Characterization of invariant NKT cell phenotype and function in Wiskott-Aldrich Syndrome (WAS)

WASp è una proteina che regola il rimodellamento dell’actina del citoscheletro nelle cellule ematopoietiche. Mutazioni nel gene che codifica per WASp (WAS) causano la Sindrome di Wiskott-Aldrich (WAS). Sebbene WASp sia coinvolto in svariate funzioni delle cellule del sistema immunitario, il suo ruolo nei linfociti invarianti NKT (iNKT) non è mai stato investigato. Difetti delle cellule iNKT potrebbero infatti contribuire allo sviluppo di alcune caratteristiche dei pazienti WAS quali le infezioni ricorrenti e l’ alta incidenza tumorale. Infatti il nostro studio ha rivelato una profonda riduzione numerica dei linfociti iNKT periferici nei pazienti WAS, direttamente correlata con la severità del fenotipo clinico dei pazienti. Per definire ulteriormente il fenotipo delle cellule iNKT prive di WASp, abbiamo esteso l’analisi ai topi was-/-. Le cellule iNKT sono significativamente ridotte anche nel timo e negli organi linfoidi periferici dei topi was-/- rispetto ai controlli wt. Inoltre l’analisi dello sviluppo delle cellule was-/- iNKT cell ha messo in luce un completo blocco maturativo allo stadio intermedio CD44+NK1.1-. In particolare, la generazione di chimere di midollo osseo, ha dimostrato un difetto maturativo intrinseco delle cellule was-/- iNKT. L’assenza di WASp non altera la stimolazione indotta dall’IL-15, che è importante nello sviluppo delle cellule iNKT. Contrariamente, WASp è coinvolto nel controllo della proliferazione omeostatica di questa tipologia cellulare. Le cellule iNKT prive di WASp presentano anche un difetto funzionale, come messo in evidenza dalla ridotta secrezione di IL-4 e IFN-γ dopo la loro attivazione in vivo. In aggiunta, saggi funzionali condotti in vitro, suggeriscono che il difetto funzionale delle cellule iNKT prive di WASp sia mediato dal TCR e che la ridotta produzione di IL-4 sia causata da un difetto funzionale intrinseco alle cellule iNKT, mentre la minor produzione di IFN-γ sembra derivare da un’interazione non efficiente tra le cellule was-/- iNKT cells e le cellule dendritiche was-/-. Nel loro insieme questi risultati dimostrano il ruolo rilevante di WASp nell’integrare segnali critici per lo sviluppo e la funzione delle cellule iNKT, e suggeriscono che i difetti di questa popolazione linfocitaria possano contribuire alla patologia della Sindrome di Wiskott-Aldrich.WAS protein (WASp) is a key regulator of actin cytoskeleton in hematopoietic cells. Mutations of WAS gene cause the Wiskott-Aldrich Syndrome (WAS). Although WASp is involved in various immune cell functions, its role in invariant NKT cells (iNKT) has never been investigated. Defects of iNKT cells could contribute to the pathogenesis of several WAS features, such as recurrent infections and high tumor incidence. Indeed, we found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was-/- mice. iNKT cell number is significantly reduced in thymus and periphery of was-/- mice as compared to wt controls. Moreover analysis of was-/- iNKT cell maturation reveals a complete arrest at the CD44+NK1.1- intermediate stage. Notably, generation of BM chimeras demonstrated a was-/- iNKT cell autonomous developmental defect. The lack of WASp does not affect IL-15 signaling, which is important in iNKT cell development. Conversely WASp is required for the control of homeostatic proliferation. was-/- iNKT cells are also functionally impaired, as suggested by the lower expansion and reduced secretion of IL-4 and IFN-γ upon in vivo activation. Furthermore, in vitro assays suggest that the functional defect of WASp null iNKT cells is TCR-mediated and indicated that the defective IL-4 production is due to a was-/- iNKT cell autonomous defect, whereas the lower IFN-γ production is caused by an inefficient crosstalk between was-/- iNKT cells and was-/- DC. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells, and suggest that defects in these cells may play a role in WAS pathology

Harnessing activin A adjuvanticity to promote antibody responses to BG505 HIV envelope trimers

