Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked

CCL16/LEC powerfully triggers effector and antigen-presenting functions of macrophages and enhances T cell cytotoxicity

AbstractThe huan CC chemokine CCL16, a liver-expressed chemokine, enhances the killing activity of mouse peritoneal macrophages by triggering their expression of tumor necrosis factor α (TNF-α) and Fas ligand. Macrophages also respond to CCL16 by enhancing their production of monocyte chemoattractant protein-1, regulated on activation, normal T cells expressed and secreted chemokines, and interleukin (IL)-1β, TNF-α, and IL-12. The effect of CCL16 is almost as strong as that of lipopolysaccharide and interferon-γ, two of the best macrophage activators. Moreover, CCL16-activated macrophages overexpress membrane CD80, CD86, and CD40 costimulatory molecules and extensively phagocytose tumor cell debris. On exposure to such debris, they activate a strong, tumor-specific, cytolytic response in virgin T cells. Furthermore, cytolytic T cells generated in the presence of CCL16 display a higher cytotoxicity and activate caspase-8 in tumor target cells. This ability to activate caspase-8 depends on their overexpression of TNF-α and Fas ligand induced by CCL16. These data reveal a new function for CCL16 in the immune-response scenario. CCL16 significantly enhances the effector and the antigen-presenting function of macrophages and augments T cell lytic activity

280 lentiviral mediated gene therapy restores b cell homeostasis and tolerance in wiskott aldrich syndrome patients

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency characterized by micro-thrombocytopenia, eczema and increased risk of infections, autoimmunity and tumors. Allogeneic hematopoietic stem cell (HSC) transplantation is a recognized curative treatment for WAS, but when a matched donor is not available, administration of WAS gene-corrected autologous HSCs represents a valid alternative therapeutic approach. Since alterations of WAS protein (WASp)-deficient B lymphocytes contribute to immunodeficiency and autoimmunity in WAS, we followed the B cell reconstitution in 4 WAS patients treated by lentiviral vector-gene therapy (GT) after a reduced-intensity conditioning regimen combined with anti-CD20 administration. We analyzed the B cell subset distribution in the bone marrow and peripheral blood by flow cytometry and the autoantibody profile by a high-throughput autoantigen microarray platform before and after GT. Lentiviral vector-transduced progenitor cells were able to repopulate the B cell compartment with a normal distribution of transitional, naive and memory B cells. The reduction in the proportion of autoimmune-associated CD21low B cells and in the plasma levels of B cell-activating factor was associated with the decreased autoantibody production in WAS patients after GT. Then, we evaluated the functionality of B cell tolerance checkpoints by testing the reactivity of recombinant antibodies isolated from single B cells. Before GT, we found a decreased frequency of autoreactive new emigrant/transitional B cells in WAS patients, suggesting a hyperfunctional central B cell checkpoint in the absence of WASp. In contrast, high frequency of polyreactive and Hep2 reactive clones were found in mature naive B cells of WAS patients, indicating a defective peripheral B cell checkpoint. Both central and peripheral B cell tolerance checkpoints were restored after GT, further supporting the qualitative efficacy of this treatment. In conclusion, WASp plays an important role in the regulation of B cell homeostasis and in the establishment of B cell tolerance in humans and lentiviral-mediated GT is able to ameliorate the functionality of B cell compartment contributing to the clinical and immunological improvement in WAS patients

B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome

Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21-CD35- and CD21low

Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients

NKp46-expressing human gut-resident intraepithelial V\u3b41 T cell subpopulation exhibits high anti-tumor activity against colorectal cancer

\u3b3\u3b4 T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine \u3b3\u3b4 IEL gut-specificity, homing and functions, \u3b3\u3b4 T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic V\u3b41 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/V\u3b41 IELs depends both on distinctive features of V\u3b41 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident V\u3b41 intestinal IELs or its induced-expression on IL-2/IL-15 activated V\u3b41 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/V\u3b41 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/V\u3b41 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/V\u3b41 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies

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

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth

Expanding the clinical and immunological phenotypes of PAX1-deficient SCID and CID patients

Paired box 1 (PAX1) deficiency has been reported in a small number of patients diagnosed with otofaciocervical syndrome type 2 (OFCS2). We described six new patients who demonstrated variable clinical penetrance. Reduced transcriptional activity of pathogenic variants confirmed partial or complete PAX1 deficiency. Thymic aplasia and hypoplasia were associated with impaired T cell immunity. Corrective treatment was required in 4/6 patients. Hematopoietic stem cell transplantation resulted in poor immune reconstitution with absent naïve T cells, contrasting with the superior recovery of T cell immunity after thymus transplantation. Normal ex vivo differentiation of PAX1-deficient CD34+ cells into mature T cells demonstrated the absence of a hematopoietic cell-intrinsic defect. New overlapping features with DiGeorge syndrome included primary hypoparathyroidism (n = 5) and congenital heart defects (n = 2), in line with PAX1 expression during early embryogenesis. Our results highlight new features of PAX1 deficiency, which are relevant to improving early diagnosis and identifying patients requiring corrective treatment