolite, and retinoic acid, an active vitamin A metabolite, augmented the induction of T cells that express both the regulatory molecule Foxp3 and the gut-homing receptor CCR9 in vitro and in vivo. In vivo, the newly generated Ag-specific Foxp3+ T cells homed to intestines. Additionally, transfer of such engineered DCs robustly suppressed ongoing experimental colitis. Moreover, CD4+ T cells from spleens of the mice transferred with the engineered DCs suppressed experimental colitis in syngeneic hosts. The data suggest that the engineered DCs enhance regulatory function in CD4+ T cell population in peripheral lymphoid tissues. Finally, we showed that colitis suppression following in vivo transfer of the engineered DCs was significantly reduced when Foxp3+ Treg cells were depleted. The data indicate that maximal colitis suppression mediated by the engineered DCs requires Treg cells. Collectively, our data support that DCs de novo overproducing both 1,25-dihydroxyvitamin D and retinoic acid are a promising novel therapy for chronic intestinal inflammation.

Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3–4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.

OR12-2 Obesity Induces Changes in T Cell Metabolism and Function

Low-grade inflammation is responsible for many obesity-associated pathologies such as insulin resistance, type 2 diabetes and reduction in protective immunity. T cells play an important role in mounting an effective immune response and the modulation of inflammation, and have been identified as a key player in obesity-associated inflammation. Altered T cell metabolism can influence T cell differentiation and function; however, it is not yet known if or how obesity influences the T cell metabolic state. We hypothesized that diet induced obesity promotes changes in T cell metabolism leading to increased pro-inflammatory T cells function. To test our hypothesis, we assessed the effect of diet-induced obesity (DIO) on T cell subpopulation number and metabolism in the visceral adipose tissue (VAT) and peripheral lymphoid tissue (spleen). To achieve that, C57BL/6 mice were placed on high fat (DIO mice) or regular chow diet (control mice) at weaning and remained on diet for 18 weeks. VAT and spleen were collected and T cells were isolated and analyzed by flow cytometry for: T cells subpopulation markers, glucose uptake, and cytokine production. In addition, the expression of almost 240 enzymes involved in several metabolic pathways were measured in CD4+ T cells by RT-qPCR. We found that proportions of Treg cells were decreased in the VAT of DIO mice while CD8+ T cells increased, indicating an inflammatory environment in the adipose tissue of the DIO mice. CD4+ T cells isolated from the spleen of DIO mice were also found to be metabolically altered with increased glucose uptake and elevated oxygen consumption rate (OCR; a marker of mitochondrial metabolism). The spare respiratory capacity, an indicator of mitochondrial reserve, of CD4+ T cells from DIO mice was found to be reduced by the fatty acids oxidation inhibitor Etomoxir, while the pyruvate transport inhibitor UK5099 was found to have no significant effect, indicating a preference toward the use of fatty acids as a mitochondrial fuel source. The metabolic qPCR panel showed slight differences in expression between control vs. DIO mice with a tendency toward a decrease in the expression of enzymes involved in oxidative phosphorylation and fatty acid transport along with an increase in enzymes that positively influence glycolysis in DIO mice. Lastly, the antidiabetic drug metformin was found to inhibit OCR and alter the expression of several activation markers in CD4+ cells in vitro and in vivo, indicating that metformin may be an effective candidate to reverse T cell metabolic dysfunction and thereby restore T cell dysfunction in obesity. Research Support: National Institutes of Health grant R01-DK106090 and Derfner Foundation Research Award 2018.

Myeloid cell‐derived IL‐6 in Chronic Lymphocytic Leukemia

First‐line therapies, like chemo immunotherapy have limitation that lead patients to build resistance to mainstream treatments for chronic lymphocytic leukemia. Traditionally, the focus has been on leukemic cells. Conversely, little attention has been brought to the influence myeloid cells have on CLL progression. This study focused on the production of IL‐6 specifically by monocytes, macrophages, and myeloid‐derived suppressor cells (MDSCs) in the microenvironment of CLL cells. Experimentation revealed an increase of myeloid cell‐derived IL‐6 production in the peripheral blood and lymphoid tissues of Eμ‐TCL1 transgenic mice. Ultimately, these findings can be the basis for exploration of new treatments that target myeloid cells, and can be presented as an alternative for patients who have acquired a resistance to current therapies for CLL.Support or Funding InformationSM was supported by the CPRIT Research Training Program RP170067This study was supported by CLL Global Research Foundation (to MTSB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HIV Replication in Humanized IL-3/GM-CSF-Transgenic NOG Mice.

