Consuelo Ortega

IL-17-producing CD8+ T lymphocytes from psoriasis skin plaques are cytotoxic effector cells that secrete Th17-related cytokines

IL‐17‐producing CD4+ T lymphocytes (Th17) are currently considered relevant participants in the pathogenesis of psoriasis skin lesions. However, little is known about the potential role of IL‐17‐producing CD8+ T cells, which are also present at the psoriatic plaque. We have addressed the functional characterization of this CD8+ subtype of T lymphocytes from psoriasis patients. Our results show that CD8+IL‐17+ cells from psoriasis‐inflamed skin tissue produce TNF‐α and IFN‐γ (Th1‐related cytokines) as well as IL‐17, IL‐21, and IL‐22 (Th17‐related cytokines) efficiently. A significant up‐regulation of the RORC transcription factor is also observed. These cells are refractory to Tregs but show a proliferative response to anti‐CD3/CD28 stimulation that is enhanced by IL‐12 and IL‐15. Blocking of TNF‐α activity inhibits TCR‐mediated activation and IL‐17 production. CD8+IL‐17+ T cells are cytotoxic cells that display TCR/CD3‐mediated cytotoxic abilities to kill target cells. Thus, CD8+IL‐17+ T cells share some key features with Th17 cells and exhibit remarkable differential abilities attributable to the CD8+ lineage of T lymphocytes, adding new insights into the functional resources of IL‐17‐producing cells from human epidermis that could be of potential interest to our understanding of the pathogenesis of psoriasis.

Characterization of gliadin-specific Th17 cells from the mucosa of celiac disease patients.

OBJECTIVES:Celiac disease (CD) is a disorder characterized by a deregulated immune response to ingested wheat gluten and related cereal proteins in susceptible individuals. It has been considered that the onset of CD is mediated by a skewed Th1 response. However, the participation of Th17 cells in the pathogenesis of the disease, a key cell population in other autoimmune disorders, has not been studied in detail. We have investigated the presence of Th17 cells in the mucosa of active CD patients and their functional implications in the pathogenesis of the disease.METHODS:T cells obtained from duodenum biopsies from 15 untreated patients and 11 control individuals were characterized by flow cytometry, immunoassays, and real-time PCR.RESULTS:We found gliadin-specific CD4+ interleukin (IL)-17A-producing T cells in the mucosa of CD patients with a phenotype consisting of TCR (T-cell receptor)αβ+ CD45RO+ CD161+ CCR6+ (C–C chemokine receptor type 6) and IL-23R+. Functional analysis showed that Th17 cells from CD patients are different from those of control individuals in terms of cytokines production. Th17 cells from CD patients, but not from controls, simultaneously express transforming growth factor-β (TGFβ). Th17 CD cells also produce interferon-γ (IFNγ), IL-21, and IL-22. The analysis of the transcription factors revealed a high expression of interferon regulatory factor-4 as a feature of gliadin-specific cells from CD patients with respect to controls.CONCLUSIONS:Gliadin-specific Th17 cells are present in the mucosa of CD patients having a dual role in the pathogenesis of the disease as they produce proinflammatory cytokines (such as IL-17, IFNγ, IL-21), mucosa-protective IL-22, and regulatory TGFβ, which actively modulates IL-17A production by T cells in the celiac mucosa.

Expression of CD94 and NKG2 molecules on human CD4(+) T cells in response to CD3-mediated stimulation.

We investigated the ability of human peripheral CD4+ cellsto express CD94 and NKG2 molecules as a consequence ofCD3‐mediated activation. Using highly purified peripheralCD4+ T cells, we found expression of both CD94 and NKG2A 15days after CD3‐mediated stimulation of cells. We also determined byreverse transcriptase‐PCR that all gene members of NKG2 family—namely,NKG2A, ‐C, ‐D, and ‐E—are sequentially expressed on CD4+cells. We found that this expression is tightly regulated by cytokines,and we identified transforming growth factor‐β1 and interleukin‐10 asthe main factors that, on CD3‐dependent stimulation, positivelycontribute to the expression of CD94 and NKG2A on CD4+cells. We also investigated the functional role of NKG2A and found thatcoligation of CD3 and NKG2A by specific monoclonal antibodies resultsin significant inhibition of interferon γ and tumor necrosis factorα production by stimulated CD4+ cells. The presence andfunction of these receptors on CD4+ lymphocytes support amore general role for NKG2 molecules, whose functions were originallythought to be confined to cytotoxic cells, in the immune system.

