Prevalence of Abs against L5P and MAP-derived ZnT8 homolog in T1D at-risk subjects and HC.

<p>Plasma samples were analyzed in duplicate for Abs directed against L5P (A) and MAP3865c_133-141 (B) epitopes. Distributions are relative to the sample sets including only the first or the last time-point collection in comparison to HC. Horizontal bars indicate means. The dotted line corresponds to the Abs positivity threshold. AUC and <i>p</i>-values relative to T1D at-risk subjects vs. HC test are given in the upper part. Specific percentage of reactivity is indicated above each distribution.</p

<i>In Vivo</i> Targeting of Intratumor Regulatory T Cells Using PEG-Modified Single-Walled Carbon Nanotubes

Recent evidence regarding the role of regulatory T cells (T_reg) in tumor development has suggested that the manipulation of T_reg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective <i>in vivo</i> targeting of intratumor T_reg or other immune cell subpopulations. Herein, we investigated the ability of ligands against T_reg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into T_reg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) T_reg compared to other reported T_reg-specific markers (folate receptor 4, CD103, and CD39). <i>Ex vivo</i> investigations showed that the T_reg targeting efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. <i>In vivo</i> investigations showed that PEG-SWCNTs armed with GITR ligands targeted T_reg residing in a B16 melanoma more efficiently then intratumor non-T_reg or splenic T_reg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor T_reg vs effector T cell (T_eff) ratio, and active targeting of markers that are enriched in intratumor vs splenic T_reg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by T_reg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus <i>ex vivo</i> and <i>in vivo</i>. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer

Overexpression of LYPW inhibits TCR signaling in DP thymocytes.

<p>A–B, Overexpression of LYPW causes reduced activation of Erk in thymocytes. Total thymocytes from TgLYPW or TgLYPW^C227S (striped bars) or their respective non-Tg littermates (white bars) were stimulated with 20 µg/ml anti-CD3 and 10 µg/ml anti-Armenian Hamster IgG1 crosslinker for 2.5 minutes. A, Graph shows phosphorylation of Erk in total thymocyte lysates assessed using the PathScan® phospho-p44 MAPK (Thr202/Tyr204) sandwich ELISA kit. Histogram shows mean and range of fold induction of at least 3 biological replicates. B, Phosphorylation of Erk in DP thymocytes was assessed by phosphoflow analysis after intracellular staining with an anti-pErk Ab. Fold induction of Erk phosphorylation was normalized within each experiment relative to the sample with the highest induction. Histogram shows mean and range of at least 3 biological replicates. C, Overexpression of LYPW causes reduced T cell activation in DP thymocytes. Thymocytes from TgLYPW or TgLYPW^C227S (grey graphs) and their respective non-Tg littermates (black graphs) were cultured in the presence of 10 µg/ml (long dashed graphs) or 25 µg/ml (solid graphs) anti-CD3, or media alone (dotted graphs), for 18 hours. Graphs shows expression of CD69 in DP thymocytes as assessed by flow cytometry analysis after staining with an anti-CD69 Ab. Median fluorescence intensity (MFI) values are indicated on each graph. Graphs are representative of at least 3 biological replicates with identical results.</p

Mice transgenic for LYP-W620 (TgLYPW) show overexpression of LYP in DP thymocytes.

<p>A, LYP protein expression in TgLYPW thymocytes. Total thymocytes from TgLYPW (lane 2) or a non-Tg littermate (lane 1) and TgLYPW^C227S (lane 4) or a non-Tg littermate (lane 3) were lysed and subjected to immunoprecipitation (IP) using an anti-HA antibody (Ab). Panel shows Western blotting using an anti-LYP Ab. Data are representative of 3 independent experiments with similar results. B, LYP phosphatase activity in TgLYPW thymocytes. Anti-HA IPs were performed from lysates of total thymocytes from TgLYPW and TgLYPW^C227S mice. Graphs show the phosphatase activity of LYP as assessed by dephosphorylation of the fluorescent substrate DiFMUP. Data are representative of 2 independent experiments with similar results. C–D, LYP-W620 transgene expression in thymocyte subpopulations. Expression of LYPW (C) or LYPW^C227S (D) was assessed by intracellular staining using a fluorophore-conjugated anti-HA Ab in DN (upper left panel), DP (upper right panel) CD4SP (lower left panel) and CD8SP (lower right panel) thymocytes from Tg mice (black graphs) and control non-Tg littermates (grey filled graphs). E, Quantification of overexpression of LYPW relative to endogenous Pep in DP thymocytes of TgLYPW mice. mRNA encoding LYP and Pep was quantified by qPCR from sorted DP thymocytes from control BALB/c (white bar) and TgLYPW (striped bar) mice, using a primer pair that amplifies both human <i>PTPN22</i> and mouse <i>Ptpn22</i> mRNAs. Graph shows relative expression levels of total <i>PTPN22</i> after normalization to the mouse housekeeping gene <i>Polr2a</i>. Data are average and SE of 3 biological replicates.</p

OT-1 thymocytes overexpressing LYP-W620 show similar antigen sensitivity as non-Tg thymocytes.

<p>Freshly isolated OT-1 transgenic thymocytes from TgLYP (black circles), TgLYP^C227S (black triangles) or control non-Tg mice (white diamonds) were co-cultured overnight together with RMA cells and the indicated doses of SIINFKL peptide (N4, left panel), of the low and very low affinity altered peptide ligands SIITFEKL (T4, middle panel) and SIIVFEKL (V4, right panel). Graphs show normalized dose-response values of peptide concentration vs the fraction of maximum numbers of residual DP (CD4 and CD8 bright) thymocytes.</p

Cell distribution in thymus of TgLYPW and TgLYPW^C227S.

