Cells Expressing MHC Class Research Articles

Immunological microenvironment in colon cancer patients with different phenotypes of cancer stem cells.

e15503 Background: The purpose of this study was to analyze the effect of the CD44+ and CD133+ co-expression in cancer stem cells (CSCs) on lymphocytic microenvironment of colon cancer (CC). Methods: 200 CC patients received surgery as the first stage of treatment. The percentage of CSCs with the expression of CD44+ and/or CD133+ markers was studied in the tumor homogenates by flow cytometry, as well as some indicators of local immunity (CD3+, CD4+, CD8+, T regs (CD4+CD25+CD127dim), CD19+, PD-1, PD-L1, Th0, Tm, CD16/56+ and immunophenotypic characteristics of tumor cells (PD-L1, MHC-ABC). Results: Gradation depending on the absence or presence of co-expression of CSC markers on tumor cells allowed identification of 11 statistically significant differences out of 17 studied parameters of tumor cells and their lymphocytic microenvironment. Co-expression of CSC markers was accompanied by higher percentage of T regs (7.3±0.4 versus 5.3±0.5%), together with lower levels of CD4+ cells. At the same time, a higher content of the total number of T-lymphocytes was noted due to CD8+ with an increase in the percentage of memory T cells and a decline in naive T lymphocytes within this subpopulation. In addition, the co-expression of CSC markers was accompanied by a lower content of PD-L1 (34.3±3.0 vs. 42.9±2.5%) on lymphocytes and its higher content on tumor cells (10.6±1.5 vs. 4.1±0.8%), while the PD-1 expression on lymphocytes was higher (38.4±3.7 versus 22.3±2.9%). The presence of CD44+CD133+ CSCs was also accompanied by lower percentage of tumor cells expressing MHC class I (60.4±4.9 vs. 79.3±7.6%), which characterized the inhibition of recognition processes, and increased levels of CD8+, perhaps, should be considered as compensatory. Conclusions: The lymphocytic microenvironment of CC in the presence of CSCs with the CD44+CD133+ immunophenotype seems to be more immunosuppressive, according to the increase in the local content of T regs and the decrease in MHC-ABC expression. Higher expression of PD-L1 on tumor cells and PD-1 on lymphocytes allows activation of the PD-1/PD-L1 interaction, which enhances the immunosuppressive and growth-stimulating properties of the tumor microenvironment, but at the same time, makes tumor cells adequate targets for immunotherapy with immune checkpoint inhibitors.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Anatomical distribution of respiratory tract leukocyte cell subsets in neonatal calves

Neonatal calves are highly susceptible to a number of diseases including those that infect via the mucosal surfaces of the respiratory and gastrointestinal tracts. In order to determine appropriate vaccine design and delivery systems, or to identify suitable immunostimulatory methods to combat these infections, a detailed understanding of the immune cell populations present at clinically relevant sites is key. Few studies have assessed the immune cell composition of the neonatal calf lung and comparisons with circulating immune cells in the blood are lacking. We describe immune cell populations present in the peripheral blood, bronchoalveolar lavage (BAL) fluid and lung tissue of young disease-free calves. Flow cytometric analysis revealed significant differences in cell subset distribution between the peripheral blood and respiratory tract, and between compartments within the respiratory tract. Notably, whereas WC1+ γδ TCR + T lymphocytes dominate the peripheral blood, both the BAL fluid and lung tissue contained a high proportion of myeloid cells which expressed CD14 and CD172a (SIRPα). Very low numbers of tissue myeloid cells expressed MHC Class II in comparison to circulating myeloid cells in the blood. Respiratory tract tissues had low frequencies of CD4+ and CD8 + T lymphocytes, which were significantly lower than in the blood. Differences in the proportion of NKp46+ natural killer cells were also observed between tissue compartments. In order to target vaccines or immunostimulatory therapeutics appropriately, these differences in immune cell populations in tissue compartments should be taken into consideration.

Regulatory T Cell Transmigration and Intravascular Migration Undergo Mechanistically Distinct Regulation at Different Phases of the Inflammatory Response.

