Development of autologous T lymphocytes in two males with X-linked severe combined immune deficiency: molecular and cellular characterization.

We report on two patients affected with severe combined immune deficiency (SCID) with an unusual immunological phenotype and a substantial number of autologous, poorly functioning T cells. Distinct mutations identified at the IL2RG locus in the two patients impaired IL-2-mediated signaling but affected T-cell lymphopoiesis differently, resulting in generation of a polyclonal or oligoclonal T-cell repertoire. These observations add to the growing complexity of the immunological spectrum of SCID in humans and indicate the need for detailed immunological and molecular investigations in atypical cases.

Cutaneous CD30+ cells in children with atopic dermatitis.

BACKGROUND: CD30 expression can be considered a marker of Th2 cells. We investigated the presence of CD30+ cells in the lesional skin of children with atopic dermatitis (AD). We also analyzed the possible relationship between CD30+ cells and serum soluble CD 30 (sCD30) levels, and IgE, soluble interleukin-2 (IL-2) receptor (sIL-2R) or soluble CD23 (sCD23) levels in the blood, and clinical score. METHODS: Ten eczematous children (4 males, 6 females; median age: 4 years and 5 months; range: 11 months to 14 years), 9 sex- and age-matched control children and an adult control group were studied. A clinical score (SCORAD index), was given to eczematous lesions. Blood was taken for the determination of IgE, sCD30, sIL-2R and sCD23 levels. Punch biopsies of lesional skin were stained with hematoxylin and eosin or incubated with anti-CD30 monoclonal antibodies. Skin prick tests (SPTs) were also performed. RESULTS: In the biopsy specimens, CD30 expression was observed in high proportions of infiltrating cells. In children with AD, total serum IgE, sCD30, sIL-2R, sCD23 and eosinophils were significantly elevated compared to controls. CD30+ cells were not associated with serum IgE, sCD30, sIL-2R, sCD23, or SPT results, score of inflammatory cells in lesional skin or clinical score. Children with AD who had high total IgE and specific IgE antibodies did not differ from those with normal total IgE and negative specific IgE in respect of age, clinical score, number of CD30+ cells, sCD30, sIL-2R and sCD23 levels, score of inflammatory cells in skin or clinical score. CONCLUSION: Our results showed remarkable numbers of CD30+ cells in the lesional skin and high sCD30 in the serum of children with AD. CD30+ cells did not correlate with systemic markers of IgE reaction.

Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: down-modulation of Bcl-2 and high expression of CD95/Fas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death.

We have studied the regeneration of T cell subsets and function after BMT in 21 children affected by combined immunodeficiency after BMT. In the first months, the striking predominance of CD4+ cells displayed the primed CD45R0+ phenotype and a high number of activated (HLA-DR+) T cells were observed. Regeneration of naive CD4+CD45RA+ cells correlated with the recovery of proliferative responses to mitogens (r = 0.64, P<0.001). Peripheral blood lymphocytes circulating after BMT undergo an increased process of in vitro cell death, resulting from two mechanisms: spontaneous apoptosis (SA), a consequence of defective production of IL-2 and down-regulation of Bcl-2 (P = 0.02 vs. healthy controls), and high susceptibility to activation-induced cell death (AICD) after restimulation with mitogens. In accordance with the role of CD95/Fas in this latter process, we have observed a high level of CD95 expression (P<0.001 vs. healthy controls), correlated with AICD (P<0.001) but not with SA, and decreasing with time after BMT (P<0.001). Both SA and AICD levels correlated with the presence of activated T cells and decreased with the progressive recovery of T cell proliferative response. Therefore, the lymphocyte hyperactivated status might explain their susceptibility to apoptosis and contribute to the genesis of immunodeficiency that follows BMT.

Development of autologous, oligoclonal, poorly functioning T lymphocytes in a patient with autosomal recessive severe combined immunodeficiency caused by defects of the Jak3 tyrosine kinase.

Defects of the common gamma chain subunit of the cytokine receptors (gamma c) or of Jak3, a tyrosine kinase required for gamma c signal transduction, result in T-B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (> 3,000/microL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+ HLA-DR+ CD62L(lo)), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in gamma c-/y and in Jak3-/- mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.

Study of spontaneous apoptosis in HIV+ patients: correlation with clinical progression and T cell loss.

