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We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-gamma (IFN-gamma) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CD1a+, HLA-DR+, CD4+, CD40+, CD80+ but CD14- cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-gamma selectively induced HLA-DR and CD86 but did not up-regulate CD1a expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-gamma could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-gamma to GM-CSF plus TNF-alpha during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-alpha mutants, we investigated the relative involvement of TNF-alpha receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-R1 triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-alpha and IL-4; second, that the effect of TNF-alpha in DC generation involves signalling via the TNF-R1 receptor; and third, that IFN-gamma counteracts some of the effects of IL-4 in DC generation.

Cell-mediated immunity and cytokines are probably involved in the pathogenesis of malaria. To investigate the role and the activity of different immune cells, we measured levels of tumour necrosis factor-(TNF-alpha), gamma interferon (IFN-gamma) and several interleukins (IL-2, IL-4, IL-6 and IL-10) in children with mild (MM) and cerebral (CM) Plasmodium falciparum malaria and compared them with those of healthy children from Guadalupe--Lobata District, St. Tomé Island, where malaria is mesoendemic. Both groups of patients had significantly higher levels of IL-6, IL-10 and TNF-alpha than controls. For IL-2, IL-4 and IFN-gamma we found no difference between the groups. However, 24 h after admission the levels of IL-10 and IL-6 were significantly higher in CM than in MM patients, although 7 days after treatment they returned to normal levels, similar to those found in control children. Therefore, TNF-alpha IL-6 and IL-10 increase during Plasmodium falciparum attacks in all children, not only in those with cerebral malaria. This finding suggests the activation of the monocyte/macrophage system during the early stage of clinical malaria.

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4+ or CD8+ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8+ and CD4+ T cells from HIV-infected and HIV- subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8+ T cells from patients proliferated consistently less than controls, while responses from CD4+ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4+ and CD8+ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-gamma) in CD4+ T cells. Compared with controls, CD4+ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-gamma, IL-4 and IL-5, while CD8+ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4+ T cells from HIV+ subjects to various costimulatory pathways are relatively intact, whereas CD8+ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8+ T cell failure might contribute to viral and neoplastic complications of HIV infection.

Protective immunity against Mycobacterium tuberculosis (MTB) in animal models is based on cell-mediated immunity (CMI), involving bi-directional interactions between T cells and cells of the monocyte/macrophage (MO/MA) lineage. Key factors include MO-derived interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha as well as T cell derived IL-2 and interferon (IFN)-gamma. These cytokines appear particularly crucial in the induction of MA-mediated elimination of mycobacteria. Several lines of evidence indicate that similar mechanisms are operating in humans. During active pulmonary tuberculosis (PTB), signs of both immune depression and immune activation are concomitantly present. Decreased tuberculin skin test reactivity in vivo and deficient IFN-gamma production by MTB-stimulated mononuclear cells in vitro are observed. On the other hand, the serum levels of several cytokines, including TNF, and other inflammatory mediators are increased and circulating MO and T cell show phenotypic and functional evidence of in vivo activation. In this review, we will discuss the evidence for three models, which could explain this apparent paradox: 1. Stimulation of the T cell-suppressive function from MO/MA; 2. Intrinsic T cell refractoriness, possibly associated with tendency to apoptosis (programmed cell death), and 3. Compartmentalization and redistribution of immune responses to the site of disease. The opportunistic behavior of MTB during human immunodeficiency virus (HIV) infection can be explained by suppression of type-1 responses at the level of antigen-presenting cells, CD4 T cells and effector macrophages. The ominous prognostic significance of intercurrent PTB during HIV infection seems primarily due to prolonged activation of HIV replication in macrophages. Supportive immune therapy during PTB could aim at correcting the type-1 deficiency either by IFN-gamma inducers (e.g. IL-12, IL-18) or by neutralizing the suppressive cytokines transforming growth factor beta (TGF-beta) and IL-10. Alternatively, inflammatory over-activity could be reduced by neutralizing TNF. Finally, anti-apoptotic therapies (e.g. IL-15) might be considered.