The development of mouse models that mimic the kinetics of Human Immunodeficiency Virus (HIV) infection is critical for the understanding of the pathogenesis of disease and for the design of novel therapeutic strategies. Here, we describe the dynamics of HIV infection in humanized NOD/Shi-scid-IL2rγnull (NOG) mice bearing the human genes for interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (NOG-EXL mice). The kinetics of viral load, as well as the frequencies of T-cells, B-cells, Natural killer cells (NK), monocytes, and dendritic cells in blood and secondary lymphoid organs were evaluated throughout the time of infection. In comparison with a non-transgenic humanized mouse (NSG) strain, lymphoid and myeloid populations were more efficiently engrafted in humanized NOG-EXL mice, both in peripheral blood and lymphoid tissues. In addition, HIV actively replicated in humanized NOG-EXL mice, and infection induced a decrease in the percentage of CD4+ T-cells, inversion of the CD4:CD8 ratio, and changes in some cell populations, such as monocytes and dendritic cells, that recapitulated those found in human natural infection. Thus, the humanized IL-3/GM-CSF-transgenic NOG mouse model is suitable for the study of the dynamics of HIV infection and provides a tool for basic and preclinical studies.

Human Interleukin-34 facilitates microglia-like cell differentiation and persistent HIV-1 infection in humanized mice

BackgroundMicroglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell’s presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration.MethodsHerein, we created a humanized bone-marrow chimera producing human “microglia like” cells in NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain.ResultsHuman interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses.ConclusionsThis humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.

Increased Peripheral CD137 Expression in a Mouse Model of Permanent Focal Cerebral Ischemia.

Various studies demonstrate that CD137 (TNFRSF9, 4-1BB) promotes atherosclerosis and vascular inflammation in experimental models via interactions with the CD137 ligand (CD137L). However, the exact role of CD137 in ischemic stroke remains unclear. In this study, we analyzed dynamic changes of peripheral CD137 expression on T cells in a mouse model of cerebral ischemia-middle cerebral artery occlusion (MCAO), as well as alternation of neurological function, infarct size and cerebral inflammatory status after inhibition of the CD137/CD137L pathway using an anti-CD137L monoclonal antibody. MCAO mice showed elevated surface expression of CD137 on T cells in both peripheral blood and lymphoid tissues during early cerebral ischemia. Remarkably, blockade of the CD137/CD137L pathway reduced the post-ischemic brain damage. Our findings indicate that enhanced CD137 costimulation occurs in early cerebral ischemia and promotes T cell activation, which in turn upregulates inflammatory immune response and possibly exerting deleterious effects on cerebral ischemia.

The thymus is relevant in the migration of mature lymphocytes.

The thymus is a primary lymphoid organ where T lymphocyte proliferation and selection takes place. The different subsets of lymphocytes leave the thymus as recent thymic emigrants. Peripheral dendritic cells migrate to the thymus. In addition to the homing of hematopoietic progenitor cells to the thymus, there is evidence for lymphocyte entry from peripheral lymphoid tissues mainly into the medulla. The entry sites are the venules in the medullary part near to the cortex with a higher endothelium. Furthermore, there are also B lymphocytes in the thymus. The thymus is not only a primary lymphoid organ but is well integrated in lymphocyte traffic as shown in several different species.

