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We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3− effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40−/− mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ−/− mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40−/−, and PKC-Ѳ−/− mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40(−/−) mice had significantly reduced numbers of CD25(−)Foxp3(low) tTreg precursors and CD25(+)Foxp3(+) mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3+ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3+CD103+CD38− stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3+CD103−CD38+ labile-phenotype Tregs in the thymus and increased autoreactive CD4+ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4+ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.

We have previously shown GM-CSF derived bone-marrow dendritic cells (G-BMDCs) can induce the selective expansion of Tregs through the surface-bound molecule OX40L; however, the physiological role of this ex vivo derived DC subset remained to be elucidated. We determined GM-CSF administration to mice induced the generation of in vivo derived OX40L+ DCs, phenotypically similar to ex vivo OX40L+G-BMDCs, in the spleen, brachial lymph nodes and liver. The generation of OX40L+ DCs correlated with increased percentages of functionally suppressive Tregs in the spleen, brachial lymph nodes, and liver of GM-CSF treated mice. DCs from GM-CSF treated mice expanded Tregs in CD4+ T-cell co-cultures in an OX40L dependent manner, suggesting OX40L+ DCs may play a role in peripheral Treg homeostasis. Furthermore, comparing the transcriptome data of OX40L+ DCs to that of all immune cell types revealed OX40L+ DCs to be distinct from steady-state immune cells and, microarray analysis of OX40L+G-BMDCs and OX40L−G-BMDCs revealed higher expression of molecules that are associated with tolerogenic phenotype and could play important roles in the function of OX40L+ DCs. These findings suggest that OX40L+ DCs may represent a unique DC subset induced under inflammatory conditions that may play an essential role in maintaining Treg homeostasis.

The immune system ensures optimum T-effector (Teff) immune responses against invading microbes and tumor antigens while preventing inappropriate autoimmune responses against self-antigens with the help of T-regulatory (Treg) cells. Thus, Treg and Teff cells help maintain immune homeostasis through mutual regulation. While Tregs can contribute to tumor immune evasion by suppressing anti-tumor Teff response, loss of Treg function can result in Teff responses against self-antigens leading to autoimmune disease. Thus, loss of homeostatic balance between Teff/Treg cells is often associated with both cancer and autoimmunity. Co-stimulatory and co-inhibitory receptors, collectively known as co-signaling receptors, play an indispensable role in the regulation of Teff and Treg cell expansion and function and thus play critical roles in modulating autoimmune and anti-tumor immune responses. Over the past three decades, considerable efforts have been made to understand the biology of co-signaling receptors and their role in immune homeostasis. Mutations in co-inhibitory receptors such as CTLA4 and PD1 are associated with Treg dysfunction, and autoimmune diseases in mice and humans. On the other hand, growing tumors evade immune surveillance by exploiting co-inhibitory signaling through expression of CTLA4, PD1 and PDL-1. Immune checkpoint blockade (ICB) using anti-CTLA4 and anti-PD1 has drawn considerable attention towards co-signaling receptors in tumor immunology and created renewed interest in studying other co-signaling receptors, which until recently have not been as well studied. In addition to co-inhibitory receptors, co-stimulatory receptors like OX40, GITR and 4-1BB have also been widely implicated in immune homeostasis and T-cell stimulation, and use of agonistic antibodies against OX40, GITR and 4-1BB has been effective in causing tumor regression. Although ICB has seen unprecedented success in cancer treatment, autoimmune adverse events arising from ICB due to loss of Treg homeostasis poses a major obstacle. Herein, we comprehensively review the role of various co-stimulatory and co-inhibitory receptors in Treg biology and immune homeostasis, autoimmunity, and anti-tumor immunity. Furthermore, we discuss the autoimmune adverse events arising upon targeting these co-signaling receptors to augment anti-tumor immune responses.

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases such as Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance.

Granulocyte macrophage colony stimulating factor (GM-CSF) is generally recognized as an inflammatory cytokine. Its inflammatory activity is primarily due its role as a growth and differentiation factor for granulocyte and macrophage populations. In this capacity, among other clinical applications, it has been used to bolster anti-tumor immune responses. GM-CSF-mediated inflammation has also been implicated in certain types of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Thus, agents that can block GM-CSF or its receptor have been used as anti-inflammatory therapies. However, a review of literature reveals that in many situations GM-CSF can act as an anti-inflammatory/regulatory cytokine. We and others have shown that GM-CSF can modulate dendritic cell differentiation to render them "tolerogenic," which, in turn, can increase regulatory T-cell numbers and function. Therefore, the pro-inflammatory and regulatory effects of GM-CSF appear to depend on the dose and the presence of other relevant cytokines in the context of an immune response. A thorough understanding of the various immunomodulatory effects of GM-CSF will facilitate more appropriate use and thus further enhance its clinical utility.

