Characterization of autoantibodies to natural killer cells in HIV-infected patients.

Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Auto-antibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/microliters) and antibody negative (141/microliters) patients (P = 0.3). However, pre-incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells (P = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo. There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo.

Progressive increase of CD7- T cells in human blood lymphocytes with ageing.

CD7 is one of the major surface antigens expressed very early during T cell ontogeny. Lack of CD7 expression on mature T cells is regarded as a classical feature of malignant T cells in certain forms of cutaneous T cell lymphoma. Previously, we identified a CD7- subset of peripheral blood T lymphocytes in normal human individuals. In this study we determined the portion of CD7- T cells in the peripheral blood of healthy volunteers ranging in age from 8 months to 90 years (n = 85) and in cord blood of full-term infants (n = 14). Furthermore, this CD7- subset was characterized in detail by the use of MoAbs and three-colour flow cytometry. In cord blood no CD7- T cells could be detected. After birth, percentage and absolute number of CD7- T cells increased with age. Independently of age, most CD7-CD3+ cells belonged to the CD4+ subpopulation. Focusing on the latter we could demonstrate the predominance of the CD45RO+CD45RA- phenotype in the CD7- subset. Furthermore, CD7- T cells contained a higher number of cells expressing activation markers and the CD57 antigen, but a reduced number of CD62L+ cells in comparison with CD7+ T cells.

Anti-lymphocyte antibodies in plasma of HIV-1-infected patients preferentially react with MHC class II-negative T cells and are linked to antibodies against gp41.

It has previously been shown that HIV-infected patients develop anti-lymphocyte antibodies. The relationship between anti-lymphocyte antibodies and antibodies against different viral antigens is unknown, and it remains controversial whether some lymphocyte subpopulations are targeted preferentially. We have set out using three-colour flow cytometry to measure antibodies against different lymphocyte subsets. Staining with anti-human immunoglobulin and two MoAbs was performed to characterize the immunoglobulin load of different lymphocyte subsets. Comparison was done between patients' antibody reactivity against HIV-1 antigens and anti-lymphocyte antibodies. We were able to demonstrate the presence of anti-lymphocyte antibodies in approximately 75% of the HIV-infected patients (n = 78) (healthy controls were all negative). MHC class II-negative T cells showed a stronger reaction with anti-lymphocyte antibodies than B cells or MHC class II-positive T cells. Patients with antibodies against CD4 lymphocytes showed a significantly higher antibody reaction with the retroviral antigen gp41 than patients without these antibodies. An association between anti-lymphocyte antibodies and antibody reactivity against other HIV-1 antigens was not noticed. In conclusion, anti-lymphocyte antibodies in HIV-1-infected patients show a preferential reactivity with T cells which lack expression of MHC class II molecules. There is an increased antibody reactivity against gp41 in patients with anti-CD4+ T cell antibodies. The association hints at a specific origin of anti-lymphocyte antibodies in HIV-1-infected patients due to cross-reactivity with viral epitopes or network phenomena. These anti-CD4 cell antibodies could be of interest in the clinical course of HIV infection.

Antibodies against CD4+ lymphocytes in plasma of HIV-infected patients are related to CD4 cell depletion in vivo.

The role of autoimmune phenomena in the pathogenesis of AIDS is not well understood. Antibodies against CD4+ lymphocytes are frequently detectable in HIV-infected patients. However, the relevance of these antibodies is unknown. In this study anti-CD4 cell antibodies in plasma of HIV-infected patients were compared to patients' CD4 cell count. Lymphocytes of a healthy donor were incubated with plasma of patients and controls. Antibodies against CD4+ lymphocytes were detected using anti-human Ig antibodies and monoclonal anti-CD4 antibodies simultaneously. The degree of staining was visualized by flow cytometry. The experiments revealed that 60% of HIV-infected patients harbored anti-CD4 cell antibodies in their plasma. Anti-CD4 cell antibodies were not detectable in plasma of healthy controls. Patients with anti-CD4 cell antibodies in their plasma presented with significantly lower numbers of circulating CD4+ lymphocytes (P = 0.025). The degree of antibody reactivity was negatively correlated to patients' CD4 cell counts (P = 0.02). In conclusion there is evidence for an association between plasma anti-CD4 cell antibodies and CD4 cell depletion. Whether this association represents an epiphenomenon or a pathogenetic relevant pathway needs to be investigated in further studies.

