P.Th.A. Schellekens

ASSOCIATION BETWEEN BIOLOGICAL PROPERTIES OF HUMAN IMMUNODEFICIENCY VIRUS VARIANTS AND RISK FOR AIDS AND AIDS MORTALITY

49 individuals seropositive for human immunodeficiency virus (HIV) antibody were studied longitudinally for the relation between in-vitro properties of their sequential HIV isolates and clinical course before and after the development of AIDS. They were classified into three groups according to the syncytium-inducing capacity, replication rate, and host range of their HIV isolates. The most rapid progression to AIDS (median 15 months) and the lowest survival rate following AIDS diagnosis (median survival 12.5 months) were observed in individuals with high-replicating, syncytium-inducing HIV isolates, followed by individuals with high-replicating, non-syncytium-inducing isolates. In contrast, most individuals with low-replicating, non-syncytium-inducing HIV isolates remained symptom-free during the study period (median follow-up until AIDS diagnosis greater than 42 months), and the few individuals from this group in whom AIDS developed were still alive at the end of the study period (median survival greater than 34 months). In addition, AIDS patients from the three groups differed with respect to their symptoms. Zidovudine treatment in the symptom-free period seemed to delay the onset of AIDS in all risk groups, although stabilisation of CD4+ cell numbers was observed only in individuals with non-syncytium-inducing HIV variants.

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.

Activation of resting T cells has been proposed to purge the reservoir of HIV-1-infected resting CD4+ T cells. We therefore treated three HIV-1-infected patients on antiretroviral therapy with OKT3, a CD3 monoclonal antibody, and recombinant human IL-2. Here we report the profound and partially long-lasting host responses induced by the OKT3 and IL-2 treatment. OKT3/IL-2 induced a strong but transient release of plasma cytokines and chemokines. The percentage CD4+ and CD8+ cells in the blood expressing the activation marker CD38 transiently increased to almost 100%, and in lymph nodes we “observed” a 10-fold increase in the number of dividing Ki67+ cells and increased numbers of apoptotic cells. Following OKT3/IL-2 treatment, a long-lasting depletion of CD4+ cells in the peripheral blood and lymph nodes occurred, suggesting the physical deletion of these cells. Increases in CD4+T cell numbers during the two year followup period were due mainly to increased memory cell numbers. CD8+ cells were also depleted in the blood, but less severely in lymph nodes, and returned to baseline levels within several weeks.

Administration of prednisolone in vivo affects the ratio of OKT4/OKT8 and the LDH-isoenzyme pattern of human T lymphocytes.

Oral administration of prednisolone (in single doses of 10, 30, or 60 mg) to healthy volunteers was found to affect the T lymphocytes in the blood with regard to binding of monoclonal antibodies and lactate dehydrogenase isoenzyme pattern. The findings indicate that these effects are dependent on the dose of the drug and the time after the administration of the drug. Prednisolone induces a T lymphocytopenia in the peripheral blood that affects OKT4-positive lymphocytes more than OKT8-positive lymphocytes, resulting in a slight decrease in the ratio OKT4/OKT8. Moreover, the lactate dehydrogenase isoenzyme pattern changes, resulting in a decrease of the H/M ratio of this enzyme. The proliferative responses of peripheral blood lymphocytes are not affected after a single dose of 10 mg. However, after administration of either 30 or 60 mg of prednisolone, the proliferative responses are decreased to a different extent, depending on the stimulus used. In vitro experiments are presented showing that any effect of prednisolone on nonstimulated lymphocytes is reversible. Based on the observed changes in OKT pattern and lactate dehydrogenase isoenzyme profile of the T lymphocytes induced by administration of prednisolone, it is concluded that the drug induces a temporary depletion from the peripheral blood, preferentially of high-reactive T lymphocytes. As a consequence, the peripheral blood compartment is enriched for T lymphocytes with a low H/M ratio of lactate dehydrogenase isoenzymes, known to be less reactive to proliferative stimuli.

Failure of lymphocyte-membrane HLA-A and -B expression in two siblings with combined immunodeficiency.

A diagnosis of partial combined immunodeficiency was made in two Turkish siblings with a history of multiple pyogenic infections and persistent candidiasis. They demonstrated severe hypo-γ-globulinemia, with B-lymphocytes, but deficient plasma cell differentiation. T-Lymphocytes were decreased in number and did not respond to antigens, but did proliferate in cultures with lectins and allogeneic cells. HLA-A and -B determinants were not detected on blood lymphocytes, but they were expressed by cultured lymphoblasts, cultured fibroblasts, and were present in serum. MLR-Stimulatory capacity was intermediate and only two of six anti-HLA-DRw7 antisera demonstrated B-cell reactivity. β-2-Microglobulin (B2M) was not detected on the surface of T-lymphocytes, but was found in cross-sectioned T-cell membranes. B-lymphocytes carried B2M normally. The absence of HLA-A and -B determinants on lymphocytes of patients with similar immunodeficiency syndromes suggests a role for HLA determinants in lymphocyte differentiation.

Separation of human lymphocyte subpopulations: I. Transformation characteristics of rosette-forming cells

Abstract Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC). In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL. In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis. It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.

