Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.     

Detection of IL-2 receptor positive tonsillar lymphocytes by monoclonal antibody and protein A coated erythrocytes

A sensitive rosette method combining staphylococcal protein A coated rabbit red blood cells and the monoclonal antibody anti-Tac was used to search for the presence of interleukin 2 (IL-2) receptor-bearing T lymphocytes in tonsils from patients with chronic tonsillitis. This method revealed the presence of Tac positive T lymphocytes in the tonsils whereas a conventional immunofluorescence technique was unable to do so. To demonstrate that Tac rosette formation resulted in a selective enrichment of IL-2 receptor-bearing T cells, we measured the proliferative response of these cells to exogenous IL-2. The response of Tac rosette positive cells to recombinant IL-2 was always higher than that of the Tac rosette negative or unselected cells, indicating that this rosette method specifically selects T cells expressing IL-2 receptor.     

IL-2-induced polyclonal proliferation of human peripheral blood lymphocytes: functional and phenotypic characteristics of proliferating cells

Unstimulated human peripheral blood lymphocytes (HPBL) were found to proliferate when cultured in vitro with interleukin-2 (IL-2). In bulk long-term cultures of HPBL cultured with IL-2, cell numbers usually doubled after 8-11 days of culture, and a 10-fold increase in cell number occurred between the second and third weeks of culture. These cells retained their ability to respond to a panel of T-cell dependent antigens, phytomitogens and allogeneic cells up to Day 21 of culture. The proliferating cells predominantly expressed the T-cell antigens (CD3, CD4 and CD8), but not antigens of natural killer (NK) cells, B cells or mononuclear phagocytic cells. The proportion of cells expressing CD3 and CD4 antigens progressively increased with length of culture. Purified lymphocytes expressing either CD4 or CD8 antigens were also found to be capable of showing a proliferative response to IL-2, especially when provided with autologous accessory cells. However, purified human peripheral blood B cells expressing the Leu 12 antigen did not respond with or without autologous accessory cells. Unlike the responses to phytomitogen, soluble antigens or allogeneic cells, the proliferative responses of HPBL to IL-2 were not inhibited by a monoclonal antibody (OK-Ia-1) to the non-polymorphic part of human class II histocompatibility antigens.     

Murine anti-human IL-6 monoclonal antibody prolongs the half-life in circulating blood and thus prolongs the bioactivity of human IL-6 in mice.

Human interleukin-6 (hIL-6) injected into mice increases antigen-specific antibody production. In the present study, we examined the possibility that anti-hIL-6 murine monoclonal antibody (MH-166) could neutralize this hIL-6 activity in vivo. Although MH166 completely neutralized hIL-6 activity in vitro, treatment in vivo with hIL-6 and MH166 combined unexpectedly increased both anti-dinitrophenyl (DNP) and anti-sheep red blood cell (SRBC) antibody production more than treatment with IL-6 alone did. To explain this phenomenon, the serum hIL-6 level was monitored following the MH166 administration. The hIL-6 level was significantly higher in the mice treated with both hIL-6 and MH166 than in the mice treated with hIL-6 alone during the 24 hr following hIL-6 administration. These results indicate that MH166 prolongs half-life of a xenogeneic hIL-6.     

Chicken-Activated-T-Lymphocyte-Antigen (CATLA) recognized by monoclonal antibody INN-CH 16 represents the IL-2 receptor

Monoclonal antibody INN-CH 16 recognizes a surface determinant exclusively present on activated chicken T lymphocytes. The present experiments suggest that this chicken activated T lymphocyte antigen (CATLA) represents the chicken IL-2 receptor or an associated structure, as (i) the kinetics of CATLA expression during T cell activation are analogous to those described for the mammalian receptor for IL-2, (ii) the IL-2 dependent proliferation of mitogen prestimulated chicken lymphocytes is competitively inhibited by INN-CH 16, and (iii) pretreatment of T lymphoblasts with INN-CH 16 drastically reduces their capacity to absorb IL-2 activity from supernatants of mitogen activated chicken lymphocytes.     

