The induction of Bim expression in human T-cell blasts is dependent on nonapoptotic Fas/CD95 signaling

The BH3-only protein Bim is required for maintaining the homeostasis of the immune system, since Bim regulates the down-modulation of T-cell responses, mainly through cytokine deprivation. Using T-cell blasts from healthy donors and also from patients with autoimmune lymphoproliferative syndromes (ALPSs) due to homozygous loss-of-function mutation of FasL (ALPS-Ic) or heterozygous mutation in the Fas/CD95 death domain (ALPS-Ia), it is shown that the induction of Bim expression during the process of human T-cell blast generation is strictly dependent on FasL/Fas-mediated signaling. The main pathway by which Fas signaling regulates the levels of Bim expression in human T-cell blasts is the death-domain- and caspase-independent generation of discrete levels of H2O2, which results in the net increase of Foxo3a levels. The present results connect the 2 main pathways described until the moment for the control of T-cell responses: death receptor-mediated activation-induced cell death and apoptosis by cytokine deprivation.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Cell cycle regulation by FasL and Apo2L/TRAIL in human T-cell blasts. Implications for autoimmune lymphoproliferative syndromes

The Fas-FasL pathway plays an important role in the homeostasis of mature lymphocytes, with defects causing autoimmune lymphoproliferative syndromes (ALPS). Human T-cell blasts are not sensitive to FasL or Apo2L/TRAIL-induced apoptosis unless they get reactivated, but either of those ligands inhibits their growth in the absence of cell death induction due to a cell cycle arrest in S-G2/M. In the present work, we have studied the mechanism(s) by which FasL or Apo2L/TRAIL regulate T-cell blast cell cycle in healthy donors and in two types of ALPS patients. Our data indicate that in human CD8+ T-cell blasts, Fas ligation, and especially Apo2L/TRAIL induce the p53-dependent decrease in cyclin-B1 levels. However, the induction of the negative cell cycle regulator p21WAF1 by FasL or Apo2L/TRAIL in either CD4+ or CD8+ T-cell blasts seems to be the main regulatory mechanism. This mechanism is dependent on caspase activation and on H2O2 generation. The increase in p21 levels by FasL or Apo2L/TRAIL is concomitant with p53 increases only in CD8+ T-cell blasts, with p21 levels maintained high for longer times than p53 levels. In CD4+ T-cell blasts p21 levels are controlled through a transient and p53-independent mechanism. The present results suggest that the etiology of ALP syndromes could be related not only to defects in apoptosis induction, but also in cell cycle regulation.Ministerio de Educación y CienciaDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

C4 gene polymorphism in primates: evolution, generation, and Chido and Rodgers antigenicity

Eleven new C4d genomic primate sequences of the fourth complement factor (C4) have been obtained. Seven of them belong to five species not yet explored for this gene: Pan paniscus (pygmy chimpanzee), Cercopithecus aethiops (green monkey), Macaca mulatta (rhesus monkey), Macaca fascicularis (cynomolgus), and Saguinus oedipus (cotton top tamarin). The New World monkeys (tamarins, four individuals) sequenced for C4 have a single C4d sequence only, which shows a B isotypic specificity and a Rodgers 3 (Rg3), Chido 1 (Ch1) antigenicity. Rg3 and Ch1 could thus be the oldest Rg/Ch specificity (at least 50 million years old) and Rg1, Rg2, Ch3, and Ch6 could be more recent human-specific antigens. Mechanisms of C4d polymorphism generation were analyzed by compiling all the presently available sequences. Examples of both point mutations and crossing-over events among C4d primate sequences could be detected. The problem of a possible trans-species inheritance of C4d polymorphism was addressed and two apparently contradicting dendrograms were obtained. One of them, constructed by using both exon and intron sequences, does not support trans-species evolution, but supports the proposed theory of extensive homogenization of the C4 genes occurring within each species, because alleles from each primate species cluster together. Another completely different dendrogram, obtained by using exon sequences only, suggests the existence of trans-species evolution for C4d polymorphism, because alleles belonging to different species cluster together in a way similar to that found for HLA class I or II alleles. However, orangutan sequences group together in both kinds of C4d sequence dendrograms and seem to have arisen from an ancestor different from that of chimpanzee, gorilla and man C4d sequences. Finally, further data have been obtained that support trans-species conservation of A-ness and B-ness and the existence of trans-specifically conserved allelic motifs, both in intronic and exonic sequences.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Peripheral blood reduction of memory (CD29+, CD45RO+, and “Bright” CD2+ and LFA-1+) T lymphocytes in Papillon-Lefèvre syndrome