Background: Primary malignant tumors arising from bone are uncommon but important malignant neoplasms which account for 0.2% of all primary cancers in adults and approximately 5% of all childhood malignancies. Aim: No comprehensive surveillance data about primary malignant bone tumors is available from this region of West Bengal in the literature. An attempt is undertaken to correlate clinicopathological findings, both histological & cytological features of primary bone tumours. Method: This observational study of clinical presentation was corroborated with cytology and histopathology of 67 cases of primary malignant bone tumors in 2005-2007 in a tertiary health care centre. Results: Among the primary malignant tumors, 39 cases (58.20%) were male and 28 (41.80%) were female. Sixty one lesions (91%) were located in the long bones and 6 (9%) involving flat and short bones. Osteosarcomas are found to be the predominant primary malignant bone tumors (44.77%), followed by Ewing’s sarcomas (20.89%), chondrosarcomas (13.43%), high grade giant cell tumor, and plasmacytoma (5.97%). Predilection for male (M: F 2:1) found in both osteosarcoma and chondrosarcoma, but it was reverse in cases of Ewing’s sarcomas. Conclusion: Osteosarcoma is the most common around knee joint and chondrosarcoma is frequent in long bones in this region. We made an attempt to correlate FNAC findings with the histopathology and it was seen that overall sensitivity of FNAC of primary malignant bone tumors was 56.71%.

Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis, and type 1 diabetes, and could also reverse ongoing EAT and experimental autoimmune myasthenia gravis. The protective effect was mediated through the induction of tolerogenic CD11C+CD8α− dendritic cells (DCs) and consequent expansion of Foxp3+ regulatory T cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic dendritic cells (DCs; GM-CSF–induced bone marrow-derived DCs [GM-BMDCs]), which directed Treg expansion in a contact-dependent manner. This novel mechanism of Treg expansion was independent of TCR-mediated signaling but required exogenous IL-2 and cosignaling from DC-bound OX40L. In this study, we observed that OX40L-mediated signaling by GM-BMDCs, although necessary, was not sufficient for Treg expansion and required signaling by Jagged1. Concurrent signaling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L+Jagged1+ BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L- and Jagged1-induced cosignaling in GM-BMDC–induced Treg expansion.

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation.

Virtually all efforts to generate an effective protection against the life-long, recurrent genital infections caused by HSV-2 have failed. Apart from sexual transmission, the virus can also be transmitted from mothers to neonates, and it is a key facilitator of HIV coacquisition. In this article, we uncover a nanoimmunotherapy using specially designed zinc oxide tetrapod nanoparticles (ZOTEN) with engineered oxygen vacancies. We demonstrate that ZOTEN, when used intravaginally as a microbicide, is an effective suppressor of HSV-2 genital infection in female BALB/c mice. The strong HSV-2 trapping ability of ZOTEN significantly reduced the clinical signs of vaginal infection and effectively decreased animal mortality. In parallel, ZOTEN promoted the presentation of bound HSV-2 virions to mucosal APCs, enhancing T cell–mediated and Ab-mediated responses to the infection, and thereby suppressing a reinfection. We also found that ZOTEN exhibits strong adjuvant-like properties, which is highly comparable with alum, a commonly used adjuvant. Overall, to our knowledge, our study provides the very first evidence for the protective efficacy of an intravaginal microbicide/vaccine or microbivac platform against primary and secondary female genital herpes infections.

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Primary tumors involving the bony skeleton of the chest wall are uncommon. After a retrospective review of the histopathology archive at our institution from Oct 08 to Mar 09 we found three malignant neoplasms affecting the rib, clavicle and the costal cartilages, one mesenchymal chondrosarcoma, one case of clear cell chondrosarcoma and an aggressive osteoblastoma each having distinctive histological diagnosis. Our limited experience in the occurrence of primary chest wall tumors showed that there are occurrences of rare bony lesions. A strong clinical suspicion, clinico-radiological correlation and histopathological confirmation are required for proper evaluation.