Parameters of immune activation/differentiation were studied in a group of newly diagnosed HIV- and HIV+ pulmonary tuberculosis (TB) patients. Compared with controls, HLA-DR expression on both CD4 and CD8 T cells from the HIV- TB patients was approximately doubled; HLA-DR on T cells from the HIV+ group was tripled. The monocytes from both groups of patients expressed abnormally high levels of the Fc gamma receptors I and III. Serum levels of tumour necrosis factor-alpha (TNF-alpha), neopterin and beta 2-microglobulin were increased in HIV- and even more so in HIV+ TB patients. The expression of HLA-DR on T cell subsets and of Fc gamma R on monocytes correlated with each other, but not with serum activation markers. This pattern of non-specific activation during TB infection may be associated with enhanced susceptibility to HIV infection.

The cellular immune responses to fractionated Haemophilus ducreyi antigens, coated on latex beads, were assessed in patients with chancroid and in controls, using an in vitro lymphocyte proliferation assay. Several fractions of H. ducreyi antigen revealed stimulating activity. However, only the molecular size ranges 91-78 kD, 59-29 kD, and 25-21 kD induced proliferation that may be specifically related to H. ducreyi infection. Lymphocytes from four HIV- patients, successfully treated for chancroid, were not stimulated by H. ducreyi antigen. In general, lymphocytes from HIV+ chancroid patients were less responsive to H. ducreyi antigen compared with those from HIV- chancroid patients. However, two HIV-infected patients showed exceptionally strong responses to high molecular weight fractions. To our knowledge this is the first report demonstrating that H. ducreyi contains specific T cell-stimulating antigens. Based on this work, further identification and purification of the T cell antigens is feasible.

The cellular immunologic and virologic status of a chimpanzee, naturally infected with a human immunodeficiency virus type 1 (HIV-1)-like lentivirus (SIVcpz-ant), was compared longitudinally with those of 3 HIV-1-infected and 5 uninfected chimpanzees for a period of 49 months. Evidence of immune deficiency was not observed in the HIV-1-infected chimpanzees, nor could virus be isolated from plasma. Virus could be isolated from plasma of the SIVcpz-ant-infected chimpanzee, but clinical signs of immune deficiency were never observed. Absolute CD4+ cell counts remained relatively stable, but NK cells fluctuated significantly over time and tended to correlate inversely with the virus titer in peripheral blood. Although only CD8+ T cells were directly demonstrated to exert a suppressive effect on viral replication in vitro, the observed fluctuation of NK cells suggests that these cells may also be involved in the interaction with lentivirus infection in this species.

The various stages of Plasmodium falciparum (sporozoites and liver stages, asexual blood stages and gametocytes) each interact in a particular way with the human immune system. Specific immunity against the liver stages is achieved through a coordinated action of CD8 T cells and specific antibodies, the latter in collaboration with NK cells and macrophages. In this reaction, interferon-gamma plays an essential role. A non-specific "concomitant" immunity against sporozoites is based on a cytokine reaction, elicited by the blood stages. In practice, the high variability in the immunogenic structures of the sporozoite precludes completely protection against recurrent infections. The spleen macrophages have a pivotal role in the immune defense against the asexual blood stages. The elimination of merozoites and parasitized red blood cells (RBC) is facilitated by specific antibodies, produced under the control of CD4 T cells. There are, however, multiple mechanisms of immune deviation, suppression and evolutionary adaptation, which inhibit a sterilizing immunity against the blood stages. Nevertheless, symptoms may be absent in exposed adults, even when parasitemia persists. This clinical resistance, however, is relatively short-lived, once exposition is interrupted. The observation that HIV infection has no adverse effect on malaria also is a remarkable but consistent finding. All these data indicate that a strong T cell-mediated immune memory is absent in human P. falciparum infections. Cerebral malaria and some other serious complications are the consequence of insufficient elimination of parasitized erythrocytes by the spleen, presumably in combination with parasite factors (particular variant surface structures) and with human host genetics (HLA type, blood group etc.). Parasitized RBC massively stick to the endothelium of the micro-vessels and non-parasitized RBC roset around the parasitized ones. Eventually, serious problems in the micro-perfusion and in the local metabolism occur and organ failure may finally ensue. The immune reaction against the surface-antigens of the sexual stage is limited and insufficient, most probably for similar reasons as in the asexual stages. Internal structures of the gametocytes, however, are highly immunogenic, but, unfortunately. Normally cannot be reached by the immune system. Based on these fundamental data, some of the perspectives of vaccination and new therapeutic tools are critically discussed.