Relationship of IL-2, IL-2R (CD25+), Soluble IL-2R and IL-4 with Disease Activity in SLE Patients

The plasma levels of interleukin-4 (IL-4), interleukin-2 (IL-2), soluble receptor of IL-2 (IL-2R) and T cell expression of IL-2 receptor chain (CD25+) were determined in an attempt to relate these parameters with disease activity in systemic lupus erythematosus (SLE). IL-4, IL-2 and sIL-2R plasma levels of SLE patients were significantly higher than those of the control group (P<0.05) while CD25+ expression was similar in both groups. Only sIL-2R levels were significantly higher (P<0.05) in active than in inactive patients.

Role for NKG2-A and NKG2-C surface receptors in chronic CD4 + T-cell responses

The participation of CD94 and NKG2 gene family members in the function of NK cells and CD8+ cytolytic cells has recently been addressed in detail. However, the role that these molecules play in the key CD4+ regulatory cells remains largely unexplored. This study has examined the expression and regulation of CD94 and NKG2 genes in purified human peripheral CD4+ cells stimulated with several agents. We found a constitutive expression of NKG2‐E in CD94‐depleted resting peripheral CD4+ cells, whereas inductions of NKG2‐A and NKG2‐C required chronic cell activation and occurred after expression of CD94. We found that CD3‐mediated stimulation induces the expression of CD94 first by day 5 of culture, followed by NKG2‐A by day 15 and finally NKG2‐C, which is not detected until 20 days after repeated stimulation. This pattern of gene expression differs sharply from that observed in purified CD8+ T cells, where mRNA from all NKG2 gene family members are detected after 5 days of stimulation. Selective activation of TCR Vβ2‐bearing cells with toxic shock syndrome toxin‐1 superantigen reveals that mRNA induction of NKG2‐A and NKG2‐C genes is significantly influenced by the presence of cytokines (IL‐10 and TGF‐β) and by the restimulation of the cells. In addition, the occupancy of the CD94/NKG2‐A receptor expressed on these superantigen‐stimulated CD4+ T lymphocytes abrogates TNF‐α and IFN‐ γ production, whereas NKG2‐C enhances production of these cytokines. Taken together our results reveal strict gene regulatory mechanisms for CD94 and NKG2 gene expression on CD4+ cells that are different from those governing the expression of these same genes in CD8+ cells. The results suggest that these genes also participate in chronic CD4+ T‐cell responses.

IL-17 producing T cells in celiac disease: angels or devils?

Celiac disease (CD) is a very common chronic condition in human beings, affecting approximately one in 100 individuals. It is an autoimmune disease with a defined environmental trigger, the gluten contained in dietary cereals, occurring in genetically susceptible individuals. The disease has a very strong HLA association. More than 90% of CD patients have HLA-DQ2, and almost all of the remaining celiac population possesses HLA-DQ8 molecules. Th17 cells seem to participate in the disease pathogenesis producing and secreting either proinflammatory or anti-inflammatory cytokines.

A novel IL2RG mutation presenting with atypical T(-)B(+)NK+ phenotype: rapid elucidation of NK cell origin.

