Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.

The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.

Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes.

Peripheral blood monocytes can be induced by stimuli such as bacterial lipopolysaccharide (LPS) to secrete an array of cytokines. We have studied the effects of interleukin 7 (IL-7) on human peripheral blood mononuclear cells (PBMC) and found that IL-7 is a relatively potent inducer of IL-6 secretion IL-6 protein levels were determined either by the B9 hybridoma growth factor assay or by enzyme-linked immunosorbent assay, and mRNA for IL-6 was analyzed by Northern hybridization. Detailed examination revealed that, among PBMC, monocytes, rather than lymphocytes, were secreting IL-6 in response to IL-7. In contrast to the low concentrations of IL-7 required to stimulate T cell growth and differentiation (as low as 0.1 ng/ml), relatively high concentrations of IL-7 were necessary to induce IL-6 secretion by monocytes (at least 10 ng/ml). An optimal concentration of IL-7 (100 ng/ml) induced monocytes to secrete 10-fold more IL-6 than an optimal concentration of IL-1 beta (10 ng/ml), and almost as much as LPS. However, significantly more IL-7 than IL-1 beta was required to induce detectable levels of IL-6. The kinetics of IL-6 secretion by monocytes were identical in response to IL-7, IL-1 beta, or LPS, with IL-6 protein detectable in culture supernatants as early as 2 h after the initiation of culture. IL-4 was found to markedly inhibit the ability of IL-7 or LPS to induce IL-6 mRNA and IL-6 secretion. In addition to promoting IL-6 production, IL-7 induced the secretion of immunoreactive IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by monocytes. IL-7 also induced monocyte/macrophage tumoricidal activity against a human melanoma cell target, an activity that may be related to the secretion of IL-1 alpha, IL-1 beta, and TNF-alpha. Finally, we used a whole blood culture system as a bridge to in vivo analysis to demonstrate that IL-7 induces cytokine secretion in the absence of culture medium, fetal calf serum, and adherence to plastic. Our data suggest that IL-7, in addition to regulating lymphocyte growth and differentiation, has potent effects on cells of the monocytic lineage. Thus, IL-7 may be an important mediator in inflammation and in the macrophage immune response to tumors.

CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40.

CD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or interferon gamma (IFN-gamma) resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-alpha and IL-6 production in the presence of GM-CSF, IL-3, or IFN-gamma, and enhanced IL-8 production stimulated by GM-CSF or IL-3. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human melanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.

Fas transduces activation signals in normal human T lymphocytes.

The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.

Characterization of human CD8+ T cells reactive with Mycobacterium tuberculosis-infected antigen-presenting cells.

Previous studies in murine models, including those using the beta2 microglobulin knockout mouse, have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). At present, little is understood about these cells in the human immune response to tuberculosis. This report demonstrates the existence of human Mtb-reactive CD8+ T cells. These cells are present preferentially in persons infected with Mtb and produce interferon gamma in response to stimulation with Mtb-infected target cells. Recognition of Mtb-infected cells by these CD8+ T cells is restricted neither by the major histocompatibility complex (MHC) class I A, B, or C alleles nor by CD1, although it is inhibited by anti-MHC class I antibody. The Mtb-specific CD8+ T cells recognize an antigen which is generated in the proteasome, but which does not require transport through the Golgi-ER. The data suggest the possible use of nonpolymorphic MHC class Ib antigen presenting structures other than CD1.

Fas ligand mediates activation-induced cell death in human T lymphocytes.

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

B-cell stimulation.

Recent studies have identified CD40 ligand (CD40L) as the critical membrane-expressed molecule responsible for T cell dependent B-cell activation. CD40L co-operates with various cytokines to induce B-cell activation, proliferation, and immunoglobulin isotype switching. Some antigens, however, can also stimulate B-cell activation and isotype switching in the absence of CD40L or T cells. Recent studies have suggested that cytokines derived from non-T cells, such as natural killer cells, macrophages and mast cells, are responsible for isotype switching in T cell independent responses.

