Lymphocyte activation in HIV-1 infection. I. Predominant proliferative defects among CD45R0+ cells of the CD4 and CD8 lineages.

OBJECTIVES AND DESIGN: The proliferative defects of CD4 and CD8 cells taken from 474 HIV-1-seropositive individuals during various stages of disease were quantitated. Phytohaemagglutinin (PHA) and soluble anti-CD3 were used in optimal mitogenic concentrations in the presence of recombinant interleukin-2 (rIL-2) and conditioned medium, and the proliferation of cells from HIV-1-seropositive donors was assessed in co-culture with HIV-1-seronegative cells in order to exclude effects of cytokine deficiency. Defects within the CD45RA+ ('unprimed') and CD45R0+ ('primed') T-cell populations were also investigated. METHODS: Quantitative immunofluorescence and double and triple labelling in flow cytometry were performed for (1) CD25 (IL-2 receptor alpha chain) expression, (2) lymphocyte and T-cell survival, and (3) blast transformation and proliferation--in relation to the original input of cells for each subpopulation. RESULTS: T cells from normal and HIV-1-seropositive donors were CD25+ at day 1. In HIV-1-seropositive patients a variable number of CD4 and CD8 lymphocytes failed to further increase CD25, and died as a sign of activation-associated lymphocyte death (AALD). Forty-two per cent of asymptomatic subjects, including 32% of those with CD4 cell counts > 400 x 10(6)/l, showed a poor blast transformation (< 30% blasts). Cells from HIV-1-seropositive donors showed poor blast responses when co-cultured with HIV-1-seronegative cells; both CD4 and CD8 cells were handicapped. In asymptomatic HIV-1-seropositive people T cells with the CD45R0+ RA- ('primed') phenotype were three to five times more vulnerable to AALD than the CD45RA+ RO- ('unprimed') cells. In patients in Centers for Disease Control and Prevention (CDC) disease stage IV both CD45R0+ and -RA+ populations were severely affected. CONCLUSIONS: This is the first quantitative analysis to demonstrate that in HIV-1 infection mitogen-stimulated CD45R0+ ('primed') T cells preferentially die upon activation. Both the CD4 and CD8 lineages are affected, as seen in animal models of graft versus host disease. AALD may explain defects of immunological memory. The analysis of AALD may be a suitable assay for studying whether antiviral drugs influence the proliferative responses of lymphocytes.

Isoprinosine potentiation of human peripheral blood mononuclear cell response to mitogens: kinetics and effect on expression of the IL-2 receptor and the activity of interleukin 2.

Isoprinosine, a synthetic purine derivative, enhanced the proliferative response of peripheral blood mononuclear cells to the lectin phytohaemagglutinin, and to the monoclonal antibody OKT3. The drug did not potentiate the activation of cells following oxidation by sodium periodate. Isoprinosine had no effect on the expression of receptors for interleukin-2. Increased interleukin-2 activity was detected in three out of nine supernatants from PHA activated mononuclear cells which were cultured in the presence of isoprinosine. Kinetic experiments involving addition or removal of the drug at various time intervals indicated that potentiation of the proliferative response required the presence of the drug at both early and late stages of cell activation. Inhibition of mononuclear cell activation by cyclosporine A was not affected by isoprinosine. These results suggest that enhancement of mononuclear cell proliferation by isoprinosine involves events in both early and late stages of the activation cycle.

Defective dendritic cell accessory function in NZB/W F1 hybrid mice.

The proliferative response of NZB/W F1 hybrid mice when activated by sodium periodate (NaIO4) was found to be markedly defective as compared to the response in control NZW mice. This defect was observed in both 4 month old female mice and in 8-10 month old male mice. To determine whether the defect was an intrinsic T cell defect or an accessory cell defect, splenic dendritic cells (DC) were purified and their ability to activate enriched T cells after NaIO4 stimulation was assessed. In mixing experiments it was observed that normal NZW dendritic cells could restore the response of NZB/W F1 hybrid T cells whereas addition of NZB/W F1 dendritic cells to NZW T cells resulted in defective 3H-thymidine incorporation after NaIO4 stimulation. These results indicate that accessory DC of NZB/W F1 mice are defective and unable to support T cell responses to the mitogen NaIO4.

Low specific IgE, IgG and lymphocyte reactivity in a group of patients developing anaphylaxis following a honey-bee sting.