Autoantibodies against ganglioside GM3 represent a portion of anti-lymphocyte antibodies in AIDS patients.

In this study we analysed the relationship between anti-lymphocytic ganglioside antibodies and anti-lymphocyte antibodies in AIDS patients. Anti-lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets. Anti-lymphocytic ganglioside antibodies were detected in 23 out of 49 AIDS patients sera (46.9%). All positive sera reacted selectively with the GM3 comigrating band from AIDS lymphocytes. Twenty two out of the 23 anti-lymphocytic GM3 positive sera also had antibodies against CD4+T cells, versus 17/26 anti-GM3 negative. Furthermore, patients with lymphocytic GM3 antibodies showed a significantly higher antibody reactivity against CD4+ T cells than patients in which these antibodies were not detected. The absorption tests revealed that preincubation of positive sera with GM3 was followed by a decrease in the reaction with target lymphocytes. These findings suggest that anti-GM3 antibodies are a portion, but not the majority, of antibodies reacting with CD4+ T cells.

CD7-negative helper T cells accumulate in inflammatory skin lesions.

Recently, we identified a particular T-cell subset in the peripheral blood of normal individuals that lack CD7 expression. In this study we determined the portion of CD7- T cells in the peripheral blood and skin of patients with various inflammatory skin diseases. We found that skin-infiltrating lymphocytes isolated from different benign and malignant skin lesions (n = 20) contain a high portion of CD7- helper T cells, whereas the number of CD7- T cells in the peripheral blood was not altered compared to healthy controls. Cell activation in vitro did not induce CD7 expression in negative T cells but increased CD7 expression in CD7-positive cells. Thus, lack of CD7 expression seems to be a stable characteristic in a major subset of skin-infiltrating lymphocytes. During long-term culture of CD7- helper T-cell clones derived from a psoriasis skin lesion, no phenotypic change in the CD7 phenotype could be monitored by sequential flow-cytometric analyses. No CD7 mRNA could be detected by Northern blot analysis, indicating transcriptional regulation of CD7 expression. The results show that CD7- T cells accumulate in certain inflammatory skin lesions without alteration of the circulating CD7- population. These cells may be identical to or derived from CD7- T cells of the peripheral blood.

Induction of nuclear contour irregularity during T-cell activation via the T-cell receptor/CD3 complex and CD2 antigens in the presence of phorbol esters.

To determine whether specific T-cell activation pathways could produce nuclear contour irregularity in normal human lymphocytes, purified T cells were stimulated in vitro and subsequently analyzed by electron microscopy. The degree of nuclear contour irregularity was determined with the use of a computerized planimeter. Stimulation via the T-cell receptor (TCR)/CD3 complex using anti-CD3 monoclonal antibodies induced Sézary-like morphology (nuclear contour indices > 6.5) in a significant portion (9% to 28%) of T cells derived from different normal donors. T-cell activation via CD2 antigens showed induction of Sézary-like nuclear morphology in a lower percentage of cells in comparison with stimulation via the TCR/CD3 complex. In contrast, mitogenic stimulation in vitro did not induce alterations of nuclear morphology in T cells. Immunoelectron microscopy showed that nuclear contour irregularity induced in vitro did not correlate with surface-antigen expression of T-cell subpopulations. The results indicate that Sézary-like morphology is associated with cell activation in normal human T cells.

Could non-insulin-dependent diabetes mellitus be attributable to a deficiency of FAD-linked glycerophosphate dehydrogenase?