It was previously shown that CD26 (DPP IV, EC 3.4.14.5) is a binding site for adenosine deaminase (ADA, EC 3.5.4.4) on T cells and that costimulation by some anti-CD26 monoclonal antibodies (mAbs) and anti-CD3 induces CD4+ T cell proliferation. The CD26 epitopes involved in costimulation, the precise sequence of the events preceding proliferation, and the response of CD8+ compared with CD4+ T cells to CD26 were not extensively studied. We therefore compared the effects of the novel TA5.9 anti-CD26 mAb, recognizing an ADA-binding epitope, and the clearly distinct anti-Ta1 reference anti-CD26 mAb for their costimulatory properties in various T cell subsets. Both purified CD4+ and CD8+ T cells proliferated upon costimulation with anti-CD3 and either anti-CD26 mAb, but anti-TA5.9 mAb induced a more potent response than anti-Ta1. Either anti-CD26 mAb, together with anti-CD3, caused a similar sequential up-regulation of CD69, CD25 (IL-2R alpha), and CD71 (transferrin receptor) expression on CD4+ and CD8+ T cells. The activation markers appeared faster on the CD45R0+ than on the CD45R0- subsets. After costimulation, CD4+ T cell cultures contained significant amounts of the Th1 cytokines IL-2, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). In CD8+ T cell cultures relatively more IFN-gamma and TNF-alpha but almost no IL-2 was measured after triggering of CD3 and CD26. Our data demonstrate that the recognition of the ADA-binding epitope is not a prerequisite for the costimulatory capacity of anti-CD26 mAbs. Both CD4+ and CD8+ T cells and their CD45R0- and CD45R0+ subsets are sensitive to various aspects of activation via CD26, but the magnitude and/or kinetics differ according to the anti-CD26 used and the T cell subset studied.

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen-presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage-related fashion. CD28- CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL-2R alpha. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co-stimulation with cell-bound anti-CD3 and B7. Nevertheless, when stimulated with plate-fixed anti-CD3, CD8+ T cells from HIV-infected subjects proliferated normally, and normal levels of IL-2R alpha and transferrin-receptor could be induced on CD28- CD8+ T cells from the patients. In addition, stimulation with plate-fixed anti-CD3 induced proliferative responses in highly purified CD28- CD8+ T cells from both HIV- and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti-CD3 redirected assay, was predominantly exerted by CD28- CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down-regulation and were slightly hyporesponsive to B7 co-stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co-stimulation via B7. CD28- CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7-independent stimulation.

In this study, the immunogenicity of a recombinant pre-S2 containing hepatitis B vaccine was determined in a cohort of 147 homosexual men. Both seroconversion rate and geometric mean titers indicated that pre-S2 was less immunogenic than HBsAg. Forty-eight subjects, including nine non-responders and seven high-responders to HBsAg, were tested for HLA Class I and II antigens to study a possible association between HLA-type and non-response. Both HLA-B8 and HLA-DR3 were clearly overrepresented in non-responders to HBsAg. Non-response to pre-S2, observed in 7/9 non responders and 6/39 responders to HBsAg, was not associated with any of the tested HLA markers.

Cryopreservation techniques have become essential in longitudinal evaluation of lymphocyte function. This study describes a convenient method for freezing of peripheral blood mononuclear cells, using a small cryocontainer. Analysis of cell function, assayed by lymphoproliferation to recall antigens and by cytotoxic capacity of activated lymphocytes, was performed in parallel on fresh and frozen-thawed cells. Although cryopreservation did affect lymphocyte function, our results indicate that this freezing method performed equally well compared to a computer controlled device. We conclude that the cryocontainer has proved to be a suitable and practical tool in clinical studies and is an economical alternative to conventional methods.

