4 research outputs found
A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes
Transient Depletion of Foxp3 Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice.
Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes
Additional file 1: Figure S1. of A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes
Characterization of α-VSA sera. In order to specify the exact target sequence of the polyclonal antisera, the semi-conserved and the variable domain of the RIFIN variants RIF40 and RIF50 and of the STEVOR proteins PFL2610w and MAL13P1.7 were separately expressed as recombinant proteins. The RIFIN antisera α-RIF40.2, α-RIF44 and α-RIF50 (A) as well as the STEVOR α-PFL2610w, α-MAL13P1.7, α-PFC0025c and α-PFA0750w (B), which were generated by immunization with a protein spanning the semi-conserved and the variable domain, were subsequently used for Western blot analysis. Furthermore, the α-PfMC-2TM-SC serum generated solely against the semi-conserved region of PFF1525c was included in the analysis to check the specificity for its protein family (C). Approximately 20 ng of each recombinant protein or lysate from 1x107 cell membranes were loaded in each lane as indicated. All four antisera, α-RIF40.2, α-RIF50, α-PFL2610w and α-MAL13P1.7, are specific to the semi-conserved region of their own antigen. Furthermore, all antisera used in this study were shown to recognize the semi-conserved region of their own protein family, but are not cross-reactive with other VSA families. Unspecific cross-reactions of the small VSA antisera with the His-tag were excluded using an unrelated Entamoeba histolytica His-tagged protein recombinantly expressed under the same conditions (His-tag control) and lysate from uninfected red blood cells (RBC lysate). The recombinant proteins of all three small VSA families tend to form multimer complexes; accordingly bands corresponding in size to monomers are labelled with a single *, dimers with ** and trimers with *** as calculated by the size of the recombinant His-tagged protein
Additional file 1: Figure S1. of A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes
Characterization of α-VSA sera. In order to specify the exact target sequence of the polyclonal antisera, the semi-conserved and the variable domain of the RIFIN variants RIF40 and RIF50 and of the STEVOR proteins PFL2610w and MAL13P1.7 were separately expressed as recombinant proteins. The RIFIN antisera α-RIF40.2, α-RIF44 and α-RIF50 (A) as well as the STEVOR α-PFL2610w, α-MAL13P1.7, α-PFC0025c and α-PFA0750w (B), which were generated by immunization with a protein spanning the semi-conserved and the variable domain, were subsequently used for Western blot analysis. Furthermore, the α-PfMC-2TM-SC serum generated solely against the semi-conserved region of PFF1525c was included in the analysis to check the specificity for its protein family (C). Approximately 20 ng of each recombinant protein or lysate from 1x107 cell membranes were loaded in each lane as indicated. All four antisera, α-RIF40.2, α-RIF50, α-PFL2610w and α-MAL13P1.7, are specific to the semi-conserved region of their own antigen. Furthermore, all antisera used in this study were shown to recognize the semi-conserved region of their own protein family, but are not cross-reactive with other VSA families. Unspecific cross-reactions of the small VSA antisera with the His-tag were excluded using an unrelated Entamoeba histolytica His-tagged protein recombinantly expressed under the same conditions (His-tag control) and lysate from uninfected red blood cells (RBC lysate). The recombinant proteins of all three small VSA families tend to form multimer complexes; accordingly bands corresponding in size to monomers are labelled with a single *, dimers with ** and trimers with *** as calculated by the size of the recombinant His-tagged protein