Induction by interleukin-7 of lymphokine-activated killer activity in lymphocytes from autologous and syngeneic marrow transplant recipients before and after systemic interleukin-2 therapy.

Therapy with recombinant lymphokines after autologous bone marrow transplantation (ABMT) is being explored as a way to prevent relapse. Lymphokine therapy may exert an antitumor effect through a variety of mechanisms, including the induction of lymphokine-activated killer (LAK) cell cytotoxicity. We tested the ability of interleukin-7 (IL-7) to induce LAK cytotoxicity in peripheral blood mononuclear cells (PBMC) from healthy subjects and from patients early after ABMT. LAK activity was defined as lysis of Daudi by PBMC after incubation with IL-7 at 10 to 100 ng/mL or IL-2 at 1000 U/mL. PBMC from four healthy subjects were cultured with either IL-7 or IL-2. IL-7 induced LAK activity in two of the four, whereas IL-2 induced LAK activity in all four. The median percent lysis (effector-to-target ratio [E:T] 40:1) with IL-7 (23%) was lower than with IL-2 (67%). PBMC were obtained from 15 patients 27 to 84 days after autologous (n = 13) or syngeneic (n = 2) bone marrow transplantation (BMT) and tested for IL-7-induced LAK activity. Eleven exhibited significant activity (10% to 77% lysis at E:T 40:1). In contrast to the results in PBMC from normal subjects, in PBMC from ABMT patients IL-7 induced LAK activity of a magnitude similar to that induced by IL-2. Studies were also performed on PBMC from eight patients who had received IL-2 after ABMT (3.0 x 10(6) U/m2/d) for 4 days by continuous intravenous (IV) infusion. In seven of the eight patients, IL-7 induced significant LAK activity, which was higher than that seen in PBMC from ABMT patients who had not received IL-2. Thus, IL-7 reproducibly induced significant LAK activity in cells obtained early after autologous or syngeneic BMT. Indeed, such LAK activity was comparable quantitatively to that induced by IL-2. Finally, IL-7 induced an even greater LAK activity in vitro in PBMC obtained after ABMT and preactivated in vivo by IL-2 therapy. The results suggest that IL-7 may have a potential immunotherapeutic role, alone or with IL-2, after ABMT.

Prospects for a better vaccine against tuberculosis.

There have been many new promising approaches to developing human vaccines against tuberculosis (TB). Advances in gene and antigen identification, availability of genome sequences, a greater understanding of immune mechanisms in resistance to TB, the development of adjuvants and delivery systems to stimulate T-cell immunity, and increased funding from public and private agencies are some of the reasons for progress in this area. Dozens of vaccine candidates have been tested in animal models in recent years, and several of these are poised to move into clinical trials in the next several years. Thus, there is renewed optimism for the potential of developing new and improved TB vaccines.

Validity of the British system for anticoagulant control using the national reagent.

The national system for anticoagulant control depends on drawing the best line obtained by visual comparison of the points representing corresponding prothrombin ratios with the British comparative thromboplastin in the local reagent. This line is then used to correct subsequent values using the local laboratory method of corresponding values in terms of the British comparative thromboplastin.A study has been made of the statistical validity of the recommended system for anticoagulant control using the Quick one-stage test by comparing the line drawn by inspection with the confidence limits of the regression line. There was little difference between the best straight line by visual comparison and the calculated line for the majority of hospitals. The recommended procedure, therefore, provides an adequate conversion of the local method to the British comparative thromboplastin without the need for calculation. The significance of aberrant points is discussed and it is suggested that when more than two of the 12 results fall in this category the standardization procedure should be repeated.

The British system for anticoagulant control and Thrombotest.