In 12 out of 32 non-insulin-dependent diabetic subjects, the activity of FAD-linked glycerophosphate dehydrogenase in T lymphocyte homogenates was abnormally low when measured by both a colorimetric and radioisotopic procedure. A comparable situation characterized by a deficient activity of FAD-linked glycerophosphate dehydrogenase in both the colorimetric and radioisotopic assays was only observed once among 26 other subjects including 11 healthy volunteers, 9 non-diabetic patients, 5 type-1 (insulin-dependent) diabetics, and 1 pancreatectomized diabetic. By analogy, it is speculated that an impaired activity of FAD-linked glycerophosphate dehydrogenase in the insulin-producing pancreatic B-cell could represent a far-from-uncommon contributive factor in the pathogenesis of non-insulin-dependent diabetes mellitus.

Systemic interferon gamma treatment in severe atopic dermatitis.

BACKGROUND: Recent results suggest decreased interferon gamma (IFN-gamma) but high interleukin 4 (IL-4) production in patients with atopic dermatitis (AD). Because the relative activities of IL-4 and IFN-gamma seem to regulate the amplitude of the IgE response we suggested a role for IFN-gamma in the treatment of AD. OBJECTIVE: The purpose of this study was to assess the efficacy of systemic IFN-gamma treatment in patients with severe AD. METHODS: Patients with severe AD (n = 14) were treated with recombinant IFN-gamma for 6 weeks. During the study only basic local therapy with steroid-free hydrophilic or emollient ointments was allowed. RESULTS: Eight patients (57%) showed marked clinical improvement during systemic IFN-gamma therapy. Four of these patients showed continuous improvement 3 months after treatment was discontinued. Mean total and antigen-specific serum IgE concentrations were not statistically different during and after treatment, whereas mean spontaneous IgE production in vitro was significantly lower after 6 weeks of IFN-gamma therapy. CONCLUSION: Our results suggest that IFN-gamma treatment may represent a novel therapeutic approach in patients with severe AD.

Relationship of antibodies against CD4+ T cells in HIV-infected patients to markers of activation and progression: autoantibodies are closely associated with CD4 cell depletion.

Antibodies against lymphocytes have been shown in human immunodeficiency virus (HIV)-infected patients, but their relevance in the pathogenesis of acquired immune deficiency syndrome (AIDS) remains controversial. We investigated increased levels of lymphocyte surface Ig and antibodies against CD4+ T cells in the plasma. The relationship to CD4 cell depletion and serological parameters were analysed. A three-colour flow cytometric method was used to detect surface Ig on the surface of patients' cells and antibodies in the plasma of the patients. We observed a high percentage of patients with increased surface Ig on CD4+ T cells (94%-47/50). Antibodies in the plasma reacting with healthy donors' CD4+ T cells were detectable in 72% (23/32) of the patients. CD4 cell-surface Ig correlated well with surface Ig on different T-cell subpopulations but not with increased surface Ig on B cells. Only one control showed elevated surface Ig, plasma antibodies against lymphocytes were not detectable. Surface Ig levels of CD4+ T cells were closely associated with the CD4 cell number in HIV-infected patients of all stages of disease (r = -0.67, P = 0.00005). Other lymphocyte subsets' surface Ig did not show a significant association to CD4 cell depletion. Surface Ig and antibodies against CD4+ T cells were not related to levels of beta 2-microglobulin, p24 antibodies or interleukin-6 (IL-6), and did not depend on hypergammaglobulinaemia. In conclusion surface Ig on CD4+ T cells is likely to have an autoantibody origin. The high prevalence and association to CD4 depletion support the view that autoimmune phenomena could be involved in the pathogenesis of AIDS.

CD7- T cells represent a subset of normal human blood lymphocytes.