Ten patients who developed severe generalized reactions following a honey-bee sting were investigated for the presence of specific IgE and IgG antibodies, and for lymphocyte reactivity following in-vitro honey-bee venom (HBV) stimulation. Five of the patients (high responders) showed high HBV-specific IgE and IgG levels, whereas the other five patients (low responders) showed low HBV-specific IgE and IgG levels. Mononuclear cells from the high responder group incorporated significant amounts of 3H-thymidine when activated with pure bee venom, whereas insignificant lymphocyte proliferation was observed in the low-responder group. It is concluded that, amongst HBV-sensitive patients, a group of low responders exists in whom the mechanism of anaphylaxis cannot be explained.

A double-blind study comparing monomethoxy polyethylene glycol-modified honeybee venom and unmodified honeybee venom for immunotherapy. I. Clinical results.

Thirty-five patients were allocated at random to immunotherapy (IT) in a double-blind way with either monomethoxy polyethylene glycol (mPEG)-modified honeybee venom (HBV) or HBV. The two groups were well matched regarding age, sex, skin sensitivity, HBV-specific serum IgE and IgG antibodies, and history of reactions after a field sting; mPEG-HBV-treated patients received doses that increased more steeply than doses of the HBV-treated patients. The maintenance dose of the former group (200 micrograms) was greater than that of the latter group (100 micrograms). During IT, both groups had the same frequency of local swellings after injections. Four patients receiving mPEG-HBV developed one mild systemic reaction (SR) during dose increase, whereas 10 patients receiving HBV demonstrated one or more of these reactions, compelling two patients to stop therapy. Following challenge with a honeybee sting after about 14 weeks of IT, six patients with SR were observed, four in the mPEG-HBV-treated group and two in the HBV-treated group. In the HBV-treated group, three patients were not challenged, one because of an insufficient IgG increase and two other patients because they dropped out of IT before reaching maintenance dose because of repeated SRs. Since the mPEG-HBV is extremely well tolerated during IT and the success rate is not significantly lower than with unmodified HBV, we suggest it as an attractive alternative to HBV for the treatment of HBV hypersensitivity. Increase of the maintenance dose may result in an even better clinical efficacy.

Depressed 3H-thymidine incorporation by measles infected mononuclear cells can be corrected by treatment with isoprinosine or 5-fluoro-2-deoxyuridine.

Incorporation of thymidine by measles infected PHA-activated lymphocytes was found to be depressed although production of interleukin 2 (IL-2) and expression of IL-2 receptors on these cells was similar to that of non-infected cells. The decrease in incorporation of 3H-thymidine by infected cells was not due to cell death and could be restored by treating the cells with isoprinosine or 5-fluoro-2-deoxyuridine. These results suggest that the depressed incorporation of 3H-thymidine by measles-infected cells is not due to inhibition of early events required for lymphocyte proliferation, but is rather due to interference in the thymidine pathway required for the synthesis of DNA.

Lack of responsiveness of beekeeper mononuclear cells to in vitro stimulation with pure bee venom.

Lymphocyte proliferation activity after in vitro bee venom (BV) stimulation was examined in a group of patients allergic to bee stings and in a group of beekeepers. Although the allergic patients responded strongly to increasing doses of BV, the beekeepers demonstrated no proliferative activity and an inability to produce interleukin-2 after BV stimulation. Removal of adherent cells or various populations of suppressor cells, including T gamma cells and OKT8 positive cells, did not influence the cellular unresponsiveness of cells of beekeepers after BV stimulation. Furthermore, cells of beekeepers, when they were trypsinized or when they were preincubated for 72 hours, did not proliferate after BV challenge. It is concluded that the lack of proliferation of lymphocytes of beekeepers and the inability to produce interleukin-2 is not due to a suppressor mechanism or to the presence of anti-idiotype antibodies coating the surface of lymphocytes of beekeepers. The mechanism behind the failure of cells of beekeepers to proliferate remains unclear.

Defective monocyte function in patients with systemic lupus erythematosus.

Peripheral blood adherent cells from patients with systemic lupus erythematosus (SLE) were shown to have markedly reduced phagocytic activity as compared to normal adherent cells or those from non-SLE patients receiving corticosteroid therapy. Both resting and phagocytosing monocytes showed decreased hexose monophosphate shunt and glycolytic activity. Mononuclear cells from SLE patients showed grossly impaired proliferative activity after NaIO4 activation. Furthermore, addition of SLE adherent cells to normal adherent cell-depleted lymphocytes decreased [3H]thymidine incorporation of the latter cells following NaIO4 treatment. Addition of normal adherent cells to SLE lymphocytes corrected the previous defect, indicating that an adherent abnormality is responsible for the defect in SLE mononuclear cell proliferation to NaIO4 activation.

Modulation of the sodium periodate (NaIO4) response in systemic lupus erythematosus (SLE): effect of interleukin-1 (IL-1), interleukin-2 (IL-2), phorbol myristate acetate (PMA), and indomethacin.