A Papillon-Lefèvre patient with characteristic chronic periodontal disease and palmoplantar keratoderma was studied over a 4-year period. An abnormal T-cell phenotype was steadily observed in peripheral blood; both low numbers of CD29+ and CD45RO+ cells and a low density surface expression of CD2 and LFA-1 molecules were found. T-cell activation through CD3, CD2 and ConA, PWM and IL-2 receptors was normal; however, there was impairment in the activation via CD28. CD2, LFA-1 and CD45 molecules were normal in charge and molecular weight. There was no tissue sequestering of T lymphocytes in periodontal lesions, but rather a relative T-cell reduction. It is suggested that an important decrease of the so-called "memory/hyperreactive" (CD45RO-positive) T cells does exist; therefore, hyperreactive T cells would not be available in sufficient numbers to leave the bloodstream through blood vessel endothelium, and the periodontium would be left without these important defenses and thus exposed to chronic infections. A disregulated factor affecting the transition from "naive" to "memory" T cells and the increase in certain surface molecules expression (i.e., CD2, LFA-1, CD29, and CD45RO) or the reversion from memory to naive T cells may be responsible for the disease pathogenesis. CD2 and LFA-1 molecule synthesis might be conjointly regulated on T lymphocytes.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Longitudinal analysis of immune function in the first 3 years of life in thymectomized neonates during cardiac surgery

Summary The purpose of this study is to evaluate the effects of neonatal thymectomy in the functional capacity of the immune system. We selected a group of 23 subjects, who had undergone thymectomy in their first 30 days of life, during an intervention for congenital heart disease. Several parameters of the immune system were evaluated during their first 3 years of life. Lymphocyte populations and subpopulations (including naive, memory and effector subpopulations), T cell receptor (TCR) Vβ repertoire, response of T cells following in vitro stimulation by mitogen, quantification of immunoglobulins, TCR excision circles (TRECS) and interleukin (IL)-7 were measured. We found that neonatal thymectomy produces long-term diminution in total lymphocyte counts, especially in naive CD4+ and CD8+ T cells. Additionally, TRECS were decreased, and plasma IL-7 levels increased. A statistically significant negative correlation was found between absolute CD4+ T cells and IL-7 (r = −0·470, P = 0·02). The patients did not suffer more infectious events than healthy control children, but thymectomy in neonates resulted in a significant decrease in T lymphocyte levels and TRECS, consistent with cessation of thymopoiesis. This could produce a compromise in immune function later in life, especially if the patients suffer T cell depletion and need a reconstitution of immune function.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Novel mutations and defective protein kinase C activation of T-lymphocytes in ataxia telangiectasia

Summary: Three ataxia telangiectasia (AT) patients have been characterized immunologically and molecularly. Patient 1 presents two nondescribed splicing mutations which affect exons 15 and 21 of the ATM gene. The maternal defect consists of a G>A transition in the first nucleotide of the intron 21 donor splicing site which results in a complete deletion of exon 21. The paternal mutation consists of an A > C transversion in the intron 14 acceptor splicing site which produces a partial skipping of exon 15. Two abnormal alternative transcripts were found, respectively, 17 and 41 nucleotides shorter. Patient 2 presents a homozygous genomic deletion of 28 nucleotides in the last exon of the gene. This deletion changes the normal reading frame after residue 3003 of the protein and introduces a premature stop codon at residue 3008 that could originate a truncated ATM protein. Patient 3, a compound heterozygote, presents a defect which consists of a G > A transition in the first nucleotide of intron 62 donor splicing site which results in a complete deletion of exon 62. The results obtained during a three year period in the proliferation assays show an impaired PMA (phorbol myristate acetate) activation in specific T lymphocyte activation pathways (CD69, CD26, CD28, CD3, PHA, PWM and Con A mediated) but not in others (CD2, ionomycin, and Ig surface receptor). The possible link among specific ATM mutations and abnormal immune responses is unknown.Ministerio de Educación y CienciaComunidad Autónoma de MadridDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

A homozygous Fas ligand gene mutation in a patient causes a new type of autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphoproliferation and autoimmune clinical manifestations and is generally caused by defective Fas-mediated apoptosis. This report describes the first homozygous FASL gene mutation in a woman with clinical and immunologic features of ALPS. T-cell blasts from the patient did not induce FasL-mediated apoptosis on Fas-transfected murine L1210 or on Jurkat cells, and activation-induced cell death was impaired. Furthermore, Fas-dependent cytotoxicity was drastically reduced in COS cells transfected with the mutant FasL. In addition, FasL expression on T-cell blasts from the patient was similar to that observed in a healthy control, despite its bearing the high-producer genotype -844C/C in the FASL promoter. Sequencing of the patient's FASL gene revealed a new mutation in exon 4 (A247E). The location of A247E in the FasL extracellular domain and the conservation of the protein sequence of that region recorded in 8 species different from humans support the essential role of FasL COOH terminal domain in Fas/FasL binding. These findings provide evidence that inherited nonlethal FASL abnormalities cause an uncommon apoptosis defect producing lymphoproliferative disease, and they highlight the need for a review of the current ALPS classification to include a new ALPS type Ic subgroup.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Phenotypic and functional evaluation of CD3+CD4-CD8- T cells in human CD8 immunodeficiency