In our earlier work, we had shown that GM-CSF treatment of CBA/J mice can suppress ongoing thyroiditis by inducing tolerogenic CD8α− DCs, which helped expand and/or induce CD4+Foxp3+ Tregs. To identify the primary cell type that was affected by the GM-CSF treatment and understand the mechanism by which Tregs were induced, we compared the effect of GM-CSF on matured spDCs and BMDC precursors in vitro. Matured spDCs exposed to GM-CSF ex vivo induced only a modest increase in the percentage of Foxp3-expressing T cells in cocultures. In contrast, BM cells, when cultured in the presence of GM-CSF, gave rise to a population of CD11c+CD11bHiCD8α− DCs (BMDCs), which were able to expand Foxp3+ Tregs upon coculture with CD4+ T cells. This contact-dependent expansion occurred in the absence of TCR stimulation and was abrogated by OX40L blockage. Additionally, the BMDCs secreted high levels of TGF-β, which was required and sufficient for adaptive differentiation of T cells to Foxp3+ Tregs, only upon TCR stimulation. These results strongly suggest that the BMDCs differentiated by GM-CSF can expand nTregs and induce adaptive Tregs through different mechanisms.

Autoantibodies directed against the skeletal muscle acetylcholine receptor (AChR) play a critical role in the pathogenesis of the autoimmune disease, myasthenia gravis (MG). The pathogenic importance of anti-AChR antibodies is substantiated clinically by the often dramatic clinical improvement that follows removal of circulating antibodies utilizing extracorporeal plasma exchange (PE). Unfortunately, the effects of PE are non-specific as immunoglobulins (IgG) and other plasma proteins are removed in addition to anti-AChR IgG. In this study, we have successfully incorporated the AChR protein purified from Torpedo californicus into a Nanodisc (ND) membrane scaffold protein/phospholipid structure. We go on to demonstrate the effectiveness of this ND-AChR complex, administered intravenously, in the in vivo down-modulation of anti-AChR antibodies and subsequent amelioration of clinical disease in the experimental murine model of MG. These results provide proof-of-principle for the in vivo antigen-specific reduction of pathogenic anti-AChR antibodies utilizing ND-AChR particles. Further development of this strategy may provide an effective, antigen-specific, and readily accessible acute therapy for exacerbating MG or myasthenic crisis.

Ig class switch recombination (CSR) is regulated through long-range intrachromosomal interactions between germline transcript promoters and enhancers to initiate transcription and create chromatin accessible to activation-induced deaminase attack. CSR occurs between switch (S) regions that flank Cμ and downstream CH regions and functions via an intrachromosomal deletional event between the donor Sμ region and a downstream S region. It is unclear to what extent S region primary sequence influences differential targeting of CSR to specific isotypes. We address this issue in this study by generating mutant mice in which the endogenous Sγ3 region was replaced with size-matched Sγ1 sequence. B cell activation conditions are established that support robust γ3 and γ1 germline transcript expression and stimulate IgG1 switching but suppress IgG3 CSR. We found that the Sγ1 replacement allele engages in μ→γ3 CSR, whereas the intact allele is repressed. We conclude that S region identity makes a significant contribution to CSR. We propose that the Sγ1 region is selectively targeted for CSR following the induction of an isotype-specific factor that targets the S region and recruits CSR machinery.

Regulatory T-cells (Tregs) play a pivotal role in immune-tolerance, and loss of their function can lead to autoimmunity. Natural Tregs generated in the thymus contribute substantially to the Treg pool in the periphery where they suppress self-reactive Teff responses. Recently, we showed that OX40L (TNFSF4) can drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low dose IL-2. Therefore, we reasoned that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Thymic Treg development is a two-step process: Strong T-cell receptor (TCR) signals combined with co-signals from the TNF receptor Super Family (TNFRSF) members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development when their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling can also play a critical role in the TCR-independent phase of tTreg development. OX40−/− mice had significantly reduced numbers of CD25−Foxp3low tTreg precursors and CD25+Foxp3+ matured tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeffs cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and matured tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 microg/mL in the control to 40 microg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 microg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.