In the peripheral blood of normal adults we identified a subpopulation of mature human T cells that lacks expression of CD7. These CD3+CD7- T cells represent 9% +/- 3.4 SD of PBMC as estimated by analyses of 38 different normal donors. The majority of CD7- T cells express TCR alpha/beta, and are of CD4 helper and CD45RO+CD45RA- "memory" phenotype as determined by three-color fluorescence analysis. We established seven CD4+CD7- T cell clones in vitro from purified CD7- blood T cells and compared these cells to CD4+CD7+ clones. Nonexpression and expression, respectively, of CD7 was a stable feature of all clones during long term culture for more than 6 mo. No CD7 mRNA could be detected by dot blot and Northern blot analysis of purified CD7- T cells and of expanded T cell clones implicating a transcriptional regulation of CD7 Ag expression. Ionomycin/TPA and PHA stimulation were not capable to induce CD7 expression in CD7- cells but up-regulated CD7 expression in CD7+ cells. Whereas CD7- cells of cutaneous T cell lymphoma exhibit a cerebriform nucleus (Sézary cells), no evidence was obtained for cerebriform nuclear morphology in normal CD7- T cells from peripheral blood. We suggest that these CD7- T cells represent a physiologic subset of mature T cells and may be the circulating counterpart of those lymphocytes that are found to be enriched in normal skin and in a variety of benign and malignant skin diseases.

Functional characterization of skin-infiltrating lymphocytes in atopic dermatitis.

Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In 3H-thymidine incorporation assays, SIL showed proliferation in response to IL-2, IL-3, IL-4, ionomycin (Io) + 12-o-tetradecanoyl-phorbol-13-acetate (TPA) and OKT3 + TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-alpha) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-alpha upon stimulation with mitogens but failed to secrete IFN-gamma. Io in combination with phorbol-ester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-alpha and low amounts of IFN-gamma. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-gamma but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.

Tumor-infiltrating lymphocytes isolated from a Ki-1-positive large cell lymphoma of the skin. Phenotypic characterization and analysis of cytokine secretion.

Tumor-infiltrating lymphocytes (TIL) were obtained from a biopsy of a patient with a Ki-1-positive large cell lymphoma of the skin. Immunohistologic studies of the large anaplastic tumor cells showed an "aberrant" T "helper/inducer" phenotype (CD30 + CD3-CD4+ CD8-IL-2R + HLA-DR+). Using cDNA probe for the constant region of the T-cell receptor (TCR) beta gene, the cells were identified by their distinct monoclonal rearrangement of T-cell receptor (TCR)-beta DNA. Tumor cells isolated from biopsies were cultured in the presence of interleukin-2 (IL-2). Outgrowing lymphocytes were cloned, expanded in vitro, and 11 clones were subjected to phenotypic analysis: ten clones showed a predominantly CD4-positive T "helper/inducer" phenotype whereas one clone expressed CD8 T "cytotoxic/suppressor" antigens. In contrast to the tumor cells, cells of all clones grown in vitro expressed the TCR-associated CD3 complex. Furthermore, cells from all clones analyzed expressed CD5, CD7, CD45RO (UCHL1), CD11a (LFA-1), CD25, and HLA-DR antigens. Cells of two of ten CD4-positive clones expressed CD45RA (2H4) in addition to UCHL1. T-cell clones isolated from the tumor and grown in vitro exhibited individual DNA restriction band patterns different from that of a DNA tumor biopsy specimen. Therefore, the authors conclude that these T-cell clones represent presumably nonmalignant TIL. All clones tested secreted interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in vitro. Nine of 11 clones were found to secrete additionally IL-2 and IL-4 upon stimulation with phytohemagglutinin (PHA) whereas two clones did not secrete detectable amounts of IL-4. Selective growth of TIL in the presence of IL-2 opens the possibility to use these cells in adoptive immunotherapy of cutaneous T-cell lymphoma (CTCL). Cytokines secreted by TIL cells in vitro (IL-2, IL-4, IFN-gamma, TNF-alpha) may be involved in their antitumorigenic activity. Moreover, these data implicate that CD4-positive TIL derived from CTCL cannot be grouped into different subsets based on the production of IL-2, IL-4, IFN-gamma, and TNF-alpha.