The proliferative activity of peripheral blood mononuclear cells (PBMN) from patients with systemic lupus erythematosus (SLE) activated by sodium periodate (NaIO4) is greatly diminished. The effect of the cytokines Interleukin-1 (IL-1) and Interleukin-2 (IL-2) on the NaIO4 reaction was investigated. Addition of IL-1 resulted in partial restoration of the reaction of SLE PBMN to NaIO4, and a similar effect was demonstrated in the presence of phorbol myristate acetate (PMA). Addition of IL-2 to NaIO4 activated SLE PBMN, however, caused a marked improvement in their proliferative activity. The presence of indomethacin resulted in only a slight increase in [3H]thymidine incorporation by the NaIO4-treated SLE cells. The results suggest that the defect in the response of SLE PBMN cells to NaIO4 is due to inadequate availability of IL-1 and IL-2. Excessive production of prostaglandin in SLE might also contribute to the defective response to NaIO4 but does not appear to play a major role.

Suppression of interleukin-2 production by human concanavalin A-induced suppressor cells.

When PHA-activated normal responder cells (R cells) were cocultured with mononuclear cells (MN cells) which had been preincubated for 48 hr in medium alone (C cells) an enhanced proliferative response was observed. This enhancement was only obtained when the R cells were cultured with allogeneic C cells or when PHA was in the cocultures for the entire culture period. This effect was due to greater production of interleukin 2 (IL-2) by irradiated C cells in the presence of allogeneic or mitogenic stimulation. Con A-treated mononuclear cells (S cells) cultured with PHA-activated allogeneic or autologous responder cells showed reduced [3H]thymidine incorporation and IL-2 production as compared to activated R cells alone. Glutaraldehyde-treated S cells (which retained the ability to absorb IL-2) did not affect the proliferative response or IL-2 production by the R cells, indicating that passive absorption of IL-2 was not entirely responsible for suppression induced by S cells. S cells, pretreated with IL-2, still inhibited R-cell activity. These results show that Con A-treated MN cells suppressed or prevented [3H]thymidine incorporation by actively inhibiting IL-2 production.

Mononuclear cell function in Mycobacterium tuberculosis infected guinea pigs.

In this study mononuclear cell function was studied in the lymph glands, spleen, and peripheral blood of Mycobacterium tuberculosis infected guinea pigs. Adherent cells from draining lymph nodes and spleens of infected animals spontaneously produced a factor which inhibited normal lymphocyte proliferative responses. As it has previously been shown that this factor activates a population of suppressor T cells, resident lymphocytes in the lymph nodes and spleen were examined and were shown to inhibit normal lymphocyte functions. It is suggested that adherent cells ingesting M. tuberculosis spontaneously release a suppressor cell activating factor (SCAF) which locally activates lymphocytes to become suppressor cells. Even at a time of overwhelming infection, peripheral blood adherent cells could not be shown to release SCAF and peripheral blood suppressor cells could not be identified. Although peripheral blood lymphocyte proliferative responses to PHA were normal in infected animals, their ability to produce the lymphokine macrophage inhibition factor was considerably reduced after the second week of infection. This dissociation between lymphocyte proliferation and lymphokine production is similar to that previously described in humans overwhelming tuberculosis.

The effect of hydrocortisone (HC) on sodium periodate and phytohemagglutinin-induced [3H]thymidine incorporation and lymphokine production by human lymphocytes.

Hydrocortisone (HC) in pharmacologically attainable concentrations was shown to inhibit mitogen-induced lymphocyte blastogenesis. When adherent and nonadherent cells were treated separately with hydrocortisone and then reconstituted, treatment of either cell population resulted in a diminished mitogen-activated response. HC-treated adherent cells produced less interleukin 1 (IL-1) than control cells, and interleukin 2 (IL-2) production by HC-treated lymphocytes was also reduced. This latter finding could not be reversed by adding IL-1-containing adherent cell supernatants to the culture systems. Leukocyte inhibiting factor (LIF) production by sodium periodate-activated mononuclear cells was also reduced after HC treatment, but could be corrected by adding IL-1-containing adherent cell supernatants to the cultures. PHA-induced LIF production, which is less dependent upon adherent cells and their products, was not affected by HC treatment. It is concluded that HC exerts its suppressive effect by independently inhibiting IL-1 production by adherent cells and IL-2 production by lymphocytes.

Laboratory evaluation of commercial interferon preparations.

The antiviral, antiproliferative and natural killer-cell (NKC) stimulatory activities of four commercial therapeutic interferon preparations were assayed in our laboratory. The antiviral and antiproliferative activities of each preparation were relatively similar, but an unexpectedly high NKC stimulatory activity was found in one of them. In-house determination of antiviral activity and evaluation of the antiproliferative and NKC stimulation potential of interferon preparations are essential before rational clinical trials of this agent are carried out.

