Comparative Study of the Immunoregulatory Capacity of In Vitro Generated Tolerogenic Dendritic Cells, Suppressor Macrophages, and Myeloid-Derived Suppressor Cells

International audienceBackground. Regulatory myeloid cell (RMC) therapy is a promising strategy for the treatment of immunological disorders such as autoimmune disease and allograft transplant rejection. Various RMC subsets can be derived from total bone marrow using different protocols, but their phenotypes often overlap, raising questions about whether they are truly distinct.Methods. In this study, we directly compared the phenotype and function of 3 types of RMCs, tolerogenic dendritic cells, suppressor macrophages, and myeloid-derived suppressor cells, generated in vitro from the same mouse strain in a single laboratory.Results. We show that the 3 RMC subsets tested in this study share some phenotypic markers, suppress T cell proliferation in vitro and were all able to prolong allograft survival in a model of skin transplantation. However, our results highlight distinct mechanisms of action that are specific to each cell population.Conclusions. This study shows for the first time a side-by-side comparison of 3 types of RMCs using the same phenotypic and functional assays, thus providing a robust analysis of their similarities and differences

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

International audienceIn a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival , which is mediated by the induction of CD8+CD45RC[lo] regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.A[a]/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.A[a]/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function

Role of the intracellular ion channels TMEM176A and TMEM176B in the immune system

International audienceIon channels represent attractive targets for the development of new therapies but still remain poorly studied in the immune system, in particular intracellular ion channels. In this work, we explored the role of two highly redundant ion channels named TMEM176A and TMEM176B that are intriguingly strongly expressed both in RORγt+ cells (ILC3, Th17) and immature dendritic cells. To investigate the role of these homologs and avoid any compensation effect, we generated a double KO (DKO) mouse using CRISPR-Cas9. Surprisingly, Tmem176a/b appeared dispensable for the function of RORγt+ cells and in the protective function of type 17 immunity during chemically-induced or infectious colitis. In contrast, antigen presentation by dendritic cells to CD4+ T cells through MHC II was selectively impaired in DKO mice. Using a real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. These results indicate that TMEM176A/B ion channels play a predominant role in adaptive immunity as new intracellular components of the intracellular MHC II machinery

Infection in Patients with Suspected Thrombotic Microangiopathy Based on Clinical Presentation

International audienceBackground and objectives - In contrast to shigatoxin-associated (STEC) causing hemolytic uremic syndrome, STEC-unrelated infections associated with thrombotic microangiopathy are less characterized. Design, setting, participants, & measurements - Our retrospective study in a four-hospital institution of 530 consecutive patients with adjudicated thrombotic microangiopathies during the 2009-2016 period studied STEC-unrelated infections' epidemiology and major outcomes (death, acute dialysis, and major cardiovascular events). Results - STEC-unrelated infection was present in 145 of 530 (27%) patients, thrombotic microangiopathies without infection were present in 350 of 530 (66%) patients, and STEC causing hemolytic and uremic syndrome was present in 35 of 530 (7%) patients. They (versus thrombotic microangiopathy without infection) were associated with age >60 years (36% versus 18%), men (53% versus 27%), altered consciousness (32% versus 11%), mean BP <65 mm Hg (21% versus 4%), lower hemoglobin and platelet count, and AKI (72% versus 49%). They were associated with more than one pathogen in 36 of 145 (25%) patients (either isolated [14%] or combined [86%] to other causes of thrombotic microangiopathy); however, no significant clinical or biologic differences were noted between the two groups. They were more frequently due to bacteria (enterobacteria [41%], [11%], and [3%]) than viruses (Epstein-Barr [20%], cytomegalovirus [18%], influenza [3%], hepatitis C [1%], HIV [1%], and rotavirus [1%]). STEC-unrelated infections were independent risk factors for in-hospital death (odds ratio, 2.22; 95% confidence interval, 1.18 to 4.29), major cardiovascular event (odds ratio, 3.43; 95% confidence interval, 1.82 to 6.69), and acute dialysis (odds ratio, 3.48; 95% confidence interval, 1.78 to 7.03). Bacteria (versus other pathogens), and among bacteria, enterobacteria, presence of more than one bacteria, and without shigatoxin were risk factors for acute dialysis. Conclusions - Infections are frequent thrombotic microangiopathy triggers or causes, and they are mostly unrelated to STEC. Infections convey a higher risk of death and major complications. The most frequent pathogens were enterobacteria, , Epstein-Barr virus, and cytomegalovirus. Podcast - This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_09_07_CJN17511120.mp3

Adoptive transfer of B cells transfers tolerance.

