The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.
More than 10 protein molecules with endo‐1,4‐beta‐glucanase activity were identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.
The HLA locus is the strongest risk factor for anti-citrullinated protein antibody (ACPA)+ rheumatoid arthritis (RA). Despite considerable efforts in the last 35 years, this association is poorly understood. Here we identify (citrullinated) vinculin, present in the joints of ACPA+ RA patients, as an autoantigen targeted by ACPA and CD4+ T cells. These T cells recognize an epitope with the core sequence DERAA, which is also found in many microbes and in protective HLA-DRB1*13 molecules, presented by predisposing HLA-DQ molecules. Moreover, these T cells crossreact with vinculin-derived and microbial-derived DERAA epitopes. Intriguingly, DERAA-directed T cells are not detected in HLA-DRB1*13+ donors, indicating that the DERAA epitope from HLA-DRB1*13 mediates (thymic) tolerance in these donors and explaining the protective effects associated with HLA-DRB1*13. Together our data indicate the involvement of pathogen-induced DERAA-directed T cells in the HLA–RA association and provide a molecular basis for the contribution of protective/predisposing HLA alleles. Autoantibodies targeting citrunillated proteins are common in rheumatoid arthritis patients. Here the authors show that vinculin (a human protein) and some microbial proteins are recognized by these antibodies and by CD4+T cells, and this response is absent in patients carrying a protective HLA allele.
Abstract Background Several methods exist for flow‐cytometric estimation of human peripheral blood CD4 + T regulatory cells (CD4 + Tregs). Methods We report our experience with the estimation of human CD4 + Tregs via three different characterizations using flow cytometry (CD25 high FoxP3 + , CD25 high CD127 low/− FoxP3 + , and CD4 + CD25 high/int CD45ROFoxP3 + ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4 + T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. Results Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4 + T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3 + non‐Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4 + CD25 high CD127 low/− cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3 + non‐Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3 + non‐Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non‐Tregs. Conclusion Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.
BALB/c mice were immunized with human islets of Langerhans and spleen cells from two mice. found to develop cell‐surface antibodies against insulin‐producing rat islet tumour RIN‐5F cells, were fused with mouse myeloma cells. Antibody‐producing hybrids were cloned on the basis of their production of surface antibodies reactive with paraformaldehyde‐fixed RIN‐5F cells by indirect immunofluorescence analysis in the fluorescence‐activated cell sorter. Among 236 primary clones, eight stable cell lines producing islet‐cell‐surface antibodies were eventually cloned. Antibody 2G3 (IgM) reacted with viable normal rat islet cells and high insulin‐producing rat islet tumour RIN5‐A2 cells, while 3G3(IgM) only reacted with RIN5‐A2 cells. AntibodyβB1 (IgG1) reacted with all islet cells tested and detected an M 1 21k component in immunoblotting experiments with RIN‐5AH cell plasma membrane proteins electrophoretically transferred to nitrocellulose filters. Antibody 7F6 (IgM) reacted with all islet and non‐islet cells tested and delected bands of M 1 66k and 27k by immunoblotting. Antibodies γB3,γB6, γC2, and 6BI (all IgM) showed varying degrees of binding to different islet cells, but reacted only weakly with non‐islet human cells. It is concluded that monoclonal antibodies against pancreatic islet cells may define specific endocrine islet‐cell‐surface determinants.
HLA-DQA1 and -DQB1 are strongly associated with type 1 diabetes (T1D), and DQ8.1 and DQ2.5 are major risk haplotypes. Next generation targeted sequencing of HLA-DQA1 and -DQB1 in Swedish newly diagnosed 1-18 year-old patients (n=962) and controls (n=636) was used to construct abbreviated DQ haplotypes, converted into amino acid (AA) residues, and assessed for their associations with T1D. A hierarchically-organized haplotype (HOH) association analysis, allowed 45 unique DQ haplotypes to be categorized into seven clusters. The DQ8/9 cluster included two DQ8.1 risk and the DQ9 resistant haplotypes, and the DQ2 cluster, included the DQ2.5 risk and DQ2.2 resistant haplotypes. Within each cluster, HOH found residues α44Q (OR 3.29, p=2.38*10<sup>-85</sup> ) and β57A (OR 3.44, p=3.80*10<sup>-84</sup>) to be associated with T1D in the DQ8/9 cluster representing all ten residues (α22, α23, α44, α49, α51, α53, α54, α73, α184, β57) due to complete linkage-disequilibrium (LD) of α44 with eight such residues. Within the DQ2 cluster and due to LD, HOH analysis found α44C and β135D to share the risk for T1D (OR 2.10, p=1.96*10<sup>-20</sup>). The motif “QAD” of α44, β57, and β135 captured the T1D risk association of DQ8.1 (OR 3.44, <i>p</i>=3.80*10<sup>-84</sup>), the corresponding motif “CAD” captured the risk association of DQ2.5 (OR 2.10, <i>p</i>=1.96*10<sup>-20</sup>). Two risk associations were related to GADA and IA-2A, but in opposite directions. “CAD” was positively associated with GADA (OR 1.56; <i>p</i>=6.35*10<sup>-8</sup>) but negatively with IA-2A (OR 0.59, <i>p</i>= 6.55*10<sup>-11</sup>). “QAD” was negatively associated with GADA (OR 0.88; <i>p</i>= 3.70*10<sup>-3</sup>) but positively with IA-2A (OR 1.64; <i>p</i>= 2.40*10<sup>-14</sup>), despite a single difference at α44. The residues are found in and around anchor pockets 1 and 9, as potential TCR contacts, in the areas for CD4 binding and putative homodimer formation. The identification of three HLA-DQ AA (α44, β57, β135) conferring T1D risk should sharpen functional and translational studies.
The DAMIC-M (DArk Matter In CCDs at Modane) experiment employs thick, fully depleted silicon charged-coupled devices (CCDs) to search for dark matter particles with a target exposure of 1 kg-year. A novel skipper readout implemented in the CCDs provides single electron resolution through multiple non-destructive measurements of the individual pixel charge, pushing the detection threshold to the eV-scale. DAMIC-M will advance by several orders of magnitude the exploration of the dark matter particle hypothesis, in particular of candidates pertaining to the so-called "hidden sector." A prototype, the Low Background Chamber (LBC), with 20g of low background Skipper CCDs, has been recently installed at Laboratoire Souterrain de Modane and is currently taking data. We will report the status of the DAMIC-M experiment and first results obtained with LBC commissioning data.
