A tryptophan metabolite made by a gut microbiome eukaryote induces pro-inflammatory T cells.

The large intestine harbors microorganisms playing unique roles in host physiology. The beneficial or detrimental outcome of host-microbiome coexistence depends largely on the balance between regulators and responder intestinal CD4+ T cells. We found that ulcerative colitis-like changes in the large intestine after infection with the protist Blastocystis ST7 in a mouse model are associated with reduction of anti-inflammatory Treg cells and simultaneous expansion of pro-inflammatory Th17 responders. These alterations in CD4+ T cells depended on the tryptophan metabolite indole-3-acetaldehyde (I3AA) produced by this single-cell eukaryote. I3AA reduced the Treg subset in vivo and iTreg development in vitro by modifying their sensing of TGFß, concomitantly affecting recognition of self-flora antigens by conventional CD4+ T cells. Parasite-derived I3AA also induces over-exuberant TCR signaling, manifested by increased CD69 expression and downregulation of co-inhibitor PD-1. We have thus identified a new mechanism dictating CD4+ fate decisions. The findings thus shine a new light on the ability of the protist microbiome and tryptophan metabolites, derived from them or other sources, to modulate the adaptive immune compartment, particularly in the context of gut inflammatory disorders.

The Effects of NDRG2 Overexpression on Cell Proliferation and Invasiveness of SW48 Colorectal Cancer Cell Line.

BACKGROUND: Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells. METHODS: SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 (MMP9) activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively. RESULTS: MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9. CONCLUSIONS: The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity.

Hybrid structural modeling of alloantibody binding to human leukocyte antigen with rapid and reproducible cross-linking mass spectrometry.

Alloantibody recognition of donor human leukocyte antigen (HLA) is associated with poor clinical transplantation outcomes. However, the molecular and structural basis for the alloantibody-HLA interaction is not well understood. Here, we used a hybrid structural modeling approach on a previously studied alloantibody-HLA interacting pair with inputs from ab initio, in silico, and in vitro data. Highly reproducible cross-linking mass spectrometry data were obtained with both discovery- and targeted mass spectrometry-based approaches approaches. The cross-link information was then used together with predicted antibody Fv structure, predicted antibody paratope, and in silico-predicted interacting surface to model the antibody-HLA interaction. This hybrid structural modeling approach closely recapitulates the key interacting residues from a previously solved crystal structure of an alloantibody-HLA-A∗11:01 pair. These results suggest that a predictive-based hybrid structural modeling approach supplemented with cross-linking mass spectrometry data can provide functionally relevant structural models to understand the structural basis of antibody-HLA mismatch in transplantation.

Single Molecule Force Spectroscopy Reveals Distinctions in Key Biophysical Parameters of αß T-Cell Receptors Compared with Chimeric Antigen Receptors Directed at the Same Ligand.

Chimeric antigen receptor (CAR) T-cell therapies exploit facile antibody-mediated targeting to elicit useful immune responses in patients. This work directly compares binding profiles of CAR and αß T-cell receptors (TCR) with single cell and single molecule optical trap measurements against a shared ligand. DNA-tethered measurements of peptide-major histocompatibility complex (pMHC) ligand interaction in both CAR and TCR exhibit catch bonds with specific peptide agonist peaking at 25 and 14 pN, respectively. While a conformational transition is regularly seen in TCR-pMHC systems, that of CAR-pMHC systems is dissimilar, being infrequent, of lower magnitude, and irreversible. Slip bonds are observed with CD19-specific CAR T-cells and with a monoclonal antibody mapping to the MHC α2 helix but indifferent to the bound peptide. Collectively, these findings suggest that the CAR-pMHC interface underpins the CAR catch bond response to pMHC ligands in contradistinction to slip bonds for CARs targeting canonical ligands.

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01.

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.

Differential Immune Reactivity Pattern of SW48 and SW1116 Colorectal Cancer Cell Lines with Colorectal Cancer Patients Sera.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. It is also known as the second leading cause of deaths as the early stage detection is not yet available by current methods. So identification of biomarkers can also be functional in early diagnosis and prognosis. MATERIALS AND METHODS: We examined sera from 60 CRC patients of different stages as a source of auto-antibody as well as two human CRC cell lines with different invasive capacities (SW48 and SW1116) as the source of antigens. The pattern of immune reactivity in immuneblotting tests between mentioned cell lines and CRC patients' sera were evaluated by ImageJ software. RESULTS: The Immune reactivity pattern of two cell lines (SW48 and SW1116) with CRC patients' sera were different in band intensities and the most immune reactivity intensity was observed in SW48 cell lysate with sera from Stage III CRC patients. CONCLUSION: Due to the humoral immune response, sera from Stage III CRC patients contained autoantibodies that demonstrated higher immune reactivity. Moreover, SW48 cell line with high aggressive behavior reacted to CRC patients' sera with greater intensity compared with less aggressive behavior cell line (SW1116). Therefore, it is required to use other techniques such as two-dimensional electrophoresis and mass spectrometry.