Multilocular cystic renal cell carcinoma (MCRCC), also known as multilocular clear cell renal cell carcinoma (RCC), is a rare cystic tumor of the kidney with an excellent outcome. It occurs in about 3.1-6% of the conventional RCC. It is usually included in the group of tumors of undetermined malignant potential with low nuclear grade. We present a case of MCRCC in a 30-year-old female patient presenting incidentally as an apparently benign-looking multicystic space occupying lesion in the upper pole of right kidney. Right-sided simple nephrectomy was performed, and on histopathologic examination it was found to be MCRCC, stage 1 with Fuhrman nuclear grade 1. Immunohistochemistry with epithelial membrane antigen and vimentin confirmed the diagnosis.

Previously, we have shown that GM-CSF-induced Bone marrow-derived dendritic cells (G-BMDCs), upon co-culture with splenic CD4+ T cells, have the capability to induce preferential proliferation of regulatory T cells (Tregs) in TCR-independent and OX40L/OX40 dependent manner. In this study we investigated the signaling involved in this Treg proliferation induced by G-BMDCs. Firstly, using splenic CD4+ T cells from OX40 deficient mice and G-BMDCs from WT mice, we confirmed the important role of OX40L/OX40 interaction in Treg expansion. Since OX40 stimulation in the absence of TCR engagement has been reported to induce the assembly of a signalosome involving PKC-θ, we investigated the requirement of PKC-θ in our Treg proliferation mechanism. Interestingly, PKC-θ deficient-CD4+ T cells showed impaired Treg proliferation upon co-culture with WT G-BMDCs. However, this defect in proliferation could be rescued upon IL-2 addition suggesting that PKC-θ was dispensable for Treg proliferation. We further demonstrated that PKC-θ was essential for T-effectors to provide IL-2, which was required for OX40L/OX40-induced Treg proliferation. Furthermore, injecting WT, OX40- deficient, and PKC-θ-deficient mice with soluble OX40L, we established that OX40L/OX40 signaling was sufficient to induce in vivo expansion of Tregs in WT and PKC- deficient mice, but not in OX40-deficient mice. Finally, our data suggested a possible involvement of TRAF1 in Tregs, downstream of OX40 activation.

We previously reported granulocyte macrophage colony-stimulating factor (GM-CSF) prevented type 1 diabetes in NOD mice through CD11c+CD8− dendritic cell (DCs) mobilization, precipitating regulatory T-cell (Treg) expansion. Furthermore, we found GM-CSF derived bone-marrow dendritic cells (G-BMDCs) can drive Treg expansion in ex-vivo CD4+splenic T-cell co-cultures through surface-bound molecule OX40L. To determine whether GM-CSF may lead to the formation of a distinct, novel CD11c+OX40L+ expressing DC subset, we generated OX40L+G-BMDCs (CD11c+OX40L+) and analyzed these cells through flow cytometry for various cell lineage markers. We determined OX40L+G-BMDCs expressed increased levels of co-stimulatory molecules CD80, CD86, PD-L1 and MHC-II in comparison to the OX40L− counterpart. Additionally, OX40L+G-BMDCs lacked expression of Ly6G, Ly6C, FceR1, surface markers indicative of granulocyte and mast cell lineage, but to our surprise, highly expressed macrophage markers F4/80, MerTK, and CD200R. To elucidate an in vivo physiological component of these OX40L+ G-BMDCs, we performed an unsupervised hierarchical clustering comparing the transcriptome of OX40L+G-BMDCs to that of all myeloid and lymphoid lineages from the Immgen database. We found that OX40L+G-BMDCs clustered most closely with macrophages and dendritic cells. Furthermore, a principal component analysis with the transcriptome of OX40L+G-BMDCs and that of macrophages and dendritic cells from the Immgen database revealed OX40L+G-BMDCs most closely clustered with macrophages. Collectively, these results suggest OX40L+ G-BMDCs may represent a distinct GM-CSF dependent, macrophage-like cell subset involved in physiological Treg homeostasis.