The relationship between the genetic variability in the V3 loop and the biological characteristics of 38 biological clones from 5 European and 7 African HIV-1 isolates belonging to 3 different subtypes (subtype A, B, and D) was investigated. Seventeen of 19 clones displaying a syncytium-inducing (SI) capacity had a positively charged amino acid located at position 11 and/or 25 in the V3 loop sequence. All 19 non-syncytium-inducing (NSI) virus clones lacked such a positive charge at the same positions (p < 0.001). The mean of net charge in the overall V3 loop sequences of the SI clones was higher than that of the NSI clones (p < 0.001). Within the same strains, the SI clones replicated faster/higher than the NSI clones in peripheral blood mononuclear cells (p < 0.01), but not in CD4+ T cell cultures (p > 0.1). All SI clones but only 5 of 19 NSI clones could replicate in human continuous T cell and monocytic cell lines (p < 0.001). A higher number of positively charged amino acid substitutions was found among the subtype D SI clones. Only one of eight autologous sera tested had the ability to neutralize the contemporaneously isolated NSI clones, but not the SI clones. This study indicates that the V3 loop amino acid sequences of HIV-1 biological clones from different origins belonging to different genetic subtypes are clearly correlated with viral syncytium-inducing capacity, cell tropism, and replication rate.

The expression and co-expression profiles of functionally important monocyte surface markers were compared between control and HIV+ individuals using combined physical gating and dim CD4 expression to delineate the monocytes. The Fc gamma RII (CD32), the MHC class II antigen HLA-DR and the adhesion molecules CD11a (LFA-1 alpha), CD18 and CD54 (ICAM-1) showed an unimodal distribution. Of these markers, CD11a and HLA-DR were up-regulated in the HIV+ subjects compared with controls. The expression levels of the adhesion molecules correlated with each other in both patients and controls. The CD11b (CR3-alpha), CD14, Fc gamma RI, and Fc gamma RIII markers were bimodally distributed. Compared with controls, monocytes from seropositives contained fewer CD14bright+ cells, an equal proportion of Fc gamma RIbright+ cells, but twice as many Fc gamma RIII+ cells. The expression level of Fc gamma RI and CD11b within their brightly positive subset increased as CD4 T cells decreased. Both in patients and controls, co-expression of bright CD11b, CD14 and Fc gamma RI was shown, whereas the Fc gamma RIII+ cells were negative or dim positive for the former triad. We conclude that the expression of two Fc gamma R (I and III), of the adhesion molecules CD11a and CD11b and of HLA-DR showed particular alterations on monocytes from HIV+ subjects. The relationship of these phenotypic observations with altered cytokine profiles and altered monocyte function is discussed.

The expression of interleukin-2 receptor (IL2R) was studied on circulating lymphocytes from HIV-infected (HIV+) and control subjects, using chain-specific monoclonal antibodies and indirect immunofluorescence flow cytometry. The IL2R alpha chain expression was decreased on CD4 and CD8 T cells from HIV+ persons compared to controls. Conversely, beta chain expression was enhanced on both T cell subsets from the patients. IL2R-subunit levels were similar on natural killer cells from patients and controls. To evaluate the function of IL2R, we investigated to what extent IL2 could induce CD69, an early activation marker of lymphocytes. A dose-dependent increase of CD69 expression was observed on T and NK cells from all subjects. The upregulation of CD69 was similar on CD4 T and NK cells from patients and controls, but was more pronounced on CD8 T cells from HIV+ compared to HIV- subjects. Based on inhibition studies, both the alpha and the beta chain contributed to the IL-2-induced CD69 expression on CD4 T cells, pointing to involvement of the high-affinity receptor. The early activation of CD8 T cells and NK cells was mainly dependent on the intermediate-affinity receptor. We conclude that significant changes in IL2R alpha and beta chain expression on circulating T cells occur after HIV infection, but that early activation through IL2 is preserved or enhanced, even in advanced stages.