Regulatory T cells (Tregs) play important roles in limiting inflammatory responses in the periphery. During these responses, Treg abundance in affected organs increases and interfering with their recruitment results in exacerbation of inflammation. However, the mechanisms whereby Tregs enter the skin remain poorly understood. The aim of this study was to use intravital microscopy to investigate adhesion and transmigration of Tregs in the dermal microvasculature in a two-challenge model of contact sensitivity. Using intravital confocal microscopy of Foxp3-GFP mice, we visualized endogenous Tregs and assessed their interactions in the dermal microvasculature. Four hours after hapten challenge, Tregs underwent adhesion with ∼25% of these cells proceeding to transmigration, a process dependent on CCR4. At 24 h, Tregs adhered but no longer underwent transmigration, instead remaining in prolonged contact with the endothelium, migrating over the endothelial surface. Four hours after a second challenge, Treg transmigration was restored, although in this case transmigration was CCR4 independent, instead involving the CCR6/CCL20 pathway. Notably, at 24 h but not 4 h after challenge, endothelial cells expressed MHC class II (MHC II). Moreover, at this time of peak MHC II expression, inhibition of MHC II reduced Treg adhesion, demonstrating an unexpected role for MHC II in Treg attachment to the endothelium. Together these data show that Treg adhesion and transmigration can be driven by different molecular mechanisms at different stages of an Ag-driven inflammatory response. In addition, Tregs can undergo prolonged migration on the inflamed endothelium.

PB1875 IMMUNE SYNAPSE PARAMETERS DETERMINE LYMPHOMA TYPE AND IMMUNE EVASION STRATEGY

Background:The mechanisms of tumor cell evasion from the immune response are various. The malignant B‐cells must have evolved strategies of avoiding or suppressing the immune system, especially the anti‐cancer effects of T‐lymphocytes by regulating the expression of molecules involved in the formation of an immunological synapse (IS). Unlike solid tumors, B‐cell lymphoma cells express MHC class II and costimulatory CD80/CD86 molecules, allowing these lymphoma cells to act as antigen‐presenting cells by themselves. A disruption in the formation of an immune synapse between a tumor B‐cell and a T‐cell indicates that the tumor B‐cells are able to use the pathways of inhibiting the T‐cell immunity that are normal.Aims:to study a molecules including in IS in different lymphomasMethods:Study included 53 lymphoma patients (male:female = 30:23) with median age 59 (34‐87). CLL was confirmed in 31 patients, all of them were treatment‐naïve.12 patients were diagnosed with MCL and 10 with SMZL. The proportion of CD80+, CD86+, FAS+, PD‐1+ and PD‐L1+ tumor cells were determined by multicolor flow cytometry (BD FACSCanto II) in peripheral blood samples. In T‐cells (CD4+ and CD8+) we determined proportion of PD‐1+ cells. We performed cluster analysis and made dendrograms with all parameters and different lymphoma types.The control group consisted of 25 donors (male:female = 12:13), the median age of 48 years (40‐64).Results:It was found that tumor B cells in CLL and MCL have a smaller proportion of FAS+ and CD86+ compared to donors (p < 0.05). B cells of CLL also have a smaller proportion of CD80+ compared to donors, MCL and SMZL (p < 0.05). The proportion of PD‐1+ B‐cells was higher with SMZL and CLL than among donors (p < 0.05). B‐cells in MCL did not significantly differ from donors (p > 0.05). The proportion of PD‐L1+ B cells was lower in the MCL and CLL groups compared to donors and SMZL.In the SMZL and MCL groups, the proportion of CD4+PD‐1+ cells was higher than in the control group. The proportion of CD4+PD‐1+ cells in the CLL was lower than in SMZL and the proportion of CD8+PD‐1+ cells did not differ significantly.Ward's method divided lymphomas into different clusters, taking into account the proportion of CD80 +, CD86 +, FAS +, PD‐1 +, PD‐L1 + tumor B‐lymphocytes, as well as the proportion of T‐helper cells and cytotoxic T‐cells with co‐expression of PD‐1. All lymphomas formed 2 major clusters. The 1st cluster included 8 SMZL of 10; 4 MCL of 12 and no CLL. The 2nd cluster was divided into two: in cluster 2a there were 13 CLL (mostly in the B and C stages according to J. Binet) of 26, as well as 4 MCL and 1 SMZL; in cluster 2b there were 13 patients with CLL (mainly in stage A), as well as 4 patients with MCL and 1 SMZL.Summary/Conclusion:Tumor B cells are different from normal B cells. Moreover, in various lymphomas, tumor B cells have their own characteristics that affect T‐cell inhibition. CLL and MCL B cells are characterized by a decrease in the co‐activation molecules of CD80/CD86, which causes T‐cell anergy. CLL and MCL B cells are also characterized by a decrease in FAS, which prevents FAS‐mediated apoptosis of tumor B cells. In contrast, for SMZL, PD‐1‐PD‐L1 is the most significant way of inhibiting T‐cells.imageIn cluster analysis, various lymphomas were distributed in separate clusters by IS parameters. CLL is clustered separately from SMZL and MCL. CLL, in turn, clustered into subgroups that differed in the stage of the disease.

Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno-histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2weeks post-seawater transfer (wpt) compared with those infected at 9wpt. At 9wpt, there were higher numbers of MHC II+ cells in heart, pancreas and pyloric caeca, IgM+ cells in heart and pancreas, and CD3+ cells in pancreas compared to those infected at 2wpt. The majority of the immune cells infiltrating PD-affected tissues were MHC II+ and CD3+ cells suggesting that antigen-presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.

Targeted overexpression of prostacyclin synthase inhibits lung tumor progression by recruiting CD4+ T lymphocytes in tumors that express MHC class II

ABSTRACTLung-specific overexpression of prostacyclin synthase (PGIS) decreases tumor initiation in murine lung cancer models. Prostacyclin analogs prevent lung tumor formation in mice and reverse bronchial dysplasia in former smokers. However, the effect of prostacyclin on lung cancer progression has not been well studied. We investigated the effects of pulmonary PGIS overexpression in an orthotopic immunocompetent mouse model of lung cancer using two murine lung cancer cell lines. Pulmonary PGIS overexpression significantly inhibited CMT167 lung tumor growth, increased CXCL9 expression, and increased CD4+ tumor-infiltrating lymphocytes. Immunodepletion of CD4+ T cells abolished the inhibitory effect of pulmonary PGIS overexpression on CMT167 lung tumor growth. In contrast, pulmonary PGIS overexpression failed to inhibit growth of a second murine lung cancer cell line, Lewis Lung Carcinoma (LLC) cells, and failed to increase CXCL9 expression or CD4+ T lymphocytes in LLC lung tumors. Transcriptome profiling of CMT167 cells and LLC cells recovered from tumor-bearing mice demonstrated that in vivo, CMT167 cells but not LLC cells express MHC class II genes and cofactors necessary for MHC class II processing and presentation. These data demonstrate that prostacyclin can inhibit lung cancer progression and suggest that prostacyclin analogs may serve as novel immunomodulatory agents in a subset of lung cancer patients. Moreover, expression of MHC Class II by lung cancer cells may represent a biomarker for response to prostacyclin.

Immunoreceptor tyrosine-based inhibitory motif–dependent functions of an MHC class I-specific NK cell receptor

Natural killer (NK) cells express MHC class I (MHC-I)-specific receptors, such as Ly49A, that inhibit killing of cells expressing self-MHC-I. Self-MHC-I also "licenses" NK cells to become responsive to activating stimuli and regulates the surface level of NK-cell inhibitory receptors. However, the mechanisms of action resulting from these interactions of the Ly49s with their MHC-I ligands, particularly in vivo, have been controversial. Definitive studies could be derived from mice with targeted mutations in inhibitory Ly49s, but there are inherent challenges in specifically altering a single gene within a multigene family. Herein, we generated a knock-in mouse with a targeted mutation in the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Ly49A that abolished the inhibitory function of Ly49A in cytotoxicity assays. This mutant Ly49A caused a licensing defect in NK cells, but the surface expression of Ly49A was unaltered. Moreover, NK cells that expressed this mutant Ly49A exhibited an altered inhibitory receptor repertoire. These results demonstrate that Ly49A ITIM signaling is critical for NK-cell effector inhibition, licensing, and receptor repertoire development.