T follicular helper (

The Wiskott-Aldrich syndrome protein is required for iNKT cell maturation and function

The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was−/− mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was−/− mice as compared with wild-type controls. Moreover analysis of was−/− iNKT cell maturation revealed a complete arrest at the CD44+ NK1.1− intermediate stage. Notably, generation of BM chimeras demonstrated a was−/− iNKT cell-autonomous developmental defect. was−/− iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon γ upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Characterization of invariant NKT cell phenotype and function in Wiskott-Aldrich Syndrome (WAS)

WASp è una proteina che regola il rimodellamento dell’actina del citoscheletro nelle cellule ematopoietiche. Mutazioni nel gene che codifica per WASp (WAS) causano la Sindrome di Wiskott-Aldrich (WAS). Sebbene WASp sia coinvolto in svariate funzioni delle cellule del sistema immunitario, il suo ruolo nei linfociti invarianti NKT (iNKT) non è mai stato investigato. Difetti delle cellule iNKT potrebbero infatti contribuire allo sviluppo di alcune caratteristiche dei pazienti WAS quali le infezioni ricorrenti e l’ alta incidenza tumorale. Infatti il nostro studio ha rivelato una profonda riduzione numerica dei linfociti iNKT periferici nei pazienti WAS, direttamente correlata con la severità del fenotipo clinico dei pazienti. Per definire ulteriormente il fenotipo delle cellule iNKT prive di WASp, abbiamo esteso l’analisi ai topi was-/-. Le cellule iNKT sono significativamente ridotte anche nel timo e negli organi linfoidi periferici dei topi was-/- rispetto ai controlli wt. Inoltre l’analisi dello sviluppo delle cellule was-/- iNKT cell ha messo in luce un completo blocco maturativo allo stadio intermedio CD44+NK1.1-. In particolare, la generazione di chimere di midollo osseo, ha dimostrato un difetto maturativo intrinseco delle cellule was-/- iNKT. L’assenza di WASp non altera la stimolazione indotta dall’IL-15, che è importante nello sviluppo delle cellule iNKT. Contrariamente, WASp è coinvolto nel controllo della proliferazione omeostatica di questa tipologia cellulare. Le cellule iNKT prive di WASp presentano anche un difetto funzionale, come messo in evidenza dalla ridotta secrezione di IL-4 e IFN-γ dopo la loro attivazione in vivo. In aggiunta, saggi funzionali condotti in vitro, suggeriscono che il difetto funzionale delle cellule iNKT prive di WASp sia mediato dal TCR e che la ridotta produzione di IL-4 sia causata da un difetto funzionale intrinseco alle cellule iNKT, mentre la minor produzione di IFN-γ sembra derivare da un’interazione non efficiente tra le cellule was-/- iNKT cells e le cellule dendritiche was-/-. Nel loro insieme questi risultati dimostrano il ruolo rilevante di WASp nell’integrare segnali critici per lo sviluppo e la funzione delle cellule iNKT, e suggeriscono che i difetti di questa popolazione linfocitaria possano contribuire alla patologia della Sindrome di Wiskott-Aldrich.WAS protein (WASp) is a key regulator of actin cytoskeleton in hematopoietic cells. Mutations of WAS gene cause the Wiskott-Aldrich Syndrome (WAS). Although WASp is involved in various immune cell functions, its role in invariant NKT cells (iNKT) has never been investigated. Defects of iNKT cells could contribute to the pathogenesis of several WAS features, such as recurrent infections and high tumor incidence. Indeed, we found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was-/- mice. iNKT cell number is significantly reduced in thymus and periphery of was-/- mice as compared to wt controls. Moreover analysis of was-/- iNKT cell maturation reveals a complete arrest at the CD44+NK1.1- intermediate stage. Notably, generation of BM chimeras demonstrated a was-/- iNKT cell autonomous developmental defect. The lack of WASp does not affect IL-15 signaling, which is important in iNKT cell development. Conversely WASp is required for the control of homeostatic proliferation. was-/- iNKT cells are also functionally impaired, as suggested by the lower expansion and reduced secretion of IL-4 and IFN-γ upon in vivo activation. Furthermore, in vitro assays suggest that the functional defect of WASp null iNKT cells is TCR-mediated and indicated that the defective IL-4 production is due to a was-/- iNKT cell autonomous defect, whereas the lower IFN-γ production is caused by an inefficient crosstalk between was-/- iNKT cells and was-/- DC. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells, and suggest that defects in these cells may play a role in WAS pathology