A newly available commercial ELISA (TRAx CD4, T Cell Diagnostics USA) for enumerating CD4+ T lymphocytes has been evaluated with blood samples of 105 HIV seropositive and 6 seronegative subjects. Results from the flow cytometric analysis were used as reference. The sensitivity and specificity of the ELISA to identify HIV seropositive subjects having less than 200 CD4+ T lymphocytes/microliters were assessed and studied using the ROC curve. The reproducibility of the ELISA test was analyzed on 40 samples. The results of the ELISA correlated well with these of the flow cytometric analysis (r = 0.79, p < 0.001). However, the ELISA test tends to overestimate the true CD4 count in HIV seropositives. This overestimation could not be explained by the aspecific contribution of monocytic CD4. The threshold for identifying HIV seropositive subjects with less than 200 CD4+ T lymphocytes with a maximum sensitivity and specificity was determined with ROC curve and equalled 400 cell equivalents with the ELISA (sensitivity and specificity were equal to 80%) and 1,450 lymphocytes/microliters with the total absolute lymphocyte count (sensitivity and specificity were equal to 75%). Using this curve, a threshold of 300 cell equivalents for the ELISA test and of 1,100 lymphocytes/microliters for the absolute lymphocyte count was shown to maximize the specificity (> 95%) without a significant loss of sensitivity.

The relationship between CD26/dipeptidyl peptidase IV, an ectopeptidase involved in T cell activation, and the binding protein for adenosine deaminase (ADAbp) was studied. Monoclonal antibodies (mAb) against CD26 and ADAbp, respectively, showed a similar binding profile on various lymphocyte subsets from the peripheral blood. The adenosine deaminase (ADA) itself blocked the binding of a specific set of anti-CD26 mAb (among these the anti-TA5.9 mAb) on lymphocytic CD26; ADA also hindered the binding of soluble CD26 to the same immobilized anti-CD26 mAb. In addition, the interaction between immobilized ADA and purified CD26/DPP IV was inhibited by the anti-TA5.9 mAb. ADA did not inhibit the specific peptidase activity of CD26. Neither soluble nor immobilized ADA was able to down-modulate CD26 on the lymphocyte surface. Our data thus confirm the identity between ADAbp and CD26 and identify some epitopes, crucial in the binding of ADA to CD26.

Infection with HIV results in a progressive depletion of CD4+ T cells and leads to significant in vivo lymphocyte phenotype changes. In this regard, the expression of HLA-DR and CD38 on CD8+ T cells has been shown to increase dramatically with disease progression. We investigated the expression of both activation markers on CD4+ T cells in HIV-1-infected subjects at different clinical stages of infection and compared the in vivo activation of CD4+ T cells with parameters of viral activity and CD8+ T cell activation. Fresh peripheral venous blood was obtained from 54 HIV-infected subjects and from 28 uninfected healthy controls. Three-colour immunophenotyping of the CD4+ T cell subset showed that the proportion of CD4+ T cells expressing HLA-DR (10% in HIV-negative controls) or CD38 (62% in HIV-negative controls) was higher in asymptomatic (P < 0.05 for CD38) and symptomatic (P < 0.001 for HLA-DR and CD38) HIV-infected subjects than in controls, whereas the proportion of CD4+ T cells expressing CD45RO (54% in controls) remained relatively unchanged. Simultaneous expression of HLA-DR and CD38 on CD4+ T cells increased from 2.3% in controls to 11% (P < 0.001) in asymptomatic and 22% (P < 0.001) in symptomatic HIV-infected subjects. This relative increase of CD38 and HLA-DR expression occurred mainly on CD4+ T cells co-expressing CD45RO. Changes in expression of HLA-DR and CD38 on CD4+ T cells correlated with similar changes on CD8+ T lymphocytes, with the presence of HIV antigen in the circulation, and with the disease stage of HIV infection.

The inability to mount a protective level (> or = 10 IU/l) of hepatitis B surface antigen (HBsAg)-specific antibodies after vaccination is presumably the consequence of a defect in the cellular immune regulation. We compared the in vitro immune responses of peripheral blood mononuclear cells (PBMC) from high, intermediate and non-responders, after stimulation with recombinant HBsAg. The absence of a proliferative response in non-responders was not reversed by removal of CD8+ T cells, indicating that HBsAg-specific CD8+ T-cell-induced suppression was not the underlying cause of non-responsiveness. Non-responders did not produce cytokines after HBsAg stimulation. High responders displayed a typical Th1-like profile since their PBMC produced interleukin-2 (IL-2) and gamma-interferon (IFN gamma) and no detectable IL-4 or IL-5 upon stimulation with HBsAg.