<p>Cells were sorted by FACS Aria <b>(sorting strategy displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s003" target="_blank">S3D and E Fig.</a>)</b> from the spleen of tolerant rats (>100 days after the graft) that had received a transfer of splenocytes from a previously tolerant recipient and adoptively transferred to sub-lethally irradiated recipients the day before the transplant. <b>(A)</b> B cells (CD45RA⁺, n = 3), T cells (TCR⁺, n = 4), CD8⁺ Tregs (CD8⁺CD45RC^low, n = 2), pDCs (mAb 85C7⁺ n = 3) Groups are compared with each other and to irradiated animals transferred with naive splenocytes (naive splenocytes, n = 5) by Log-rank (Mantel-Cox) Test <i>p</i> <0.05*; p<0.01**; p<0.001***. <b>(B)</b> Wild type (WT) and B cell-deficient <i>Igm</i> knockout (KO) rats were treated with AAV-FGL2 (n = 8 and 3, respectively), AAV-null (n = 5) or untreated (NT, n = 3), and analyzed for graft survival. <b>(C)</b> Splenocytes from adoptively-transferred tolerant rats were depleted in CD45RA⁺ B cells (CD45RA⁻ cells) or not (splenocytes) and transferred to new irradiated recipients. Log-rank (Mantel-Cox) Test p<0.01**. <b>(D) Left:</b> A fraction of the transferred tolerogenic CD45RA⁺ B cells was tested for inhibition of CFSE-labeled CD4⁺CD25⁻ T cell proliferation in response to allogeneic LEW.1W cDCs, pDCs (stimulator/effector ratio of 1:4) or anti-CD3 at day 6 of culture. Shaded grey: naive CD45RA⁺ B cells n = 3, black line: tolerogenic CD45RA⁺ B cells n = 4. <b>Right</b>: Representative histogram of one proliferation assay of CD4⁺CD25⁻ T cells with allogeneic pDCs and CD45RA⁺ B cells from naive (shaded grey) or splenocyte-transferred tolerant rats (black line). <b>(E)</b> Graft infiltrating cells were analyzed for the presence of CD45RA⁺ cells in graft of rats transferred with B cells, at days 100 after the graft, as compared with syngeneic grafts (n = 3).</p

Splenocytes from AAVFGL2-treated rats with long-term surviving grafts transfer donor alloantigen-specific long-term graft survival in an iterative manner.

<p>Splenocytes from long-term AAV-FGL2-treated recipients were injected <i>i</i>.<i>v</i>. into sub-lethally irradiated recipients (LEW.1A) the day before heart allotransplantation (LEW.1W). Graft survival was evaluated by palpation through the abdominal wall. Total splenocytes (1x10⁸ cells) from long-term (≥120 days) AAV-FGL2-treated rats were adoptively transferred (1^st-transferred, n = 5), and then total splenocytes (10⁸ cells) were iteratively transferred to 2^nd- (n = 6), 3rd (n = 4), 4^th (n = 3), 5^th (n = 3) and 6^th (n = 3) LEW.1A recipients receiving LEW.1W hearts. Third-party grafts were from Brown-Norway origin and adoptive transfer of splenocytes from LEW.1W-transplanted animals did not inhibit acute rejection (third party, n = 3, performed in animals that received a second adoptive transfer). Splenocytes from naive non-transplanted rats did not inhibit acute rejection (naive splenocytes, n = 5) and non-irradiated non-transferred recipients (no treatment, n = 6) also showed acute rejection. Irradiation alone without cell transfer delays graft survival but does not prevent graft from rejection (irradiated, n = 5). All groups were compared to irradiated animals transferred with naive splenocytes by Log-rank (Mantel-Cox) Test (<i>p value</i> ***<0.001).</p

Over-expression of FGL2 <i>in vivo</i> prolongs cardiac allograft survival.

<p><b>(A)</b> Cardiac graft recipients received intravenously 3x10¹²vector genomes/kg of AAV-FGL2 (▲ n = 8), or non coding AAV (▼ n = 5, 2 different experiments), and received a heterotopic transplant 30 days later. Graft survival was evaluated by palpation through the abdominal wall. Log-rank (Mantel-Cox) test ***<i>p<0</i>.<i>001</i> for AAV-FGL2 vs. AAV null controls. <b>(B)</b> Left: Relative proportion of dividing CD4⁺CD25⁻ T cells at day 6 in the presence of different concentrations of recombinant human FGL2-GST was evaluated by CFSE dilution by gating first on DAPI^- live cells and then on TCR⁺CD4⁺ cells <b>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s002" target="_blank">S2B Fig.</a>).</b> The negative control was purified rat IgG at 10 μg/ml (n = 4, ** <i>p</i><0.01). Right: Representative histogram of relative proportion of dividing CD4⁺T cell in the presence of 10μg/ml FGL2-GST protein (black line) or IgG control (grey).</p

Alloantibody production was suppressed after AAV-FGL2 treatment and adoptive transfer of splenocytes.

<p>Sera were collected from naive rats, or at the moment of rejection from rats treated with AAV-control or AAV-FGL2 (rejecting at < 30 days or > 120 days after transplantation) or receiving adoptive transfers (> 120 days after transplantation). Levels of donor-specific IgG1, IgG2a, and IgG2b antibodies were evaluated by cytofluorimetry and normalized to serum from naive rats (MFI / MFI syngeneic). Two way Anova, Bonferroni post test <i>p value</i> * <0.5; ** <0.01; ***<0.001.</p

Generation of Immunodeficient Rats With Rag1 and Il2rg Gene Deletions and Human Tissue Grafting Models

International audienc