8O - 89Zr-labeled anti-PD-L1 CX-072 PET imaging in human xenograft and syngeneic tumors

Background: Immune checkpoint inhibiting antibodies have antitumor activity across several tumor types, but can also elicit immune-mediated adverse events (imAEs). CX-072 is an investigational antibody prodrug (Probody™ therapeutic), reactive to the murine and human programmed death-ligand 1 (PD-L1) immune checkpoint. CX-072 can be activated to a fully avid antibody by tumor-associated proteases that remove the masking peptides blocking the antigen-binding domain, which may limit peripheral PD-L1 binding and therefore imAEs. CX-072 was radiolabeled with zirconium-89 (89Zr) to study its biodistribution and tumor- versus lymphoid tissue-targeting properties using positron emission tomography (PET). Methods: CX-072, non-specific Probody™ therapeutic control (PbCtrl) and CX-072 parental antibody (CX-075) were radiolabeled with 89Zr. Human PD-L1-expressing MDA-MB-231 tumor-bearing BALB/c nude mice received 10 μg 89Zr-CX-072, 89Zr-PbCtrl or 89Zr-CX-075 (∼5 MBq) supplemented with 0, 40 or 240 µg of respective unlabeled antibody. Biodistribution was also evaluated in C57BL6/J mice bearing PD-L1-expressing MC38 syngeneic colon adenoma tumors. Mice underwent PET scans 1, 3 and 6 days post intravenous injection (pi), followed by ex vivo analysis. Results: In MDA-MB-231 tumors, 89Zr-CX-072 and 89Zr-CX-075 showed comparable uptake of 8.7 ± 1.0%ID/g and 8.8 ± 2.9%ID/g respectively for the 10 µg total protein dose, which is 2.3-fold higher compared to 89Zr-PbCtrl. Autoradiography showed 89Zr-CX-072 localization in PD-L1-positive tumor tissue areas on immunohistochemistry. Activated CX-072 was detected by capillary electrophoresis immunoassay mainly in MDA-MB-231 tumor, with limited amounts found in spleen. Flow cytometry confirmed PD-L1 expression in lymphoid tissues of C57BL6/J mice, while 89Zr-CX-072 uptake in these tissues was similar to 89Zr-PbCtrl and significantly lower compared to 89Zr-CX-075. Conclusions:89Zr-CX-072 accumulates in PD-L1-expressing tumors with minor uptake in murine peripheral lymphoid tissues, confirming that the CX-072 structure limits uptake in non-tumor, PD-L1-expressing tissues. We developed clinical grade 89Zr-CX-072 to study its whole-body distribution in patients. Legal entity responsible for the study: Dept. of Medical Oncology, University Medical Center Groningen (UMCG), The Netherlands. Funding: CytomX Therapeutics Inc. Disclosure: I. Popova, B. Howng, O. Vasiljeva: Employee: CytomX Therapeutics Inc. E.G.E. de Vries: Research funding: CytomX was made available to her institution (UMCG); Advisory boards: Pfizer, Daiichi Sankyo; Other funding: Amgen, Bayer, Chugai, G1 Therapeutics, Genentech/Roche, Nordic Nanovector, Regeneron, Synthon, all payments to UMCG. All other authors have declared no conflicts of interest.

Defective Autophagy in T Cells Impairs the Development of Diet-Induced Hepatic Steatosis and Atherosclerosis.

Macroautophagy (or autophagy) is a conserved cellular process in which cytoplasmic cargo is targeted for lysosomal degradation. Autophagy is crucial for the functional integrity of different subsets of T cells in various developmental stages. Since atherosclerosis is an inflammatory disease of the vessel wall which is partly characterized by T cell mediated autoimmunity, we investigated how advanced atherosclerotic lesions develop in mice with T cells that lack autophagy-related protein 7 (Atg7), a protein required for functional autophagy. Mice with a T cell-specific knock-out of Atg7 (Lck-Cre Atg7f/f) had a diminished naïve CD4+ and CD8+ T cell compartment in the spleen and mediastinal lymph node as compared to littermate controls (Atg7f/f). Lck-Cre Atg7f/f and Atg7f/f mice were injected intravenously with rAAV2/8-D377Y-mPCSK9 and fed a Western-type diet to induce atherosclerosis. While Lck-Cre Atg7f/f mice had equal serum Proprotein Convertase Subtilisin/Kexin type 9 levels as compared to Atg7f/f mice, serum cholesterol levels were significantly diminished in Lck-Cre Atg7f/f mice. Histological analysis of the liver revealed less steatosis, and liver gene expression profiling showed decreased expression of genes associated with hepatic steatosis in Lck-Cre Atg7f/f mice as compared to Atg7f/f mice. The level of hepatic CD4+ and CD8+ T cells was greatly diminished but both CD4+ and CD8+ T cells showed a relative increase in their IFNγ and IL-17 production upon Atg7 deficiency. Atg7 deficiency furthermore reduced the hepatic NKT cell population which was decreased to < 0.1% of the lymphocyte population. Interestingly, T cell-specific knock-out of Atg7 decreased the mean atherosclerotic lesion size in the tri-valve area by over 50%. Taken together, T cell-specific deficiency of Atg7 resulted in a decrease in hepatic steatosis and limited inflammatory potency in the (naïve) T cell compartment in peripheral lymphoid tissues, which was associated with a strong reduction in experimental atherosclerosis.

CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.

CRL4DCAF2 is required for mature T-cell expansion via Aurora B-regulated proteasome activity

The proliferation of T cells in peripheral lymphoid tissues requires T cell receptor (TCR)-mediated cell cycle entry. However, the underlying mechanism regulating cell cycle progression in mature T cells is incompletely understood. Here, we have identified an E3 ubiquitin ligase, CRL4DCAF2, as a critical mediator controlling M phase exit in activated T cells. DCAF2 expression is induced upon TCR stimulation and its deficiency attenuates T cell expansion. Additionally, DCAF2 T cell-specific knockout mice display impaired peripheral T cell maintenance and reduced severity of various autoimmune diseases. Continuous H4K20me1 modification caused by DCAF2 deficiency inhibits the induction of Aurkb expression, which regulates 26S proteasome activity during G2/M phase. CRL4DCAF2 deficiency causes M phase arrest through proteasome-dependent mechanisms in peripheral T cells. Our findings establish DCAF2 as a novel target for T cell-mediated autoimmunity or inflammatory diseases.

Embelin impairs the accumulation and activation of MDSCs in colitis-associated tumorigenesis

ABSTRACT Myeloid-derived suppressor cells (MDSCs) are a major component of the immunosuppressive tumor microenvironment and has been recognized as a contributing factor for inflammation-related cancers. We previously showed that embelin has potent anti-inflammatory and anti-tumor effects in a colitis-associated cancer (CAC) model. Here, by using this model, we assessed the effect of embelin on the accumulation and suppressive function of MDSCs. We have demonstrated that embelin substantially reduced accumulation of MDSCs in the peripheral lymphoid organ and tumor tissue of CAC-bearing mice. Embelin impaired immunosuppressive activity of MDSCs by reducing the generation of reactive oxygen species (ROS) and arginase 1 level, leading to restored T cell responses. In tumor milieu, embelin increased the infiltration of CD8+ T cells, NK cells and mature dendritic cells whilst depleted the regulatory T cells. Moreover, embelin could directly interfere with the generation and function of MDSCs in vitro. These effects of embelin on MDSCs were mediated largely via limiting C/EBPβ and STAT3 signaling. Our findings support the hypothesis that embelin may be a promising pharmacologic agent in regulating MDSC-mediated immune tolerance in colorectal cancer.

A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40-54/H-2Db-Ig dimer, MOG35-55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and "self-marker" CD47-Fc and encapsulate inhibitory cytokine (TGF-β1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65-79% of MOG35-55-specific CD4+ and 46-62% of MOG40-54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule-dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide-reactive T cells; reduced MOG peptide-reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-β1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell-mediated autoimmune diseases.

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Human Bone Marrow-Derived Myeloid Dendritic Cells Show an Immature Transcriptional and Functional Profile Compared to Their Peripheral Blood Counterparts and Separate from Slan+ Non-Classical Monocytes.

The human bone marrow (BM) gives rise to all distinct blood cell lineages, including CD1c+ (cDC2) and CD141+ (cDC1) myeloid dendritic cells (DC) and monocytes. These cell subsets are also present in peripheral blood (PB) and lymphoid tissues. However, the difference between the BM and PB compartment in terms of differentiation state and immunological role of DC is not yet known. The BM may represent both a site for development as well as a possible effector site and so far, little is known in this light with respect to different DC subsets. Using genome-wide transcriptional profiling we found clear differences between the BM and PB compartment and a location-dependent clustering for cDC2 and cDC1 was demonstrated. DC subsets from BM clustered together and separate from the corresponding subsets from PB, which similarly formed a cluster. In BM, a common proliferating and immature differentiating state was observed for the two DC subsets, whereas DC from the PB showed a more immune-activated mature profile. In contrast, BM-derived slan+ non-classical monocytes were closely related to their PB counterparts and not to DC subsets, implying a homogenous prolife irrespective of anatomical localization. Additional functional tests confirmed these transcriptional findings. DC-like functions were prominently exhibited by PB DC. They surpassed BM DC in maturation capacity, cytokine production, and induction of CD4+ and CD8+ T cell proliferation. This first study on myeloid DC in healthy human BM offers new information on steady state DC biology and could potentially serve as a starting point for further research on these immune cells in healthy conditions as well as in diseases.