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

NCR1 is an activating receptor expressed on a subset of canine NK cells

Defining NK cells has been challenging in many veterinary species. Although several groups have described putative NK cell populations, there is still no consensus on a definition of NK cells in the dog. In the present study, canine NK cells are characterized as CD3−GranzymeB+ cells, further divided into a NCR1+ and a NCR1− subset. All dogs examined displayed both subsets in blood, although of quite variable magnitude. Following vaccination an increase was observed in the CD3− NCR1− cell population in blood, but not in the CD3− NCR1+ population. Non-B non-T cell cultures stimulated with IL-2 and IL-15 were dominated by CD3−GranzymeB+ cells after approximately 2 weeks and a large proportion of the CD3−GranzymeB+ cells expressed NCR1. IL-12 stimulation lead to a further upregulation resulting in an almost uniform expression of NCR1. The cultured cells expressed MHC class II, showed a variable expression of CD8 and were negative for CD4 and CD21. The cultures were able to kill known NK cell targets, and NCR1 was shown to be a major activating receptor. A large proportion of the NCR1+ cells, but none of the NCR1− cells, produced IFNγ in response to IL-12 stimulation. These results show that NCR1 defines two subsets of canine NK cells, likely to represent different activation stages, and that NCR1 acts as an activating receptor on canine NK cells.

652. Culture and Characterization of Tumor-Infiltrating Lymphocytes of the Syrian Hamster

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has developed into a promising treatment modality for metastatic cancer. However, preclinical animal models for studying TIL therapy are limited to mice and therefore we set out to culture and characterize TIL from another rodent species, the Syrian hamster. First, we examined the presence of CD4+ and CD8+ cells in six implantable syngeneic hamster tumors representing various histologies. A tumor type-dependent pattern of lymphocyte infiltration was observed, with CD8+ cells ranging from 0.08% to 0.93% (of all tumor cells) and CD4+ cells ranging from 0.17% to 3.25%. To establish TIL cultures, excised tumor fragments were cultured in high-dose human interleukin-2 (IL-2) for 10 days. Flow cytometric analyses of Day 10 TIL cultures revealed a predominance of CD4+ cells over CD8+ cells in all tumor types studied. In effector/target assays the TIL cultured from HapT1 (pancreatic adenocarcinoma) and RPMI 1846 (melanoma) exhibited tumor-specific cytolytic activity. In addition, MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, suggesting that cytotoxic CD8+ T-cells were responsible for the observed cytolytic activity. Only partial abrogation was seen with HapT1 TIL suggesting the presence of cytotoxic CD4+ T-cells in the TIL population, which is supported by the observation that HapT1 tumor cells express MHC Class II. To our knowledge, this is the first time tumor-infiltrating lymphocytes of the Syrian hamster have been cultured and characterized. Thorough immunophenotyping of hamster tumors and TIL cultures requires the development of new hamster-specific antibodies in addition to the currently available ones used in this study. In conclusion, our data supports the use of the Syrian hamster as a novel platform for testing TIL protocols in a species other than mouse or human.

Mouse pancreatic beta cells express MHC class II and stimulate CD4(+) T cells to proliferate.

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4(+) and CD8(+) T cells have been shown to mediate beta-cell killing. While CD8(+) T cells can directly recognize MHC class I on beta cells, the interaction between CD4(+) T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA-DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4(+) T cells with T-cell receptors that recognize beta-cell antigens. Acute infiltration of CD4(+) T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN-γ increased MHC class II gene expression, and blocking IFN-γ signaling in beta cells inhibited MHC class II upregulation. IFN-γ also increased HLA-DR expression in human islets. MHC class II(+) beta cells stimulated the proliferation of beta-cell-specific CD4(+) T cells. Our study indicates that MHC class II molecules may play an important role in beta-cell interaction with CD4(+) T cells in the development of type 1 diabetes.