Adoptive immunotherapy with interleukin-2 (IL-2) results in diminished IL-2 production by stimulated peripheral blood lymphocytes

We examined the responses of peripheral blood lymphocytes (PBL) to a panel of T-cell mitogens in patients receiving adoptive transfers of tumor-infiltrating lymphocytes and continuous infusions of interleukin-2 (IL-2) for treatment of advanced cancer. All patients showed diminished proliferative responses to soluble and alloantigens, lectins, anti-CD3, and IL-2 during therapy. The non-major histocompatibility complex (MHC)-restricted cytolytic activities of PBL were increased by treatment and were further augmented by IL-2in vitro. The expression of 55-kd low-affinity IL-2 receptors (IL-2R) by PBL increased during treatment but functional IL-2R were simultaneously down-regulated. Proliferative responses were partially restored to pretreatment levels when PBL were costimulated with recombinant IL-2 and mitogens. Lectin stimulation of PBL produced little IL-2 secretion during treatment, while IFN-gamma secretion persisted. We conclude that infusions of IL-2 down-regulate the expression of functional IL-2R, decrease the secretion of IL-2, and lead to decreased mitogen responses by PBL.     

CD36 is rapidly and transiently upregulated on phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes. Analysis by a new monoclonal antibody (UN7)

Abstract: The monoclonal antibody (mAb) UN7, clustered as an anti-CD36 mAb, has been used to test the cell surface expression of CD36 on peripheral blood lymphocytes (PBL) following mitogenic stimulation. CD36, scarcely expressed on resting cell membranes, was rapidly upregulated on PBL after phytohemagglutinin (PHA) stimulation. The antigen was detected on the cell surface after 15 min of stimulation, increased rapidly by 60 min and peaked between 3 and 12 h, declining thereafter. The inhibition of protein synthesis by cycloheximide did not modify the PHA-induced expression of CD36. Neither the anti-CD3 0KT3 mAb nor the anti-CD2 BIL 2.29 and 9.1 mAbs induced any significant upregulation of the molecule. The addition of anti-CD28 15E8 mAb or IL-2 or IFN-γ to PHA or anti-CD3 or anti-CD2 mAbs did not influence the pattern of CD36 expression. The phorbol-2-myr-istate-13-acetate (PMA), alone or in combination with ionomycin, was unable to activate the expression of CD36, while it inhibited the PHA-induced upregulation. The PHA-induced upregulation of CD36 was partially inhibited by the addition of LY294002 or wortmannin, while not affected by that of calphostin C. Thus, CD36 was found to be early and transiently upregulated by PHA stimulation on PBL. The rapid modulation of the molecule was not related to new protein synthesis, but was probably due to the insertion into the plasma membrane of a presynthetized protein pool.     

Inhibition of lymphocyte proliferation by a monoclonal antibody (RFT2) against CD7.

The effect of the monoclonal antibody RFT2, directed against CD7, on T cell proliferation in unseparated peripheral blood mononuclear cell populations induced by various stimulants was investigated. The addition of RFT2 to cell cultures inhibited the T cell proliferation induced by tuberculin PPD and OKT3 but not by phytohaemagglutinin, concanavalin A or phorbol myristic acid; RFT2 had to be present during the first 24 h of culture in order to elicit inhibition; inhibition of proliferation was not due to down regulation of interleukin-2 receptor on the surface of T cells; and suppressive effects could be transferred by mononuclear cells pre-treated with RFT2. These results are of particular relevance in view of the known in vivo suppressive effect of RFT2 in humans.     

Characterization of a monoclonal rat anti-mouse interleukin 2 (IL-2) receptor antibody and its use in the biochemical characterization of the murine IL-2 receptor