Regulatory T-cells (Treg) play a pivotal role in suppressing self-reactive effector T-cells (Teff) and help maintain the critical balance between self-tolerance and autoimmunity. Restoration of homeostatic balance between Teff/Treg cells has been shown to ameliorate many autoimmune diseases including type-1 diabetes. Recently, we have reported a novel strategy for the selective expansion of Tregs using OX40L (a TNF-superfamily ligand) and Jagged (JAG)-1 (a Notch family ligand) treatment independent of canonical TCR-signaling, but mediated through specific interaction between OX40L-OX40 and JAG1-Notch3 preferentially expressed on Tregs. OX40L-JAG1 treatment of NOD mice with soluble OX40L and JAG1 increased proliferation of peripheral Tregs and delayed the onset of diabetes. In the present study, we show that OX40L treatment significantly increased Tregs in the thymus by inducing proliferation of thymic Tregs independent of TCR-stimulation upon addition of exogenous IL-2. OX40−/− mice had reduced numbers of thymic Tregs compared to wild type (WT) controls. The reduction in thymic Treg numbers likely due to impaired ability of proliferation in response to OX40L. However, we did not observe any change in IL-2 sensitivity of thymocytes in OX40−/− mice. Mechanistic studies suggested that OX40L-IL-2 induced thymic Treg proliferation was mediated through NF-kB and AKT-mTOR-S6 signaling. Further, phenotypic characterization studies revealed a significant decrease in TIGIT+Treg population, often associated with Treg activation, in both thymus and spleen of OX40−/− mice compared to WT controls. Taken together, these results suggest a critical role for OX40L/OX40 signaling in the development as well as function of Tregs.

To prepare a novel Bispecific Antibody (BsAb) as a potential targeted therapy for T1D, we produced a “functionally inert” monoclonal antibody (mAb) against Glucose transporter-2 (GLUT-2) expressed on β-cells to serve as an anchoring antibody. The therapeutic arm is an agonistic mAb against Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), a negative regulator of T-cell activation expressed on activated CD4 + T-cells. A BsAb was prepared by chemically coupling an anti-GLUT2 mAb to an agonistic anti-CTLA-4 mAb. This BsAb was able to bind to GLUT2 and CTLA-4 in vitro , and to pancreatic islets, both in vitro and in vivo. We tested the safety and efficacy of this BsAb by treating Non-Obese Diabetes (NOD) mice and found that it could delay the onset of diabetes with no apparent undesirable side effects. Thus, engagement of CTLA-4 on activated T cells from target tissue can be an effective way to treat type-1 diabetes.

It is difficult to predict which urothelial neoplasm would subsequently recur or progress to muscle invasive tumours or produce metastasis.The aim and objective of the study were to evaluate the scope of immunohistochemical expression of p53 in human urothelial neoplasms with regard to grade, stage and outcome of the patients. Eighteen consecutive patients were taken and urothelial tumour samples were obtained from transurethral resection or surgical excision. Histopathological examinations were performed and the grading was done according to the WHO/ISUP consensus classification of urothelial neoplasms. Immunohistochemical staining for p53 was performed on formalin fixed paraffin embedded tissue sections with appropriate positive and negative control. It was found 3 patients with papillary urothelial neoplasm of low malignant potential (PUNLMP), 5 cases of papillary low grade urothelial carcinoma, 10 patients with papillary high grade urothelial carcinoma including 2 cases of invasive urothelial carcinoma. All three PUNLMP cases showed negative results. Four out of 5 low grade papillary urothelial carcinoma had nuclear p53 accumulation, while all of the 10 papillary high grade carcinoma had high p53 index. The finding of negative p53 staining in PUNLMPs and high p53 index in high grade papillary urothelial carcinomas and invasive carcinomas support the notion that mutation of p53 gene might be unrelated to the development of urothelial neoplasms but definitely play a crucial role in progression of the malignancy.

The optimal upper limit of the normal range for prostate specific antigen (PSA) is suggested, but review of literature reveals that, malignancy of prostate can occur below that range and some benign prostatic diseases are occasionally associated with higher levels. The aim of the study is for early detection of prostatic malignancy. We tried to evaluate digital rectal examination (DRE), estimation of PSA and transrectal ultrasound (TRUS) guided core needle biopsy and histopathological examination in the patients. Seventy-two consecutive patients with lower urinary tract symptoms were taken in this retrospective study from January 2005 to February 2006. PSA level was measured by automated chemilumininescence system. Prostatic biopsies were taken for histopathological examination and stained with haematoxylin and eosin. Gleason grading was applied in case of adenocarcinoma of prostate. For detection of malignancy, sensitivity, specificity, predictive value for positive test and of negative test, percentage of false negatives and false positives, p-values, confidence interval and kappa statistical calculations were done. It was found 19 cases with PSA level4 ng/ml had benign diseases of prostate and one person having PSA level4 ng/ml had adenocarcinoma of prostate. Seven DRE positive cases had benign disease of the prostate and 5 DRE negative patients had adenocarcinoma of prostate. When compared, serum total PSA value alone and combined PSA and DRE, the later combined approach was found to be more useful. We recommend the study of DRE, PSA and TRUS guided core needle biopsy for detection of prostatic cancer at localised and potentially curable stage.