Using a novel anti-CD26 (or anti-dipeptidyl peptidase IV) monoclonal antibody, we showed that the absolute numbers and the proportions of T4 and T8 cells expressing CD26 were significantly lower in HIV-infected persons than in controls. The absolute number of CD26+ T4 cells decreased according to disease progression, whereas the number of CD26+ T8 cells was low throughout all clinical stages. These trends were similar in CD26 dim and bright positive T-cell subsets. In both controls and HIV-positive subjects, the CD26 bright positive T cells were restricted to the CD45RO+ subset and preferentially co-expressed CD25 but largely lacked HLA-DR and CD38. Recall antigen-responsive cells from seronegative individuals were shown to co-express CD26 and CD45RO. The deficient CD26 expression on T8 cells from HIV-infected subjects could be normally upregulated after in vitro stimulation. In contrast to decreased T-cell-bound CD26, the enzymatic activity of plasma CD26/dipeptidyl peptidase IV was unchanged in HIV-infected patients compared with controls. We conclude that HIV infection leads to a deficient in vivo co-expression of CD26 bright and CD45RO on T cells. We speculate that this deficiency might play a part in the decrease of immunological memory during HIV infection.

The CD26 activation antigen (Ag) which is expressed on a subpopulation of human T cells has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). In this paper, we describe the antibody binding profile of CD26/DPP IV, purified from human peripheral blood lymphocytes. The purified molecule binds to the anti-Ta1, anti-1F7 and anti-134-2C2 monoclonal antibodies (mAb), reported to react with cell-bound CD26 Ag. Among unclustered mAb recognizing T cell antigens, two, anti-BT5/9 and anti-TA5.9 were found to react with purified and cell-bound CD26 Ag. The classification of the BT5/9 Ag, the functional properties of the BT5/9+ T cell subset, as well as the in vivo effect of anti-BT5/9 mAb administration, are re-interpreted in the light of its specificity. Applying the anti-TA5.9 mAb in three color FACS analyses, we demonstrated that CD26+bright cells co-express CD45RO but not HLA-DR and CD38.

Objectives: To examine the effect of ditiocarb (DTC) treatment on immunological parameters of HIV infection. Immunophenotyping included CD4+ T-cell counting and the analysis of activation markers on CD8+ T cells. Anti-CD3-induced proliferation and anti-CD3-mediated cytotoxicity were monitored as indexes of T-cell function. In addition to the clinical evolution, HIV antigen and anti-p24 levels were monitored during treatment., Design: In this double-blind, placebo-controlled study, 50 HIV-seropositive patients belonging to all clinical disease stages were randomized to treatment with DTC or placebo and followed for 4 months., Methods: Immunophenotyping on whole blood was performed by flow cytometry, using combinations of anti-CD8 with anti-CD4, anti-HLA-DR, anti-CD38, anti-CD45RO and anti-CD57. Patient lymphocytes were freshly assayed for cytolytic capacity against OKT3-coated targets. T-cell proliferation was measured after 3 days of OKT3-stimulation., Results: No effect was observed on CD4 and CD8+ T-cell counts or on CD8+ T-cell activation markers, except for a selective increase in HLA-DR expressing CD8 cells in the DTC-treated group. Decline in anti-CD3-induced T-cell proliferation and rise in anti-CD3-mediated T-cell cytotoxicity were observed in the DTC and placebo groups. No effect on HIV antigen and anti-p24 antibody titres was observed. The incidence of clinical complications was similar in each group., Conclusion: No beneficial immunomodulatory effect of DTC was demonstrated.