Anti-murine interleukin 2 (IL-2) receptor monoclonal antibodies (mAb) were made from rats immunized with murine cytotoxic lymphocytes. One mAb, designated M7/20, strongly inhibited the proliferation of both IL-2 dependent CTLL-2 cells and concanavalin A (Con A)-induced T-cell blasts. Inhibition was linearly dependent on the concentrations of both M7/20 and IL-2. Utilizing FACS analysis, M7/20 was shown to bind selectively to mitogen-activated T lymphocytes and, to a lesser degree, to activated B lymphocytes. 125I-Labeled M7/20 binding assays indicated that 48-hr Con A-induced T-cell blasts possessed 89,000 binding sites/cell with a Kd of 1.2 X 10(-9) M. Competitive binding analyses indicated that M7/20 and IL-2 occupy the same or overlapping cell surface sites. Preliminary biochemical characterization of M7/20 immunoprecipitates of detergent extracts from both surface-iodinated and internally D-[3H]glucosamine-labeled T lymphoblasts indicated that the murine IL-2 receptor is an N-glycosylated 58,000-Da glycoprotein. Together these results suggest that mAb M7/20 binds at or near the IL-2-binding epitope on the murine IL-2 receptor and, thus, upon manipulation may act as an IL-2 agonist.     

Limiting dilution analysis of in vivo-activated (IL-2 responsive) peripheral blood lymphocytes in HIV-1-infected subjects

The progression of infection with human immunodeficiency virus, type 1 (HIV-1), is associated with a loss of helper T cell function, but the mechanism for this loss (e.g., decreased absolute number of helper cells, altered function of helper cells, or both) has not been delineated. Many studies have suggested that T-cell production of and/or responsiveness to the T cell growth factor interleukin-2 (IL-2) declines over the course of HIV-1 infection. Using a highly quantitative 6-day limiting dilution assay (LDA), we investigated whether the number and the proliferative capacity of circulating IL-2 responsive cells in patients with AIDS differ from those in patients in earlier stages of HIV-1 infection (asymptomatic or AIDS-related complex) and healthy seronegative individuals. The frequency of IL-2 responsive cells declined progressively in asymptomatic seropositive subjects, those with ARC, and those with AIDS. In contrast, the proliferative capacity of individual IL-2 responsive cells, as reflected by the magnitude of thymidine uptake per precursor, was reduced only in patients with frank AIDS and was normal in asymptomatic subjects and in those with ARC. These results suggest that the development of AIDS in the setting of HIV-1 infection may reflect a combination of qualitative as well as quantitative changes in lymphocyte function. They also suggest that analysis of lymphocyte responsiveness to IL-2 may provide a useful approach to prediction of the development of AIDS in individuals infected with HIV-1.     

HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals

T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5- tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.      

Trypanosoma cruzi-induced suppression of human peripheral blood lymphocytes activated via the alternative (CD2) pathway.

Coculture of blood forms of Trypanosoma cruzi with human peripheral blood mononuclear cells stimulated with anti-CD2(2) and anti-CD2(3) monoclonal antibodies, i.e., via an antigen-independent pathway of T-lymphocyte activation, resulted in marked immunosuppression compared with that in parallel cultures in which parasites were absent. This effect was evidenced by a decreased lymphoproliferative capacity, a significant reduction in the proportion of cells expressing interleukin-2 receptors, and a significant diminution in the cell surface density of this receptor.     

The effect of C-reactive protein on the PHA-induced proliferation of human peripheral blood lymphocytes

The effect of CRP purified from ascites fluid originated from cancer patients on human peripheral blood lymphocytes was investigated. CRP was purified using 4-step procedure including absorption on Sepharose 4B, phosphocholine-Sepharose, DE-52 ion exchange chromatography and gel filtration on Sephacryl S-200. CRP was able either to inhibit or to enhance PHA-induced lymphocyte proliferation in vitro. The effects of added CRP depended on the time of its addition, dose of CRP and lymphocyte donor.     

Prospective 5-year study of peripheral blood CD4, CD8, and CD19/CD20 lymphocytes and serum Igs in children born to HIV-1 women. The P(2)C(2) HIV Study Group