IL-33 promotes MHC class II expression in murine mast cells.

Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of FcεRI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis.

MC32 tumor cells acquire Ag-specific CTL resistance through the loss of CEA in a colon cancer model

We previously reported that MC32 cells resist carcinoembryonic antigen (CEA) DNA vaccination by losing their antigen presentation to Ag-specific CTLs in the context of MHC class I antigens in a colon cancer therapeutic model. In this study, we selected 2 tumor cells, MC32-S2–2 and MC32-S4–2, which have the ability to form tumors in CEA DNA vaccine-immunized mice. Wild type MC32 cells grew significantly less in CEA-immunized mice (with Ag-specific CTL lytic activity) than in control mice (with no Ag-specific CTL lytic activity). However, MC32-S2–2 and MC32-S4–2 cells grew at a similar rate in both control and CEA-immunized mice, confirming their resistant status against CEA DNA vaccination. MC32-S2–2 and MC32-S4–2 cells were not susceptible to lysis by CEA-specific CD8+ T cells. Moreover, when MC32-S2–2 and MC32-S4–2 cells were used as stimulating agents of CEA-specific immune cells for IFN-γ production, these cells failed to stimulate the induction of Ag-specific IFN-γ, suggesting a loss of tumor cell recognition by Ag-specific immune cells. However, MC32-S2–2 and MC32-S4–2 cells expressed MHC class I antigens in a manner similar to that of wild type MC32 cells. Finally, Western blot assay confirmed that in MC32-S2–2 and MC32-S4–2 cells, CEA expression remained absent but mouse CEA was expressed. Taken together, these data show that MC32 cells may also be able to achieve resistance to CEA-specific CTLs by antigen loss in this model.

Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen(-/-) and Ldp27(-/-)) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag-stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow-derived dendritic cells infected with LdCen(-/-) and Ldp27(-/-) parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan-Leishmania species vaccines.

Immune parameters in the intestine of wild and reared unvaccinated and vaccinated Atlantic salmon (Salmo salar L.)

Forming a barrier to the outside world, the gut mucosa faces the challenge of absorbing nutrients and fluids while initiating immune reactions towards potential pathogens. As a continuation to our previous publication focusing on the regional intestinal morphology in wild caught post smolt and spawning Atlantic salmon, we here investigate selected immune parameters and compare wild, reared unvaccinated and vaccinated post smolts.We observed highest transcript levels for most immune-related genes in vaccinated post smolts followed by reared unvaccinated and finally wild post smolts, indicating that farming conditions like commercial feed and vaccination might contribute to a more alerted immune system in the gut. In all groups, higher levels of immune transcripts were observed in the second segment of mid-intestine and in the posterior segment. In the life stages and conditions investigated here, we found no indication of a previously suggested population of intestinal T cells expressing MHC class II nor RAG1 expression.

The ThPOK transcription factor differentially affects the development and function of self-specific CD8+T cells and regulatory CD4+T cells

The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-β-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells.

Gone over to the dark side

Gone over to the dark side

Helper T cells down-regulate CD4 expression upon chronic stimulation giving rise to double-negative T cells

Double-negative T (DNT) cells are αβTCR+CD3+CD4−CD8−NK1.1− cells that constitute a small but significant proportion of the αβTCR+ T cells. Their developmental pathway and pathological significance remain unclear. In the present study, we utilized chronic in vitro stimulation of CD4+ T cells to mimic immune hyper-activation of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, conditions characterized by DNT cells accumulation. After approximately 4–5 rounds of stimulation, the CD3+CD4− population became apparent. These cells did not express CD8, NK1.1, γδTCR, or B220, exhibited a highly proliferative effector phenotype, and were dependent on T cell receptor (TCR) stimulation for survival. Moreover, CD3+CD4− cells expressed MHC class II-restricted αβTCR, indicative of their origin from a CD4+ T cell population. The results presented herein illustrate a novel method of DNT cell generation in vitro and suggest that immune hyper-activation could also be implicated in the genesis of the disease-associated DNT cells in vivo.