In Vitro Reactivity of Human Lymphocytes after Cryopreservation Using a Programmed Cooling Device

Abstract. In this article a technique is described for the cryopreservation of human lymphocytes employing a programmed cooling device. A preset temperature curve was used which compensated for the heat exchange during crystallization. The influence of this method of lymphocyte preservation on a variety of the in vitro functions of these cells was evaluated by means such as the lymphocyte transformation test, CML and investigations for lymphocyte membrane markers. All the lymphocyte functions that were tested were found to be retained in full.

Low T cell reactivity to combined CD3 plus CD28 stimulation is predictive for progression to AIDS: correlation with decreased CD28 expression

In 219 HIV‐1‐infected men of the Amsterdam cohort we measured CD4+ T cell numbers and in vitro T cell responses to CD3 MoAbs with or without CD28 costimulation and phytohaemagglutinin (PHA). The value of these markers was estimated for disease progression within 4 years. CD28 expression on T cells has been related to T cell responses. CD28 costimulation considerably enhanced T cell reactivity (≈8–10‐fold) with lower coefficients of variation compared with reactivity to CD3 MoAb alone (median 5 versus 20). T cell reactivity to CD3 plus CD28 MoAb was decreased during HIV‐1 infection and was besides CD4+ T cell numbers the only independent predictor for progression to AIDS. Compared with the group with high CD4+ T cell numbers the relative risk (RR) for the group with intermediate levels was 2.28, with low levels 5.20. In the groups with intermediate and low CD3 plus CD28 responses the RR was 2.04 and 4.16, respectively. The combined RR for both was 4.65 and 21.63. The independence of this marker was confirmed when the group with low CD4+ T cell numbers was subdivided into groups with high, intermediate and low T cell responses. The expansion of CD8+CD28− T cells was already apparent in HIV− homosexual men, but CD8+CD28+ T cells specifically decreased in patients with AIDS. CD28 expression on T cells correlated moderately with T cell responses to CD3 plus CD28 MoAb. T cell reactivity to CD3 MoAb in the presence of CD28 MoAb is a stronger prognostic marker than T cell reactivity to CD3 MoAb alone.

Prednisolone and cyclosporin a exert differential inhibitory effects on T-cell proliferation in vitro

To elucidate the mechanisms of action of prednisolone (pred) and cyclosporin A (CyA), we have investigated the effects of these immunosuppressive drugs on the proliferative response of human peripheral blood lymphocytes (PBMN) induced by various stimulants, well defined with regard to their monocyte dependence. We found that pred-induced inhibition of monocyte-dependent proliferative responses could be reversed by the addition of exogenous interleukin 2 (IL-2). Monocyte-independent proliferative responses were not affected by pred. These findings suggest that pred inhibits IL-2 production and subsequent lymphocyte proliferation at the level of the signal derived from the monocyte. In contrast, CyA inhibited monocyte-dependent as well as monocyte-independent proliferative responses of human PBMN. This inhibition could not be reversed by the addition of exogenous IL-2. We have previously demonstrated that CyA does not affect the expression of receptors for IL-2. Taken together, these data indicate that CyA-mediated inhibition does not primarily occur at the level of the IL-2 system, but rather affects the proliferative mechanism of the T cell itself. These data clearly demonstrate that pred and CyA act at distinct sites of the triggering process.

Human Cytotoxic Lymphocytes after Alloimmunization in vitro

The development of short-term cell culture systems has greatJy contributed to our insight into cell-mediated immunity. It has become apparent that after in vivo immunization, lymphoid cells develop which are, as effector cells, capable, without the help of antibody or complement, of destroying tissue culture target cells which carry the same antigen(s) to which the donor of the lymphoid cells has been immunized. Such cell-mediated cytotoxicity has been demonstrated in allograft immunity, graft versus host (GVH) reactions, delayed type hypersensitivity, autoimmunity and tumor immunity. More recently, it has become apparent that in vitro immunization of normal lymphoid cells to certain antigens can also occur when the cells are co-cultured with certain stimulator cells which carry these antigens. Such in vitro immunized effector cells display a similar cytotoxicity on target cells carrying antigens in common with the stimulator cells. This article is confined to our studies with human peripheral blood lymphocytes, in relation to the development of in vitro cytotoxicity to alloantigens after immunization in vitro. Hirschhom et al. (1965) were the first to report that human lymphocytes, co-cultured with allogeneic fibroblasts, developed a cytotoxic potential towards these cells. When cells of a human lymphoid cell line are being used as stimulators in a mixed lymphocyte culture (MLC) and also as target cells in a subsequent cytotoxicity assay, a similar target cell destruction is observed

Etiopathogenetic Studies in a Patient with Whipple’s Disease

A patient is presented with Whipples disease. Before treatment, Haemophilus influenzae type e, sensitive to tetracycline was cultured from multiple small intestinal biopsies. This isolated micro-organism was structurally similar to the one observed in the tissue. All further culture experiments during and after treatment proved negative except for one biopsy from which a tetracycline-resistant H. influenzae type-e mutant was isolated. The immunological disturbances, mainly characterized by cutaneous anergy, in absence of major humoral or in vitro lymphocytic impairment, regressed during treatment together with clinical remission of the disease. These findings are considered in favour of the secondary nature of the immunological abnormalities.