BACKGROUND: Peripheral blood CD4(+) and CD8(+) T cells, CD19(+)/20(+) B cells, and serum Igs are known to be altered by the progression of pediatric HIV-1 infection, but their evaluation as predictors of survival needs further definition. OBJECTIVE: To determine the natural history of these immune factors and their importance in predicting survival, we studied 298 HIV-1 vertically infected (HIV-1(+)) children over a 5-year period. METHODS: These immune factors and serum HIV-1 RNA levels were measured in two groups: (1) a birth cohort of children enrolled up to age 28 days postnatally, including 93 HIV-1(+) and 463 HIV-1 uninfected infants (HIV-1(-)), and (2) an older cohort of 205 HIV-1(+) children enrolled after the age of 28 days, who were classified as survivors or nonsurvivors. RESULTS: In the birth cohort HIV-1(+) children had significantly lower CD4(+) T-cell counts, higher CD8(+) T-cell counts, and lower CD19(+)/20(+) B-cell counts and higher IgG, IgA, and IgM levels than HIV-1(-) children. In the older cohort survivors had significantly higher CD4(+) and CD8(+) T-cell and CD19(+)/CD20(+) B-cell counts and higher IgG, lower IgA, and lower IgM levels than did nonsurvivors. In univariable analysis factors affecting survival in the older cohort were baseline CD4(+) and CD8(+) T-cell and CD19(+)/20(+) B-cell counts and IgG and HIV-1 RNA levels (all P <.05). In multivariable analysis high baseline CD4(+) T-cell count and low baseline HIV-1 RNA load remained important. CONCLUSION: The longitudinal mean profiles of CD4 and CD8 T-cell and CD19/20 B-cell counts and serum IgG levels helped to describe the natural progression of HIV-1 disease in children. However, only baseline CD4 T-cell count independently predicted survival.     

Increased expression of complement receptor type 1 (CR1, CD35) on human peripheral blood T lymphocytes after polyclonal activation in vitro

The receptor for C3b and C4b—complement receptor type 1 (CR1, CD35)—is present on a variety of cell types including erythrocytes, phagocytic cells, B lymphocytes and a small sub-population of T lymphocytes. The function of the receptor varies according to the different cell types, but a T lymphocytes the function is as yet not known. The present study concerns the influence of polyclonal stimulation on CR1-expressing T lymphocytes. Incubation with PHA resulted in a dose-dependent increase in the number of CR1-positive T lymphocytes. The CR1-expression T lymphocytes were found in both the CD4- and the CD8-positive subpopulation, but a significant stimulatory increase was only found in the CD4-positive population. A significant increase in the number of CR1-expressing T lymphocytes was found when monocytes were present during stimulation, indicating an importance of monocytes and/or monocyte products. However, the increase was not regulated by arachidonic acid metabolites of the cyclo-oxygenase pathway as indomethacin failed to inhibit the increase. Neither did rIL-Iα, rIL-1β, rTNFα nor rIL-6 alter the number of CR1-expressing T lymphocytes. The results of this study indicate a role for CR1 on T lymphocytes in the regulation of the immune system.     

A monoclonal antibody against the human IL-2 receptor binds to paraformaldehyde-fixed but not viable frog (Xenopus) splenocytes

Others have reported that a monoclonal anti-human IL-2 receptor antibody (anti-CD25) specifically binds a membrane receptor on Xenopus laevis PHA-induced and paraformaldehyde-fixed splenic blasts. In this paper, we present evidence suggesting that this binding is an artifact of membrane damage. Specifically, significant binding of anti-CD25 could only be achieved if the lymphoblasts were acid-washed and/or paraformaldehyde-fixed prior to being incubated with the fluoresceinated antibody. For example, in a representative experiment 95% of paraformaldehyde-fixed blasts, about 19% of acid-washed but not fixed blasts, but fewer than 2% of viable (untreated) blasts were positive for the CD25 epitope. Paraformaldehyde is known to alter membrane permeability. The DNA dye propidium iodide (PI) was used to demonstrate that the acid washing procedure also causes membranes to become permeable. Flow cytometric analyses of acid-washed PHA-induced splenic blasts doubly stained with the anti-CD25 antibody and PI showed that only 1.5% of the cells that were positive for CD25 did not stain with PI. Additionally, the anti-CD25 antibody, which immunoprecipitated a molecule from human lymphoblasts of between 50 and 60 kDa, did not immunoprecipitate any surface molecules from 125I-labeled Xenopus splenic blasts. Since binding of anti-CD25 to Xenopus splenic blasts appears to occur only after membrane damage, the antibody may be recognizing a cross-reactive internal epitope that is not involved in ligand binding on the cell surface.