Increased P-35, EBI3 Transcripts and Other Treg Markers in Peripheral Blood Mononuclear Cells of Breast Cancer Patients with Different clinical Stages.

PURPOSE: Currently, cancer as a major problem around the world threatens human health and has a high incidence in developing countries. Many reports have indicated that patients suffering from cancer demonstrate decreased antitumor immune responses as well as a high prevalence of T regulatory population. It has been reported that Foxp3(+)Tregs exert suppression by cell contact-dependent mechanisms which are mediated by soluble factors such as immunosuppressive cytokines like IL-10, TGF-ß and IL-35. Consequently there is a great need to identify prognostic and diagnostic biomarkers of regulatory T cells for vaccine and drug development. METHODS: In this study IL-10, TGF-ß, IL-35 and Foxp3 mRNA gene expression has been measured in peripheral blood of 40 breast cancer patients and 40 normal age-matched women using quantitative real-time PCR (qRT-PCR) method with Master Mix reaction containing SYBER Green. GAPDH gene was used as housekeeping gene. RESULTS: Our data demonstrated a significant up-regulation of IL-10, TGF-ß, P35, EBI3 and Foxp3 gene expression in patients' peripheral blood compared to normal healthy controls (p<0.05). CONCLUSION: The data suggests that the immune system is suppressed in breast cancer patients, which may be due to elevated Treg cells population. These results may be useful for diagnostic or therapeutic purposes. However it may require more investigations.

Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica.

BACKGROUND: Antigenic similarities between Neisseria lactamica as a commensal species and N. meningitidis serogroup B (NmB) as an important cause of meningitis infection have been considered for the development of an effective vaccine based on their common proteins to prevent life-threatening bacterial meningitis. OBJECTIVES: The main aims of this study were to determine whole proteome profiles of N. lactamica strains and to compare them with whole proteome profile of a reference strain of NmB for identification of some of common proteins between the two species. MATERIALS AND METHODS: We compared the whole proteomic profiles of N. lactamica strains and a reference strain of NmB. Lysates from bacterial strains were resolved by two-dimensional gel electrophoresis (2-DE), followed by Coomassie Brilliant blue staining. Some of the protein spots were excised from the gel and subjected to matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. RESULTS: The analysis of Coomassie-stained gels using ImageMaster 2D Platinum software identified approximately 800 reproducible protein spots in the range of pI 4.5 - 9.5 and M r of 8 - 100 kDa for each 2-DE gel of the studied bacterial strains. By comparing proteome maps of 2-DE gels, more than 200 common protein spots were recognized between the two species. Forty-eight common protein spots between the studied bacterial strains were identified by MALDI-TOF/TOF-MS. The results indicated that among the protein spots identified by MOLDI-TOF/TOF mass spectrometry, the groups of proteins included cell surface, energy metabolism, amino acid transport and metabolism, coenzyme metabolism, defense, multifunctional cellular processes, DNA, RNA and protein synthesis, ribosomal structure, regulatory functions, replication, transcription, translation, unknown and hypothetical proteins with unknown function. We found that N. lactamica strains have a proteome profile somewhat similar to each other and slightly different with NmB. CONCLUSIONS: These results show the usefulness of proteome analysis in successful identification of the common proteins between N. lactamica strains and NmB. This proteomics analysis is the starting point in the path of knowledge development about whole proteome profiles of N. lactamica strains.

Concomitant Increase of OX40 and FOXP3 transcripts in peripheral blood of patients with breast cancer.

BACKGROUND: Regulatory T cells (T-regs) have an important role in cancer by suppression of protective antitumor immune responses. Regulatory T cells express the forkhead/winged helix transcription factor (FOXP3) and OX40 molecules which have important regulatory roles in the immune system. OBJECTIVE: To evaluate FOXP3 and OX40 transcripts in the peripheral blood mononuclear cells of women with breast cancer. METHODS: Blood samples from 40 women with histologically-confirmed infiltrating ductal carcinoma of the breast and 40 healthy volunteer women without a history of malignancy or autoimmune disorders were collected. The abundance of FOXP3 and OX40 gene transcripts were determined by quantitative real-time PCR (qRT-PCR). RESULTS: There was a significant positive correlation between FOXP3 and OX40 gene expression in women with breast cancer in a stage dependent manner. CONCLUSION: This finding emphasizes the importance of T-regs as predominant targets for breast cancer immunotherapy.