<p>Table shows the basic statistics (average±SD) of cell counts in transgenic animals and non transgenic littermates.</p

Polyclonal thymocytes undergo similar levels of negative selection in the presence of absence of transgenic LYP-W620.

<p>A, Lethally irradiated Rip-mOva mice (continuous graphs and black symbols) or C57BL/6 mice (dotted graphs and crossed symbols) were reconstituted with bone marrow harvested from Vβ5xLYPW (circles) or Vβ5 control (diamonds) mice. 10 weeks after the reconstitution the mice were infected with a strain of <i>Listeria monocytogenes</i> expressing Ovalbumin (Lm-Ova). Splenocytes were harvested at 8 days after the infection and briefly <i>in vitro</i> re-stimulated with titrated doses of SIINFEKL peptide. Afterwards, the cells were intracellularly stained for IFNγ. Peptide-dose response curves showing the frequency of IFNγ producing CD8⁺ T cells as fraction of maximum response are presented. B, TgLYPW, TgLYP^C227S and control non-Tg mice contain similar numbers of low avidity auto-reactive T cells. RipxTgLYPW (left panel, black circles), RipxTgLYPW^C227S (right panel, black triangles) and control Rip mice (left and right panels, black diamonds) were infected with Lm-Ova and 4 weeks later challenged by a strain of <i>Vesicular stomatitis virus</i> expressing Ova (VSV-Ova). On day 6 after the primary (left side of each panel) or the secondary infection (right side of each panel) blood was drawn from the mice and PBMC were briefly re-stimulated with SIINFEKL peptide. The number of IFNγ producing CD8⁺ T cells was determined by intracellular cytokine staining. Panels show the frequency of Ova-specific T cells.</p

LYPW overexpression does not alter thymic repertoire and autoimmune phenotype of Skg mice.

<p>A–B, Overexpression of LYPW does not alter Vβ repertoire or numbers of CD4⁺Foxp3⁺ thymocytes in Skg/WT or Skg/Skg mice. Left panel shows average and SE % Vβ positive CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ thymocytes from Skg/WT (A) or Skg/Skg (B) TgLYPW (striped bars, n = 3 for Skg/WT and n = 6 for Skg/Skg) and control non-Tg littermates (white bars, n = 3 for Skg/WT and n = 5 for Skg/Skg) as assessed by flow cytometry analysis after staining with anti-Vβ3, -Vβ5 and -Vβ8 antibodies. Right panel shows mean and range of CD4⁺Foxp3⁺ (first and second bar) and total (third and fourth bars) thymocytes from the same Skg/WT (A) or Skg/Skg (B) TgLYPW mice (striped bars) or non-Tg littermates (white bars). C, Overexpression of LYPW does not alter the course of mannan-induced arthritis and the frequency of Th17 cells in peripheral lymph node (LN) of arthritic Skg mice. Left panel shows arthritis score (measured as ankle swelling in mm) of Skg/Skg TgLYPW mice (black circles, n = 6) and littermates non-Tg Skg/Skg mice (white circles, n = 5) followed-up for 40 days after a single i.p. injection of 20 mg mannan dissolved in 200 µl PBS. One month following mannan-injection, LN cells from Skg/Skg TgLYPW mice (black circles, n = 15) or non-Tg Skg/Skg littermates (white circles, n = 16) were stimulated with 20 ng/ml PMA and 2 mM ionomycin for 5 hours. Right panel shows % Th17⁺ cells of the CD4⁺ T cell population as assessed by flow cytometry analysis after intracellular staining with an anti-IL17 antibody.</p

The real-life management of glucose homeostasis abnormalities in pediatric onco-hematological diseases: data from a national survey

Glycemic abnormalities are a frequent finding in pediatric oncological patients, both during treatment and after its discontinuation. Moreover, impaired glucose tolerance (IGT), impaired fasting glycemia (IFG) and diabetes mellitus (DM) are not rarely diagnosed in non-oncological hematological diseases. To explore the current pediatric Italian approach to the diagnosis and the management of the glycemic alterations in this clinical setting and, thus, to identify and enforce current clinical needs, we submitted an online 23-items survey to all the Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) centers, and surveys were descriptively analyzed. Thirty-nine AIEOP centers were involved in the study. In 2021, among 75278 children and adolescents affected by an oncological or a hematological disease, 1.2 and 0.65% developed DM, while IGT or IFG were widespread in 2.3 and 2.8%, respectively. The main causes of DM were the use of corticosteroids in patients with cancer and the iron overload in patients with thalassemia. Venous fasting plasma glycemia was the most used tool to detect glycemic abnormalities. The performance of oral glucose tolerance test (OGTT) was extremely limited, except when IFG occurred. Despite the diagnosis of DM, ∼45% of patients with cancer and 30% of patients with one hematological disease did not receive an appropriate treatment. In the other cases, insulin was the drug of first choice. Emerging technologies for diabetes care (glucose sensors and insulin pumps) are not largely used yet. The results of our study support the standardization of the care of the glycemic abnormalities during or after onco-hematologic diseases in the pediatric age. Despite the scarce data in pediatric literature, proper guidelines are needed.</p