Autoimmune thyroid diseases (AITD) are one of the most common organ-specific autoimmune disorders, of which Hashimoto's thyroiditis (HT) and Graves' disease (GD) are 2 of the most common clinical expressions. HT is characterized by hypothyroidism that results from the destruction of the thyroid by thyroglobulin-specific T cell-mediated autoimmune response. In contrast, GD is characterized by hyperthyroidism due to excessive production of thyroid hormone induced by thyrotropin receptor-specific stimulatory autoantibodies. Cytokines play a crucial role in modulating immune responses that affect the balance between maintenance of self-tolerance and initiation of autoimmunity. However, the role of cytokines is often confusing and is neither independent nor exclusive of other immune mediators. A regulatory cytokine may either favor induction of tolerance against thyroid autoimmune disease or favor activation and/or exacerbation of autoimmune responses. These apparently contradictory functions of a given cytokine are primarily influenced by the nature of co-signaling delivered by other cytokines. Consequently, a thorough understanding of the role of a particular cytokine in the context of a specific immune response is essential for the development of appropriate strategies to modulate cytokine responses to maintain or restore health. This review provides a summary of recent research pertaining to the role of cytokines in the pathogenesis of AITD with a particular emphasis on the therapeutic applications of cytokine modulation.

Earlier, we have shown that GM-CSF-exposed CD8α− DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3− expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25− effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity.

Aim: The aim of the study was to investigate the expression and function of the IG20 gene in thyroid cancer cell survival, proliferation, and apoptosis. Methods: We determined the expression levels of the major isoforms of IG20 by quantitative RT-PCR in normal and thyroid tumor tissues/cell lines. We evaluated the functional consequence of IG20 knockdown in WRO (follicular carcinoma) and FRO (anaplastic carcinoma) thyroid cancer cell lines by measuring spontaneous, TNFα-related apoptosis-inducing ligand (TRAIL), and TNFα-induced apoptosis. Results: The IG20 gene expression levels were higher in benign and malignant thyroid tumors and in WRO and FRO cells relative to normal tissues. Predominantly, MADD and DENN-SV isoforms of IG20 gene were expressed. IG20 knockdown resulted in increased spontaneous, TRAIL-, and TNFα-induced apoptosis in WRO, but not FRO, cells. FRO cell resistance to apoptosis is likely due to caspase-8 deficiency. Conclusion: IG20 knockdown renders WRO cells more susceptible to spontaneous, TRAIL-, and TNFα-induced apoptosis and thus demonstrates the prosurvival function of the IG20 gene in thyroid cancer. These observations, combined with overexpression of IG20 noted in thyroid tumor tissues, may suggest a potential role in thyroid cancer survival and growth and indicate that IG20 may be targeted either alone or in conjunction with TRAIL or TNFα treatment in certain thyroid cancers.

Activation-induced deaminase (AID) is the master regulator of class switch recombination (CSR) and somatic hypermutation (SHM), but the mechanisms regulating AID function are obscure. The differential pattern of switch plasmid activity in three IgM(+)/AID(+) and two IgG(+)/AID(+) B cell lines prompted an analysis of global gene expression to discover the origin of these cells. Gene profiling suggested that the IgG(+)/AID(+) B cell lines derived from germinal center B cells. Analysis of SHM potential demonstrates that the IgVkappa domains are inducibly diversified at high rate during in vitro culture. The mutation spectra focused to A:T base pairs, revealing a component of the hypermutation program that occurs preferentially during phase 2 of SHM. The A:T error spectra were analyzed and were not characteristic of polymerase eta activity. A differential pattern of three consensus motifs used for A:T base substitutions was observed in WT and Poleta-, Msh2- and Msh6-deficient B cells. Strikingly, mutations in our B cell lines recapitulated the mutable motif profile for Poleta and Msh2 deficiency, respectively, and suggest that an additional pathway for the generation of A:T mutations in SHM is conserved in mouse and human.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 microg/mL in the control to 40 microg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 microg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.

