Iria Gómez-Touriño

Posttranslational Modification of HLA-DQ Binding Islet Autoantigens in Type 1 Diabetes

Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis–restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

Discovery of a selective islet peptidome presented by the highest-risk HLA-DQ8trans molecule

HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLA-DQ8trans–binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLA-DQ8trans. The selective processing and presentation of HLA-DQ8trans–binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.

Galectin-1 synthesis in type 1 diabetes by different immune cell types: Reduced synthesis by monocytes and Th1 cells

Galectins are a group of β-galactoside-binding mammalian lectins that play important roles in the regulation of the immune response by promoting T cell tolerance, blunting Th1 and Th17 responses and suppressing autoimmune inflammation. However, the synthesis of these molecules by different T helper (Th) subsets and in the context of human type 1 diabetes (T1D) has not yet been studied. Our results show that Th17 polarising conditions induce the synthesis of higher levels of galectin-1 compared to Th1-polarised lymphocytes. In the context of human diabetes, peripheral blood mononuclear cells (PBMCs) from T1D patients, either unstimulated or after stimulation, secreted significantly lower amounts of galectin-1 in vitro compared to healthy donors. The reduced galectin-1 synthesis observed in this autoimmune disease occurs in a dominant pro-inflammatory cytokine milieu and it is mainly due to the lower synthesis by monocytes. Surprisingly, CD4(+) T helper cells from these patients secreted similar levels of galectin-1 compared to healthy donors, probably mediated by Th17 cytokines. In conclusion, CD4(+) T helper lymphocytes from T1D patients produce normal levels of the immunoregulator galectin-1 but its reduced synthesis by monocytes helps to maintain a skewed pro-inflammatory response.

T cell receptor β-chains display abnormal shortening and repertoire sharing in type 1 diabetes

Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2 × 108TCRB sequences of circulating naive, central memory, regulatory and stem cell-like memory CD4+ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4+ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease.T cell receptors are generated by somatic gene recombination, and are normally selected against autoreactivity. Here the authors show that CD4 T cells from patients with autoimmune type 1 diabetes have shorter TCRβ sequences, broader repertoire diversity, and more repertoire sharing than those from healthy individuals.

Erratum to: Nucleofection of whole murine retinas.

The online version of the original article can be found under doi:10.1007/s10616-012-9509-3.

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.