A study has been made to determine whether a reliable comparison can be made between the British Comparative Thromboplastin and Thrombotest using the nationally adopted standardization procedure. The method recommended appeared sufficiently satisfactory and statistically valid for the comparison between the British Comparative Thromboplastin and Thrombotest. The degree of diversion in the location of the ;best lines', which should have been in an identical position at each centre, suggests that the standardization procedure at individual hospitals with Thrombotest is not usually a sufficiently reliable basis for clinical anticoagulant dosage. The degree of diversion almost certainly arises from technical error in the standardization procedure, and it is suggested that Thrombotest should be calibrated against the British Comparative Thromboplastin only in specialist coagulation centres thoroughly conversant with both techniques.

Risks of lung cancer, chronic bronchitis, ischaemic heart disease, and stroke in relation to type of cigarette smoked.

In a case control study of over 12 000 inpatients aged 35-74, risk of lung cancer, chronic bronchitis, and, particularly in those aged 35-54, ischaemic heart disease was positively associated with the number of manufactured cigarettes smoked daily and was negatively associated with long term giving up. Risk of stroke was not clearly related to smoking. Among manufactured cigarette smokers, lung cancer risk tended to be lowest in those who had always smoked filter cigarettes. This pattern was, however, evident only in men who additionally smoked pipes, cigars or handrolled cigarettes and in women, not being seen in men who smoked only manufactured cigarettes. Risk of lung cancer was not clearly related to time of switch to filter cigarettes. A markedly lower risk of chronic bronchitis was seen in men, but not women, who smoked filter rather than plain cigarettes. Heart disease risk did not vary by type of cigarette smoked 10 years before admission, but, compared with those who had never smoked filter cigarettes, those who had ever smoked filter cigarettes had a higher risk in men and a lower risk in younger women. Compared with the general population, markedly more controls were ex-smokers, suggesting incipient disease, whether or not smoking related, may alter smoking habits, thus affecting the interpretability of the findings. Control smokers were also relatively much more likely to report smoking plain cigarettes than expected. This comparison, not made in other studies relating risk to type of cigarette smoked, indicates that great care must be taken in verifying validity of reported smoking habits. While our findings are compatible with other evidence that risk of lung cancer and chronic bronchitis is probably reduced by switching from plain to filter cigarettes, they underline the difficulties in obtaining valid evidence from epidemiological studies.

Radical versus modified radical mastectomy for breast cancer.

A prospective randomised trial (534 patients, 1969-75) was designed to determine whether radical mastectomy conferred advantages over modified radical mastectomy for breast cancer in terms of total survival, local recurrence, distant metastasis, and disease-free interval. The results showed no significant difference in outcome as regards these variables between the two treatments.

New conjugate vaccines for the prevention of pneumococcal disease in developing countries.

Current pneumococcal conjugate vaccines (PCVs) are highly effective in preventing serotype-specific pneumococcal disease; however, they are relatively expensive and complicated to produce. Furthermore, PCVs do not cover all disease-causing pneumococcal serotypes. While current PCVs are available in industrialized countries and with external assistance in some low-income countries, alternative, more intrinsically affordable pneumococcal vaccines are essential for achieving more widespread use and coverage in resource-limited settings, where vaccines are often inaccessible and need is greatest. This review article describes a number of approaches to develop new PCVs designed to meet this urgent need.

Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle.

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

Regulation of immune responses by the ligands for CD27, CD30, and 4-1BB.

Activated T cells express a number of cell surface proteins that play an important role in cell contact-dependent interactions with cells in the immune system, including B cells, macrophages and other T cells. Among these T cell-expressed proteins are members of the TNF ligand family, which includes TNF, lymphotoxin (LT)-alpha and -beta, and the ligands for CD27, CD30, CD40, 4-1BB, OX-40, and Fas. The recent cloning of a number of these ligands has paved the way for a detailed analysis of the role of these molecules in immune responses. Initial studies have suggested that most, if not all, of these ligands co-stimulate T-cell proliferation. It is becoming increasingly apparent, however, that members of the TNF ligand family play unique and critical roles in both the development of the immune system and in immune responses to specific pathogens.