We have earlier demonstrated that CD11b+CD11c+ dendritic cells (DCs) derived ex vivo from bone marrow precursors with GM-CSF (GM-BMDCs) can selectively expand natural Foxp3+ regulatory T-cells (Tregs) in a contact dependent manner through OX40L/Jagged-1 co-signaling. However, an ‘in vivo’ counterpart of these GM-BMDCs is not known. In contrast BMDCs derived ex vivo using FLT3L (FL-BMDCs) are considered to be phenotypical and functional equivalents of physiological steady-state DCs. In addition, mice lacking GM-CSF or its receptor do not show a severe defect in DC development while FLt3L deficiency severely affects the physiological development of all types of DCs. To determine the role of GM-CSF in DC-development in vivo, we treated Flt3L deficient mice with GM-CSF and found no evidence of CD11c+ DC development. In contrast, we saw near complete reconstitution of DCs in these mice with Flt3L treatment. Surprisingly, we could successfully generate CD11c+ GM-BMDCs from bone marrow precursors isolated from these Flt3L deficient mice. Interestingly, these GM-BMDCs also express surface markers associated with precursor cells, macrophages and granulocytes. To address these various contradictions, we propose that G-BMDCs generated ex vivo represent a myeloid precursor population rather than fully differentiated DCs. These ‘precursors’ require other cytokines for their terminal differentiation; thus, Flt3L is critical for their differentiation into steady-state DCs.

Type 1 diabetes (T1D) is a T-cell-mediated disease characterized by destruction of pancreatic beta cells. Unfortunately, most treatment options for T1D are non-specific, with many side effects. Regulatory cells T (Tregs) play a crucial role in the maintenance of immune homeostasis against self-antigen. Many studies have revealed that Tregs in patients with various autoimmune diseases, including T1D, are diminished in number and/or dysfunctional. The therapeutic potential of Tregs has not been fully understood because generating large numbers of Tregs has remained a challenge. Recently, we reported that bone marrow precursor cells differentiated with GM-CSF (BMDCs) are able to selectively expand Tregs ex-vivo. Further investigation revealed that this expansion is TCR-independent but requires OX40/OX40L interaction. Thus, here we wanted to study whether treatment with soluble OX40L can expand Tregs in-vivo and in the NOD mice. Surprisingly, treatment with OX40L caused rapid onset of T1D in 12 wk but not 6 wk old mice. Analysis of spleens and lymph nodes showed an increase in Tregs only in the 6 wk old OX40L group. Thymic analysis revealed an increase in Tregs in both the 6 and 12 wk old OX40L groups. Interestingly, OX40L specifically induced the stable Tregs subset in 6 wk old mice, but the labile Treg subset in the 12 wk old mice. Based on these findings, we propose that OX40L can specifically induce different subsets of Tregs based on the cytokine milieu.

Adenoid cystic carcinoma is a relatively rare epithelial tumor of the salivary glands accounting for about 5-10% of all salivary gland neoplasms. Approximately, 31% of salivary gland neoplasms affect minor salivary glands particularly the palate. It involves tongue in only 19.8% of cases and even rarely the dorsum of the tongue. We report such a rare case that affected dorsum of the tongue in a 45-year-old-female patient.

Immunoglobulin class-switch recombination occurs in both frogs and mammals. A new study shows that the recognition mechanism used for the targeting of switch sequences might be evolutionary conserved.

Rhinosporidiosis is a chronic granulomatous disease caused by fungus Rhinosporidium seeberi affecting most commonly the anterior nares. Urethral involvement is extremely rare. We present such a case in a 61-year-old man with intermittent bleeding per urethra and fleshy mass coming through the urethral meatus. On histological examination of hematoxylin-eosin-stained smears, the lesion showed numerous sporangia mixed with inflammatory infiltrates and occasional giant cells, beneath the urethral squamous epithelium. The mass was excised and fulguration of the base with diathermy was offered for cure.