In vitro spontaneous apoptosis (SA) of lymphocytes was studied in HIV infection to evaluate possible clinical and prognostic correlations, in a transsectional study of 101 individuals in different clinical categories and in a prospective longitudinal study of 18 asymptomatic individuals (mean follow-up, 17.2 months). The rate of SA was higher in HIV+ patients than in healthy controls (p < 0.001) and was higher in patients with AIDS than in the other HIV+ individuals (p < 0.001). It was inversely correlated with the peripheral blood CD4+ (R -0.61; p < 0.001) and CD8+ (R -0.46; p < 0.001) cell numbers. In a group of long-term survivors (LTS), it was significantly lower than in a control group of asymptomatic HIV+ patients with a similar number of circulating CD4+ lymphocytes but a shorter follow-up (p < 0.02). In the five asymptomatic HIV-infected individuals who showed a clinical progression, peaks of SA rates above the normal range before the clinical event were much more frequent than in those who remained asymptomatic (p < 0.0001), even though they were fairly homogeneous as far as CD4+ cell count and viral load were concerned. The median levels of SA rates were moreover correlated with the rate of total T cell loss (R -0.46; p 0.053). This study suggests that evaluation of the SA levels may provide a predictive factor for clinical and immunological progression of HIV-related immunodeficiencies and strengthen the hypothesis for the role of this phenomenon in the pathogenesis of this progression.

In vitro cell death of activated lymphocytes in Omenn's syndrome.

Omenn's syndrome (OS) is characterized by erythrodermia, hepatosplenomegaly, lymphadenopathy, hypereosinophilia and elevated IgE levels associated with increased susceptibility to severe infections. Peripheral blood T cells, though usually present in normal number, show an activated phenotype (including an increased expression of CD95/Fas), a Th2 pattern of cytokine secretion and defective proliferative response to mitogens. In this report, we demonstrate that T cells from patients with OS undergo an excessive cell death in vitro resulting from two mechanisms. First, a substantial number of peripheral blood lymphocytes from OS patients die in unstimulated cultures (p = 0.009 vs. healthy controls). This spontaneous apoptosis is associated with reduced expression of bcl-2 gene product (p < 0.05) and can be prevented by addition of interleukin (IL)-2 (which also prevents down-modulation of bcl-2), while is independent from CD95 signaling. Second, lymphocytes from OS patients are highly susceptible to activation-induced cell death (AICD) induced with mitogens. This mechanism is largely independent from IL-2, while it can be significantly inhibited blocking CD95 with an IgG2b monoclonal antibody (mAb). The dependence of AICD from signals transduced via CD95 was confirmed showing that cross-linking CD95 with an IgM mAb induces a higher cell death in purified CD4+ CD45R0+ cells from OS patients than in controls (comparable for CD95 expression). Both mechanisms of cell death observed in this study result from lymphocyte hyperactivation occurring in vivo in these patients and may contribute to functional T cell defects of OS.

CD3-CD4+ cells with a Th2-like pattern of cytokine production in the peripheral blood of a patient with cutaneous T cell lymphoma.

A case of cutaneous T cell lymphoma associated with mild eosinophilia and rise of IgE levels is reported. A population of CD3-CD4+ cells was observed in the peripheral blood. After activation, these purified CD3-CD4+ cells showed a Th2 pattern of cytokine production, secreting higher levels of IL-5 and IL-4 and lower levels of IFN-gamma compared to the patient's and controls' CD3+CD4+ cells. Moreover, high levels of IL-5 and soluble CD30, a marker of Th2 cell activation, were detected in the patient's serum.

CD8+CD28- T cells in vertically HIV-infected children.

To evaluate whether vertical HIV infection interferes with the expression of CD28 on T lymphocytes, 25 HIV-infected children and 29 seroreverted children born to HIV+ mothers were studied. The percentage of CD28- cells among CD8+ T lymphocytes was higher in HIV-infected children than in controls (P < 0.001). In fact, in HIV-infected children, this percentage was elevated from the first year of life, while in healthy seroreverted children, the proportion of CD28- cells among CD8+ cells rose progressively with age (r = 0.49; P = 0.008). In HIV+ children, the CD8+ CD28-, but not CD8+ CD28+ cell proportion was significantly correlated with immunological markers of disease progression, such as CD4+ cell loss (r = -0.65; P < 0.001) and the level of in vitro spontaneous lymphocyte apoptosis (r = 0.53; P = 0.03).

Missense mutations in the Fas gene resulting in autoimmune lymphoproliferative syndrome: a molecular and immunological analysis.

Programmed cell death (or apoptosis) is a physiological process essential to the normal development and homeostatic maintenance of the immune system. The Fas/Apo-1 receptor plays a crucial role in the regulation of apoptosis, as demonstrated by lymphoproliferation in MRL-lpr/lpr mice and by the recently described autoimmune lymphoproliferative syndrome (ALPS) in humans, both of which are due to mutations in the Fas gene. We describe a novel family with ALPS in which three affected siblings carry two distinct missense mutations on both the Fas gene alleles and show lack of Fas-induced apoptosis. The children share common clinical features including splenomegaly and lymphadenopathy, but only one developed severe autoimmune manifestations. In all three siblings, we demonstrated the presence of anergic CD3+CD4-CD8- (double negative, [DN]) T cells; moreover, a chronic lymphocyte activation was found, as demonstrated by the presence of high levels of HLA-DR expression on peripheral CD3+ cells and by the presence of high levels of serum activation markers such as soluble interleukin-2 receptor (slL-2R) and soluble CD30 (sCD30).