To the Editor: IL-2R g chain is the signaling component of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, cytokine receptors that are essential in the ontogeny and function of NK and T cells. Mutations in the gene encoding for the g chain of the interleukin-2 receptor (IL-2RG), mapped to X chromosome, accounts for 50% of diagnosed SCID. X-Linked SCID is characterized by early-onset of severe infections, facilitated by the absence of T and NK lymphocytes (T BþNK phenotype) [1,2]. However, different mutations in the IL-2RG gene have been reported associated with a phenotype variant T BþNKþ [3]. Here we describe a novel mutation of the IL-2RG gene consisting of a triple insertion (ACC) at exon 5, in which NK cells were found in peripheral blood, representing a burden for initial patient diagnosis. The patient was an 8-month-old male born full term to a non-consanguineous couple. Patient’s grand-grand mother was of gypsy origin. There was no family history consistent with immunodeficiency. Since 5months of age the patient suffered several episodes of diarrhea and failure to thrive. At 8 months of age, he was admitted to the hospital due to a severe diarrhea and dehydration. Staphylococcus auricularis, Staphylococcus epidermidis, and Candida parasilopsis were identified in blood cultures. Candida albicans and Pseudomona aeruginosa were detected in stool’s cultures. Immunological findings showed hypogammaglobulinemia IgG: 41mg/dl (reference range 165–590mg/dl); IgA: 5mg/dl (15–50mg/dl); IgM: 25mg/dl (30–135mg/dl). Lymphocyte counting showed CD3þ: 8 cells/ml (reference range 2,000–5,900 cells/ml); CD19þ: 1,664 cells/ml (610–2600 cells/ml); CD16þCD56þ: 353 cells/ml (160–950 cells/ml). Despite the observed T BþNKþ phenotype, IL-2Rgc chain deficiency was investigated, as it is the most frequent cause of SCID. DNA sequence analysis of the IL-2RG gene revealed a triple insertion (ACC) at exon 5, resulting in the inclusion of a histidine in the gc chain sequence. This pathological variant p. His242_Pro243 insHis has not been previously described. Results from the female-carrier studies demonstrated that this mutation appeared de novo in the patient’s mother, as shewas the only carrier female within the family. All other family females including five sisters, the grand mother as well as the grand-grand mother of the patient, were found free of the mutation. In spite of the presence of NK cells in the peripheral blood of our patient, no signs of maternal lymphoid engraftment were evident. Maternal T cell engraftment has been reported in up to 50% of diagnosed SCID. However, as it is occurs in our patient, some of them never develop graft versus host disease [4]. Nevertheless, the assessment of the origin of the circulating NK cells was approached bymeans of a flow cytometric strategy.We reasoned that if NK cells detected were from maternal origin, they should constitutively express at the cell surface, as NK cells from healthy donors, IL2Rgc chain (CD132) and IL-21R chain (CD360). Conversely, both markers should be absent from patient’s resting B cells. Multicolor flow cytometry assay confirmed co-expression of CD132 and CD360 on the NK cell population, showing the maternal origin of NK cells. The patient received hematopoietic stem cell transplantation from an HLA-identical, non-related donor, which outcome was the immunological reconstitution and successful clinical recovery. In conclusion, we presented here a novel IL-2Rgc mutation, appearing de novo in the patient’s mother and presenting NK cells in the peripheral blood. An original and informative approach allowed us to rapidly elucidate the maternal origin of NK avoiding other laborious molecular analysis and facilitating the achievement of an accurate diagnose and appropriate therapy for this patient.

A novel phenotype variant of severe congenital neutropenia caused by G6PC3 deficiency

Severe congenital neutropenia type 4 (SCN4) is associated with mutations in the G6PC3 gene. To date, all patients bearing the p.Gly260Arg variant of the G6PC3 gene show heart defects. Here, we present a case of the p.Gly260Arg variant in a patient who did not have structural or functional heart anomalies. Treatment with granulocyte colony‐stimulating factor recovered the absolute neutrophil count and neutrophil functional competence. Pediatr Blood Cancer 2013; 60: E29–E31.

Interleukin-21 overexpression dominates T cell response to Epstein-Barr virus in a fatal case of X-linked lymphoproliferative syndrome type 1.

ABSTRACT Interleukin-21 (IL-21) is a cytokine whose actions are closely related to B cell differentiation into plasma cells as well as to CD8+ cytolytic T cell effector and memory generation, influencing the T lymphocyte response to different viruses. X-linked lymphoproliferative syndrome type 1 (XLP-1) is a primary immunodeficiency syndrome that is characterized by a high susceptibility to Epstein-Barr virus. We observed in a pediatric patient with XLP-1 that IL-21 was expressed in nearly all peripheral blood CD4+ and CD8+ T cells. However, IL-21 could not be found in the lymph nodes, suggesting massive mobilization of activated cells toward the infections target organs, where IL-21-producing cells were detected, resulting in large areas of tissue damage.

Antiadenohypophysis autoantibodies in patients with nongluten-related gastroenteropathies.

To investigate the presence of antipituitary antibodies (APA) in the serum of patients undergoing gastroenteropathies (GEP) other than celiac disease (CD).