Objective: To study the expression of the activation markers human leukocyte antigen (HLA)-DR and CD38 antigen on CD8+ T-lymphocytes in HIV-infected subjects and HIV-negative controls., Design: Two- and three-colour flow-cytometric analysis., Methods: Fresh peripheral venous blood was obtained from 16 HIV-infected subjects, representing four different stages of HIV disease, and from six HIV-negative controls. Three-colour lymphocyte immunophenotyping was performed using peridinyl chlorophyll-A protein (PerCP)-conjugated anti-CD8 monoclonal antibody (MAb) in combination with anti-HLA-DR (phycoerythrin) and anti-CD38 (fluorescein isothiocyanate) MAb., Results: The relative percentage of the lymphocyte populations thus defined differed between HIV-negative and HIV-positive subjects and between HIV-infected subjects at different clinical stages of disease. Simultaneous expression of HLA-DR and CD38 within the CD8 T-lymphocyte compartment increased from 8% in controls to 49% in asymptomatic HIV-infected subjects (P less than 0.005). Symptomatic patients differed from asymptomatic seropositives by a further increase in the HLA-DR+ CD38+ CD8 subset. In AIDS patients, the HLA-DR+ CD38- CD8 subset decreased (P less than 0.05) and the HLA-DR- CD38+ CD8 subset increased (P less than 0.05), compared with the other HIV disease stage patients., Conclusion: There is a stage-associated pattern of HLA-DR and CD38 expression on CD8 T-lymphocytes during HIV infection; specific phenotypic patterns may have functional correlates in the host response to the virus.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti-Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

Using fresh whole blood or isolated lymphocytes, the activity of in vivo generated cytotoxic T-lymphocytes (CTL) was measured as the OKT3-specific lysis of HL-60 targets, in a cross-sectional study of 53 HIV (+) patients. CTL activity in the entire HIV(+) group was two to three times higher than in HIV(-) controls, with WHO stage 3 (=pre-AIDS) patients showing the highest cytolytic function. The whole-blood CTL assay was validated and its practical and theoretical advantages are discussed. Within the CD8(+) cells, the number and proportion of the CD45RO(+) "memory" subset were significantly increased in HIV(+) subjects. The HLA-DR(+) subset rose most spectacularly in the asymptomatic stage of the infection, while the CD38(+) subset was the only one still significantly rising between the pre-AIDS and the AIDS stage. CTL activity was most closely correlated with T8 cells expressing the CD38 marker. In the context of CTL, CD38 thus seems to reflect activation rather than immaturity. Lymphocytes from HIV(+) subjects with a high OKT3-specific lytic capacity also destroyed normal lymphoblasts to a significant extent, pointing to their possible involvement in an autodestructive process. Our data thus suggest the importance of T8 cytolytic function and/or T8 subtyping in the immunopathogenesis and the prognosis of HIV infection.

The activity of both cytotoxic T lymphocyte (CTL) and natural killer (NK) cells were measured cross-sectionally in 43 subjects seropositive for HIV, in 27 HIV- blood donors and in 24 HIV- persons from the Outpatients Clinic for sexually transmitted diseases. CTL activity was evaluated using the HL-60 cells coated with OKT3 as the targets and freshly separated peripheral blood lymphocytes as the effectors. In 20 out of 43 HIV+ subjects, CTL activity was significantly enhanced in comparison to the HIV- subjects. This lytic activity correlated positively with the percentages of CD3+ HLA-DR+, of CD8+ CR3- and of CD57+ CD16- lymphocytes, and was greatly reduced after elimination of CD8+, of HLA-DR+ or of CD57+ cells. The median CTL activity seemed to increase from CDC group II to CDC group IV (Centers for Disease Control classification), but to return back to control levels in those patients with a history of opportunistic infections. NK function in HIV+ subjects was not significantly different from that in the blood donors. In seropositive patients, NK activity correlated positively with the percentages of both CD16+ CD57+ and of CD8+ CR3+ cells and was strongly diminished after elimination of CD16+ or of CD57+ cells. There was no significant change in NK function according to the clinical stage. The data show that circulating CD8+ HLA-DR+ CD57+ T cells in HIV+ subjects are activated cytotoxic T cells and point to progressive (over) activation of this T cell compartment until the onset of opportunistic infections.

We studied immune function in Belgian haemophiliacs treated with Factor VIII from volunteer donors. No patient had clinical evidence of immune deficiency. We found a decrease in T-helper cells (p less than 0.0005), in the ratio of T-helper over T-cytotoxic/suppressor cells (1.72 +/- 0.47 versus 2.24 +/- 0.82 in controls, p less than 0.005) and in lymphocyte responsiveness to mitogens (p less than 0.05). These findings could not be linked to the amount of F VIII received over the last year, the time since last F VIII administration, circulating immune complexes (54% positive patients, 7% positive controls, p less than 0.005), increased ALT levels, antibodies to cytomegalo -virus (85% of the patients, 45% of the controls, p less than 0.005), antibodies to Epstein-Barr virus, nor to the presence of HLA-DR 5 which was found in 56% of the haemophiliacs (20% of the overall Belgian population, p less than 0.005). Either F VIII induces long lasting immunological alterations unrelated to AIDS, or haemophilia is itself associated with such changes.