CD70 expression on T-cell subpopulations: study of normal individuals and patients with chronic immune activation.

CD70, the ligand of CD27, is a member of the TNF family, which includes molecules essential in the regulation of lymphocyte growth and survival. It is absent on resting lymphocytes but can be induced in vitro with activating stimuli. To extend information about its expression by different T-cell subpopulations, and its regulation in normal and pathologic conditions, highly purified T-cell subpopulations were studied: CD70 expression depended both on the activating stimulus and on the T-cell subset analyzed. PMA + Ionomycin induced CD70 on the large majority of CD8+ cells, but only on a minority of CD4+ cells (P < 0.002), and among these, preferentially on the CD45R0+ subset compared with the CD45RA+. The presence of CD4+ lymphocytes in cell cultures containing mixtures of T-cell subsets inhibited CD70 expression on CD8+ cells. On the contrary, stimulation with allogeneic cells induced CD70 expression also on CD4+ cells. Moreover, CD70 was found to be expressed by chronically in vivo activated T-cells, in conditions characterized by allogeneic and autoimmune reactions. These data suggest a possible role of CD70 in the pathogenesis of immune dysregulation; interestingly, this role may not be simply restricted to bind to, and signal through, CD27, since cross-linking of CD70 enhances the proliferative response induced by the stimuli used to elicit its expression.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Defective expression of HLA class I and CD1a molecules in boy with Marfan-like phenotype and deep skin ulcers.

We report the case of a boy with low expression of HLA class I molecules on peripheral blood mononuclear cells, which is associated with immunodeficiency. The patient, who had a Marfan-like phenotype, had chronic deep skin ulcers and sinobronchiectasis. Immunohistologic examination of the ulcerated skin showed a dense perivascular infiltrate composed of normal mature lymphocytes and macrophages. All cells in the infiltrate showed an apparently normal expression of HLA class I molecules, but intraepidermal dendritic Langerhans' cells were negative for CD1a, an antigen that is a highly specific marker for these cells and is abundantly expressed in some self-healing forms of cutaneous lesions. It is therefore speculated that a defective expression of CD1a molecules can contribute to the chronic persistence of deep skin ulcers, which have already been reported in association with defective expression of HLA class I molecules.

CD4+ cells from patients with Common Variable Immunodeficiency have a reduced ability of CD40 ligand membrane expression after in vitro stimulation.

BACKGROUND: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production. This has been variably attributed to intrinsic B-cell defects or to T-cell disfunctions. Recently, it has been reported that the expression of the CD40 Ligand (CD40L), a T-cell surface molecule that plays a critical role in the cell-contact-mediated helper signals provided to B-cells, is defective in a subset of patients with CVID. METHODS: To demonstrate that the defective expression is due to intrinsic functional abnormalities of CD4+ lymphocytes, CD4+ cells were purified from eight patients with CVID and eight age-paired controls, stimulated with PMA+Ionomycin, and studied for CD40L expression by flow cytometry using specific monoclonal antibodies. RESULTS AND CONCLUSIONS: The percentage of CD4+ cells expressing CD40L after optimal stimulation was correlated with age both in patients with CVID (r: 0.74; p: 0.04) and in healthy controls (r: 0.73; p: 0.04). The percentage of CD40L+ cells was reduced in patients with CVID compared to that of controls (p: 0.02 when data are paired for age) with a reduced density of expression (p: < 0.01). The defect was variable in different patients and in some cases it was marginal.

A case of hypereosinophilic syndrome is associated with the expansion of a CD3-CD4+ T-cell population able to secrete large amounts of interleukin-5.

Interleukin-5 (IL-5) is the major soluble factor able to mediate hypereosinophilia. We report a case of hypereosinophilic syndrome in which the presence of a population of CD3-CD4+ cells able to overproduce IL-5 was shown. The lack of CD3 and TCRAB membrane expression on otherwise phenotypically normal mature T lymphocytes together with the absence of detectable TCRBV mRNA and clonal rearrangement of TCRB gene suggested that the abnormal lymphocyte population was the expression of a peripheral T-cell lymphoma with an indolent clinical course. Peripheral blood lymphocytes enriched in this population were able to secrete high levels of IL-5 but not IL-4, and no IL-2 or interferon-gamma, when stimulated in vitro with phytohemagglutinin and phorbol myristate acetate. The serum contained eosinophil survival factors whose activity was partially neutralized by a specific antihuman IL-5 antibody. This observation further emphasized the relationship between hypereosinophilic syndrome. IL-5, and T-cell lymphoproliferative disorders.

Ontogeny of CD40L [corrected] expression by activated peripheral blood lymphocytes in humans.

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.