Gut Microbiota Confers Resistance of Albino Oxford Rats to the Induction of Experimental Autoimmune Encephalomyelitis.

Albino Oxford (AO) rats are extremely resistant to induction of experimental autoimmune encephalomyelitis (EAE). EAE is an animal model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS), with established autoimmune pathogenesis. The autoimmune response against the antigens of the CNS is initiated in the peripheral lymphoid tissues after immunization of AO rats with CNS antigens. Subsequently, limited infiltration of the CNS occurs, yet without clinical sequels. It has recently become increasingly appreciated that gut-associated lymphoid tissues (GALT) and gut microbiota play an important role in regulation and propagation of encephalitogenic immune response. Therefore, modulation of AO gut microbiota by antibiotics was performed in this study. The treatment altered composition of gut microbiota in AO rats and led to a reduction in the proportion of regulatory T cells in Peyer’s patches, mesenteric lymph nodes, and in lymph nodes draining the site of immunization. Upregulation of interferon-γ and interleukin (IL)-17 production was observed in the draining lymph nodes. The treatment led to clinically manifested EAE in AO rats with more numerous infiltrates and higher production of IL-17 observed in the CNS. Importantly, transfer of AO gut microbiota into EAE-prone Dark Agouti rats ameliorated the disease. These results clearly imply that gut microbiota is an important factor in AO rat resistance to EAE and that gut microbiota transfer is an efficacious way to treat CNS autoimmunity. These findings also support the idea that gut microbiota modulation has a potential as a future treatment of multiple sclerosis.

Novel strategies to assess the synaptic interface of primary human T cells from peripheral blood and lymphoid tissue

Abstract Our current understanding of the kinetics and structural features of T cell synaptic interfaces has largely been determined through the use of glass supported artificial bilayers and in vitro-derived T-cell clones or lines. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues in unclear, given the significant difficulty in obtaining sufficient amounts of these cells for use on conventional glass-supported planar bilayers. Moreover, emerging data suggest that tissue resident T cells and T cells from lymphoid organs are significantly different in their phenotype and functional activity from those in peripheral blood, further solidifying the need to better understand the features of T cell synaptic interfaces in primary human T cells. To this end, we developed a new mini scale approach exploiting bilayers built into multichannel flow slides that allows us to perform imaging studies of T cell/bilayer interfaces with as few as 105 cells primary T cells isolated from human peripheral blood and lymphoid tissues without sacrificing the quality of the analysis. Our results indicate that while primary peripheral blood CD8+ T cells can share synaptic interface properties similar to in vitro derived T cell clones and lines, we found significant differences in the structure of the synaptic interface formed by primary lymphoid tissue compared to peripheral blood T cells, consistent with different efficiencies of these T cells.

TCF/LEF family transcription factors in peripheral Treg homeostasis

Abstract CD4+Foxp3+ regulatory T cells (Tregs) are key players in preventing lethal autoimmunity and excessive inflammation. However, the homeostatic dynamics of Tregs in peripheral lymphoid and non-lymphoid tissues remains poorly defined. We here report that two TCF/LEF family transcription factors, LEF1 and TCF1, play critical roles in this process. Our studies led to a revised “three-stage” model of Treg peripheral differentiation, delineated by the gradient expressions of LEF1 and TCF1. We found that LEF1− TCF1− subset was bona fide effector Tregs and exhibited a core gene signature shared by various tissue Tregs. Transcriptome and conditional knockout analyses revealed that TCF1 and LEF1 may each control segregated clusters of Treg genes and regulate Treg differentiation in a “division of labor” manner. Together, these data provide novel insights into transcriptional regulation of Treg homeostasis and function.