SARS-CoV-2 mRNA Vaccines: Immunological Mechanism and Beyond

To successfully protect against pathogen infection, a vaccine must elicit efficient adaptive immunity, including B and T cell responses. While B cell responses are key, as they can mediate antibody-dependent protection, T cells can modulate B cell activity and directly contribute to the elimination of pathogen-infected cells. In the unprecedented race to develop an effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the respiratory disease coronavirus disease 2019 (COVID-19), messenger RNA (mRNA) vaccines have emerged as front runners thanks to their capacity for rapid development and ability to drive potent adaptive immune responses. In this review article, we provide an overview of the results from pre-clinical studies in animal models as well as clinical studies in humans that assessed the efficacy of SARS-CoV-2 mRNA vaccines, with a primary focus on adaptive immune responses post vaccination

<i>Slamf1</i>,<i>5</i>,<i>6</i>^Δ/Δ mice exhibit deficiencies in NKT cell development but no overt defects in CD4 T cell, CD8 T cell, or B cell development.

<p>(A) Surface expression and (B) MFI of SLAM, CD84, and Ly108 on peripheral CD4 T cells, CD8 T cells, and B cells from WT and SLAMf receptor triple gene disruption mice. (A-B) Two independent experiments are shown, with 2–3 mice per group. (C) Flow cytometry plots and (D) graphs of CD4⁺ T cell, CD8⁺ T cell, and B220⁺ B cell frequencies in spleens of WT and SLAMf receptor triple gene disruption mice. (C-D) Data is representative of two independent experiments, with 4 mice per group. (E) Flow cytometry plots and (F) graphs of B220^- CD3⁺ CD1d Tetramer⁺ NKT cells in spleens and livers of WT and SLAMf receptor triple gene disruption mice. (E-F) Data are representative of 2 independent experiments, with 4 mice per group.</p

NKT cells from <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ mice have higher levels of 2B4 expression and lower functional secretion of cytokines.

<p>(A) Histograms and (B) graphs of 2B4 surface expression on B220^- CD3⁺ CD1d Tetramer⁺ NKT cells in spleens and livers of WT and <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ mice. (C) Histograms and (D) graphs of 2B4 surface expression on splenic CD4 and CD8 T cells in WT and <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ mice. (A-D) Data shows two independent experiments, with 4 mice per group. (E) Flow cytometry plots of representative WT and <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ NKT cells (gated on B220^- CD3^int CD1d Tetramer⁺ cells) after <i>in vivo</i> stimulation with α-GalCer for 45 minutes. Expression of IL-4 and IFN-γ are shown. (F) Frequencies of IL-4 and IFN-γ expression by NKT cells (gated on B220^- CD3^int CD1d Tetramer⁺ cells). (E-F) Data represents two independent experiments, with 3 mice per group.</p

Lack of defects in germinal centers generated in <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ mice after immunization with HIV envelope trimer protein.

<p>(A) Flow cytometry plots and (B) graphs of CD19^- CD4⁺ CD44⁺ CXCR5⁺ BTLA⁺ Tfh cells, CD19^- CD4⁺ CD44⁺ CXCR5⁺ PD1⁺ GC Tfh cells, and CD4^- CD19⁺ Fas⁺ GL7⁺ GC B cells in draining popliteal lymph nodes of of WT and <i>Slamf1</i>,<i>5</i>,<i>6</i> ^Δ/Δ mice at 8 days post immunization with HIV Envelope (YU2 gp140-F) protein. (A-B) Data represents two independent experiments, with 3–4 mice per grouop. (C) Endpoint titers and Area Under Curve (AUC) analyses of HIV Env (YU2-gp140-F) specific serum IgG at 15 days post immunization with YU2-gp140-F. Data represents one experiment, with 4 mice per group.</p

Genotyping and phenotyping of CRISPR gene edited mice.

<p>Genotyping and phenotyping of CRISPR gene edited mice.</p