Stabilized aggregates of human IgG were prepared over a wide range of molecular weights. These fractions with increasing molecular weight were adjusted to the same molarity or the same protein concentration and were tested in the solid phase C1q and monoclonal rheumatoid factor immune complex assays. At constant molarity results were linearly correlated with the size of the aggregates. At constant protein concentration, results were also linearly correlated with the size of the aggregates in the lower molecular weight fractions, although the number of aggregates in the fractions decreased with increasing molecular weight. It is concluded that results in these assays can only be compared with respect to concentration if the immune complexes have identical sizes. Consequently, we studied the relative affinity of C1q and monoclonal rheumatoid factor for antigen/antibody immune complexes of different sizes or different antigen/antibody ratios, using model immune complexes composed of tetanus toxoid/anti-toxoid and streptolysine O/anti-streptolysine O. Compared to C1q, monoclonal rheumatoid factor was found to have higher affinity for smaller complexes, and for complexes with higher antigen/antibody ratio. Immune complex containing sera of 5 patients with connective tissue diseases, and one normal serum, were fractionated on a Sepharose 4B column. The binding patterns of the different fractions to solid phase C1q and monoclonal rheumatoid factor were quite variable and in only one of these sera monoclonal rheumatoid factor had the expected preference for small complexes and C1q for large complexes. Moreover a remarkably high binding to C1q and/or monoclonal rheumatoid factor of the monomeric IgG fraction was found in 4 out of 5 patient sera.

Soluble purified monoclonal and polyclonal rheumatoid factor, total serum complement, and soluble C1q all inhibit the detection of model tetanus toxoid/anti-toxoid immune complexes in the solid-phase C1q assay. The binding of these immune complexes to solid-phase monoclonal rheumatoid factor is less inhibited by soluble C1q and by total serum complement, but clearly decreased by soluble monoclonal or polyclonal rheumatoid factor. Serum complement does not reduce the size of these model complexes. We recommend the use of low ionic strength EDTA (10 mM) to partly neutralize the complement-mediated inhibition. This procedure is shown to be superior to currently used higher EDTA concentrations and to the use of IgG-Sepharose.

We studied the direct effects of the hormone calcitriol on the activation and proliferation of pure T lymphocytes and their subsets. Calcitriol inhibited the proliferation of T lymphocytes stimulated in the absence of monocytes with phytohemagglutinin (PHA) and either a monocytic culture supernatant or a combination of monocyte-derived interleukin 1 and interleukin 6. This inhibition was not influenced by the concentration of the stimulating agents. The minimal effective concentration of calcitriol was 10(-10) M. In contrast, the interleukin 2 (10 U/ml)-driven growth of PHA-stimulated T lymphocytes was not significantly altered by calcitriol at 10(-8) M. The hormone had also no influence on the T lymphocyte proliferation induced by a combination of PHA and the anti-CD28 monoclonal antibody 9.3. Pure T lymphocytes, after incubation for 5 days with PHA and monocytic factors, expressed a high level of transferrin receptors. This phenomenon was strongly suppressed on both CD4 and CD8 subsets when 10(-8) M calcitriol had been present during the culture. Moreover, the proliferation of pure CD4 cells was directly inhibited by calcitriol in similar conditions as for unseparated T lymphocytes. We conclude that T lymphocytes and their CD4 subset are direct targets for the inhibitory effect of calcitriol.

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.

Patients with multiple myeloma have transcortin levels lower than normal. This is due in essence to a subgroup of patients producing IGG heavy chains with lambda light chains. Patients producing IGG with predominantly kappa light chains have almost normal transcortin levels. On the other hand, the binding activity of the steroid binding beta globulin (SB beta G) of the kappa type of multiple myeloma is significantly higher than the steroid binding of the lambda type of multiple myeloma. The serum levels of vitamin D binding protein (DBP) fall in the normal range.