Derechos reservados de Haematologica. Ferrari Storti Foundation http://www.haematologica.org/Background: Human CD8 immunodeficiency is characterized by undetectable CD8(+) lymphocytes and an increased population of CD4(-)CD8(-) (double negative) T lymphocytes. Design and methods: We hypothesized that the double negative subset corresponds to the cellular population that should express CD8 and is committed to the cytotoxic T lymphocyte lineage. To assess this, we determined the phenotype and function of peripheral blood mononuclear cells and/or magnetically isolated double negative T lymphocytes from two CD8-deficient patients. To analyze the expression and co-localization with different organelles, 293T cells were transfected with plasmids bearing wild-type or mutated CD8α. Results: CD8α mutated protein was retained in the cytoplasm of transfected cells. The percentages of double negative cells in patients were lower than the percentages of CD8(+) T cells in healthy controls. Double negative cells mostly had an effector or effector memory phenotype whereas naïve T cells were under-represented. A low concentration of T-cell receptor excision circles together with a skewed T-cell receptor-V repertoire were observed in the double negative population. These data suggest that, in the absence of CD8 co-receptor, the thymic positive selection functions suboptimally and a limited number of mature T-cell clones would emerge from the thymus. In vitro, the double negative cells showed a mild defect in cytotoxic function and decreased proliferative capacity. Conclusions: It is possible that the double negative cells are major histocompatibility complex class-I restricted T cells with cytolytic function. These results show for the first time in humans that the presence of the CD8 co-receptor is dispensable for cytotoxic ability, but that it affects the generation of thymic precursors committed to the cytotoxic T lymphocyte lineage and the proliferation of mature cytotoxic T cells.Ministerio de Educación y CienciaMinisterio de Sanidad y ConsumoDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Innate Lymphoid cells groups 1 and 3 in the epitelial compartment of functional human intestinal allografts

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103(+) cells) principally due to reduced CD3(+) CD8(+) cells, while CD103(+) CD3(-) cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3(-) IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3(-) IELs of the grafts expressed CD56, NKp44, interleukin (IL)-23 receptor, retinoid-related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3(-) CD127(+) cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3(-) IELs in grafts produced significantly more interferon (IFN)-γ and IL-22, and a double IFNγ(+) IL-22(+) population was observed. Epithelial cell-depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3(-) IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56(+) , NKp44(+) , CCR6(+) , CD127(-) , cytotoxicity, and IFNγ secretion), ILC3 (CD56(+) , NKp44(+) , IL-23R(+) , CCR6(+) , RORγt(+) , and IL-22 secretion), and intermediate ILC1-ILC3 phenotypes (IFNγ(+) IL-22(+) ). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.Instituto de Salud Carlos IIIUnión EuropeaFundación Mutua MadrileñaDepto. de CirugíaFac. de MedicinaTRUEpu

Hemodialysis-Associated Immune Dysregulation in SARS-CoV-2-Infected End-Stage Renal Disease Patients

Fondos FEDER ; Ayuda Formación del Profesorado FPU19/06393 ; Contrato de Investigaciçon “Rio Hortega” (CM19/00120)Patients on hemodialysis show dysregulated immunity, basal hyperinflammation and a marked vulnerability to COVID-19. We evaluated the immune profile in COVID-19 hemodialysis patients and the changes associated with clinical deterioration after the hemodialysis session. Recruited patients included eight hemodialysis subjects with active, PCR-confirmed SARS-CoV-2 infection, five uninfected hemodialysis patients and five healthy controls. In SARS-CoV-2-infected hemodialysis patients TNF-α, IL-6 and IL-8 were particularly increased. Lymphopenia was mostly due to reduction in CD4+ T, B and central memory CD8+ T cells. There was a predominance of classical and intermediate monocytes with reduced HLA-DR expression and enhanced production of pro-inflammatory molecules. Immune parameters were analysed pre- and post-hemodialysis in three patients with COVID-19 symptoms worsening after the hemodialysis session. There was a higher than 2.5-fold increase in GM-CSF, IFN-γ, IL-1β, IL-2, IL-6, IL-17A and IL-21 in serum, and augmentation of monocytes-derived TNF-α, IL-1β and IL-8 and CXCL10 (p < 0.05). In conclusion, COVID-19 in hemodialysis patients associates with alteration of lymphocyte subsets, increasing of pro-inflammatory cytokines and monocyte activation. The observed worsening during the hemodialysis session in some patients was accompanied by augmentation of particular inflammatory cytokines, which might suggest biomarkers and therapeutic targets to prevent or mitigate the hemodialysis-related deterioration during SARS-CoV-2 infection.European CommissionInstituto de Salud Carlos IIIMinisterio de Ciencia e Innovación (España)Comunidad de MadridDepto. de MedicinaDepto. de Inmunología, Oftalmología y ORLTRUEpubDescuento UC