Histoplasmosis refers to an infection caused by histoplasma capsulatum, a dimorphic fungus. We report a rare case of cutaneous histoplasmosis in an immunocompromised patient. The diagnosis was made by the cytopathologic examination of skin scraping smear, which showed numerous intracellular and extracellular periodic acid-schiff positive rounded yeast cells. The patient showed dramatic response with itraconazole and amphotericin B. We opine that skin scrape cytology can be useful in establishing the diagnosis of the disease, specially when the other facilities are not readily available.

Experimental autoimmune thyroiditis, a murine model of Hashimoto's thyroiditis is characterized by the infiltration of the thyroid by self-reactive CD4+ T cells. Our earlier studies have shown that GM-CSF treatment of experimental animals can suppress ongoing thyroiditis through the induction of tolerogenic CD8a- dendritic cells (DCs) expressing low levels of pro-inflammatory cytokines. Antigen presentation by these tolerogenic CD8a- DCs resulted in an expansion of IL-10 secreting CD4+Foxp3+ regulatory T-cells (T-regs) which suppressed the effector T-cell function. To understand the underlying mechanism we studied the effect of GM-CSF on matured splenic DCs as well as on bone marrow (BM) DC precursors in vitro. In spite of increased surface expression of programmed death ligand 2 (PDL-2), a negative regulator of T-cell activation, and increased indole-deoxygenase (IDO) secretion, splenic DCs exposed to GM-CSF induced only a modest increase in Foxp3 expression in T-cells. In contrast, BM cells when cultured in the presence of GM-CSF gave rise to a population of CD11c+CD11bhigh DCs with lower levels of expression of pro-inflammatory cytokines IL-12 and IL-6 which induced Foxp3 expression in a very high percentage of T-cells in a contact dependent manner. Our studies strongly suggest that the tolerogenic effect of GM-CSF is mediated by a special class of CD8a- DCs that differentiate from BM precursors. NIH 1RO1AI058190

GM-CSF is a potent dendritic cell (DC) growth factor that plays an essential role in DC differentiation. We examined the potential of GM-CSF to impart tolerogenic potential to CD8a+ and CD8a- sub-populations of DCs. We show that GM-CSF selectively modulates myeloid CD8a-, and not plasmacytoid or CD8a+, DCs in vivo. GM-CSF treatment rendered CD8a- DCs tolerogenic which was characterized by the diminished production of inflammatory cytokines IL-12 and IL1β by these DCs. Self antigen, mouse thyroglobulin (mTg), presentation by GM-CSF exposed CD8a- DCs, relative to control CD8a- or other DCs, caused a significant increase in the frequency of Foxp3+ T cells among CD4+ T cells from mTg-primed mice. The ability of GM-CD8a- DCs to induce Foxp3+ T cells was severely abrogated upon addition of IL-12, and not IL1β, into the culture and indicated that reduced IL-12 production rendered them tolerogenic. Further, we studied the effects of treatment with various TLR ligands to either augment or abolish the effects of GM-CSF on CD8a- DCs. Interestingly, addition of TLR ligands such as LPS and CpG further augmented the ability of GM-CSF exposed CD8a- DCs to induce Foxp3+Tregs. These results indicated that exposure of GM-CD8a- DCs to TLR4 and TLR9 ligands can enhance their tolerogenic potential and strongly suggested that pro-inflammatory immune stimuli may confer an enhanced antigen specific tolerogenic effect on GM-CSF exposed CD8a- DCs. NIH 1RO1AI058190

Impairment in dendritic cell (DC) functions is implicated in defective immune regulation that leads to type-1 diabetes (T1D) in both humans and the established non-obese diabetic (NOD) mouse model. In this study, we examined the roles of DC modulators such as GM-CSF and Flt3-L to affect DC function and prevent T1D development in NOD mice. Both GM-CSF and FLT3-L could suppress the development of T1D if treatment was initiated earlier in life prior to the development of clinical diabetes. However, unlike Flt3-L, GM-CSF was far more effective in suppressing T1D even when administered at later stages of insulitis. Ongoing periodic treatment of NOD mice with GM-CSF prevented the development of T1D and the animals remained healthy until 3 months after cessation of the treatment. Adoptive transfer of CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D in NOD-SCID mice, and this suppression was associated with enhanced IL-10 and TGF-β1 production. Moreover, adoptive transfer of GM-CSF-exposed DCs to naive NOD mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. These results indicate that primarily the GM-CSF renders DCs tolerogenic resulting in subsequent Treg induction and delayed onset of T1D in NOD mice. NIH 1RO1AI058190