Interleukin-4 promotes the expansion of skin-infiltrating lymphocytes from atopic dermatitis in vitro.

Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.

Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities.

The in vitro production of interferon-gamma (IFN-gamma) in 19 atopic dermatitis (AD) patients was compared with that of 12 controls. IFN-gamma production by phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) was profoundly diminished in AD patients, whereas the proliferative response was similar to that of control PBMC. The addition of 40 U/ml of interleukin-2 (IL-2) to the cultures failed to restore IFN-gamma production. Similarly, removal of adherent cells also had no effect. Reduced IFN-gamma secretion was observed after stimulation with the CD3 monoclonal antibody OKT3, ionomycin + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with high levels of IL-2 (200 U/ml). There were increased proportions of CD4+ T helper/inducer cells and decreased proportions of CD8+ T cytotoxic-/suppressor cells and CD16+ natural killer (NK) cells in AD patients. This resulted in an increased CD4/CD8 ratio as compared with controls, but no correlation was observed between numbers of T cell subpopulations and IFN-gamma generation. However, a significant correlation was found between IFN-gamma generation in vitro and IgE serum concentration in AD patients. The data suggest that the decreased production of IFN-gamma by AD patients is due to intrinsic differences in capacity to produce this cytokine and is not the result of differences in regulatory cell interactions. Moreover, the findings indicate that decreased production of IFN-gamma may be an important factor in the pathogenesis of this disease.

Selective alterations in natural killer cell subsets in patients with atopic dermatitis.

We examined the immunophenotypic characteristics of natural killer (NK) cell subsets in patients with severe atopic dermatitis (AD), rhinitis allergica (RA) and in healthy controls. Expression of CD16, CD56 and CD57 antigens on peripheral blood lymphocytes was evaluated by simultaneous double immunocytofluorometry. Our results showed significantly lower percentages of cells with CD16, CD56 and CD57 surface antigens in patients with AD. Furthermore subdivision of the AD group into two subgroups, AD1 and AD0 (with and without antigen-specific IgE antibodies against potent inhalative allergens, i.e. mite, grass, rye, birch, cat) revealed that patients of subgroup AD1 showed a more prominent decrease compared to that of subgroup AD0. Moreover, we found a significant negative correlation between the percentage of CD56 + CD16 + NK cells and total IgE levels in serum, which were significantly higher in patients of subgroup AD1 than in AD0. NK cell activity was deficient in patients with AD but there was no difference between both subgroups. These data indicate that considerable heterogeneity in immunologic regulation may exist in patients with AD with regard to their NK cell subsets.

Cytokine release from cultured peripheral blood mononuclear cells of patients with severe atopic dermatitis.

Peripheral blood mononuclear cells (PBMC) from 14 patients with severe atopic dermatitis (AD) and 11 healthy donors were tested for their capacity to produce tumour necrosis factor-alpha (TNF-alpha) after PHA stimulation and compared with their in vitro production of interferon-gamma (IFN-gamma). The mean TNF-alpha production in AD patients did not differ vis-à-vis controls. However, a significant portion of patients with AD which was defective in generating IFN-gamma in vitro showed in addition significantly a decreased production of TNF-alpha. No correlation could be found between TNF-alpha and neopterin production in either group, whereas there was a close overall correlation between the amount of TNF-alpha and IFN-gamma detectable in culture supernatants of patients and controls. Furthermore, a significant correlation was found between TNF-alpha and IFN-gamma generation in vitro and serum IgE concentration in AD. Based on cytokine production in vitro and IgE concentration in vivo, patients with severe AD could be divided into two groups. Furthermore, 3 AD patients with normal IFN-gamma generation and low serum IgE concentration but suffering from eczema herpeticum formed a subgroup which showed an increased TNF-alpha production in vitro. The data suggest alterations in cytokine production in a subgroup of patients with AD which bear a reciprocal relationship to abnormal IgE regulation.