Induction of suppressor cells after activation of human peripheral blood mononuclear cells with sodium periodate.

Pretreatment of normal human peripheral blood mononuclear cells (MN) with sodium periodate (NaIO4) resulted in the induction of suppressor cells. The mitogenic response of fresh allogeneic and autologous cells to phytohaemagglutinin (PHA), Concanavalin A (Con A), pokeweed mitogen (PWM), various antigens and in mixed lymphocyte culture was suppressed when NaIO4 pretreated cells were present. PWM-induced plaque-forming-cell responses were also suppressed by NaIO4-pretreated cells. Treatment of cells with mitomycin C before the NaIO4 treatment abolished the suppressive activity. A ratio of 1 : combination that resulted in the strongest suppression. Supernatants from NaIO4-pretreated cell cultures were not inhibitory.

Mechanism of suppression of lymphocyte proliferation by Concanavalin A-activated mononuclear cells.

In this study, the mechanism by which Concanavalin A (Con A) pre-treated lymphocytes suppress mitogen-induced proliferation of responder cells was investigated. Responder-cell proliferation could only be suppressed when these cells were co-cultured with the Con A pre-treated cells but not with their supernatants, nor in chambers where the suppressor cells were separated from the responder cells by a millipore filter. Suppression could not be mediated by heat-killed Con A-activated cells or lysates from these cells. Trypsinization of the Con A-induced suppressor cells resulted in loss of suppressor activity which could be restored if the cells were allowed to recover overnight. Trypsinization of the responder cells, however, before their co-culture with the Con A pre-treated cells did not affect suppression. Addition of cytochalasin B to the co-culture resulted in reduced suppression and cycloheximide treatment of the suppressor cells abolished their activity. The results indicate that for optimal suppression to occur, cell-to-cell contact is required and viable, intact Con A-inducible suppressor cells, actively synthesizing protein are essential. Furthermore, suppression may be mediated via a membrane receptor on the suppressor cell as manipulations of normal membrane function may abolish suppression.

Hyperimmunoglobulinaemia associated with absent suppressor cells, aplastic anaemia and trisomy 8.

A child with aplastic anaemia and trisomy 8 in bone marrow cells was observed to have extremely high levels of circulating immunglobulins. Concanavalin A (con A)-inducible suppressor cells were assessed in the patient's peripheral blood by their ability to suppress both the proliferative response of normal allogeneic cells activated in a two-way mixed lymphocyte culture with and without added phytohaemagglutinin (PHA) and the ability of normal allogeneic cells to produce the lymphokine leucocyte inhibitory factor when activated by PHA. Con A consistently failed to induce suppressor cells from the patient but not from control lymphocytes. It is suggested that the lack of suppressor cells may account for uncontrolled B-cell proliferation and associated hypergammaglobulinaemia.

Effects of levamisole on in vitro suppressosr cell function in normal humans and patients with systemic lupus erythematosus.

Levamisole-treated mononuclear cells from normal volunteers or from patients with systemic lupus erythematosus (SLE), when cultured with normal allogeneic lymphocytes, did not significantly influence the ability of the latter cells to proliferate when stimulated with either phytohemagglutinin (PHA) or concanavalin A (Con A). Levamisole, furthermore, did not influence the ability of Con A to induce suppressor cell function from normal mononuclear cells. When mononuclear cells from patients with SLE were treated with therapeutic concentrations of levamisole in the presence of Con A, the drug was unable to restore the ability of the Con A to induce normal suppressor cell function. SLE cells treated with a high concentration of levamisole (60 micrograms/ml) in the presence of Con A did show some suppressor cell activity on normal autologous mononuclear cells activated by Con A, but not by PHA. Although the significance of this latter finding is unclear it appears that levamisole at therapeutic concentrations does not influence in vitro suppressor cell function of normal or SLE mononuclear cells.

Desensitization of patients with bee sting allergy using pure bee venom.

Forty patients who had previously experienced severe systemic reactions after a bee sting were desensitized using pure bee venom. A modified 'Rush' regimen was employed whereby patients received two injections a week and reached maximal desensitization in 5 weeks. Eleven patients have subsequently been stung again and have developed no generalized reaction. Although this form of desensitization is considered to be highly effective in protecting sensitive patients, both generalized and local side-effects were frequent. Maintenance desensitizing injections are required every month for an indefinite period. It is concluded that desensitization with pure been venom should be undertaken only in highly selected sensitive patients, and should be performed under strict control.