Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retarets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8⁺ and CD4⁺ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V11, in particular CD8⁺ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.     

CD58/LFA-3 and IL-12 provided by activated monocytes are critical in the in vitro expansion of CD56+ T cells

A small proportion of human CD3⁺ T lymphocytes are known to co-express CD56, an antigen usually restricted in its expression to natural killer (NK) cells. Whereas the in vivo function of CD3⁺ CD56⁺ T cells remains unknown, we and others have previously shown that both in vitro and in vivo, these cells can mediate a significantly greater degree of MHC-unrestricted cytotoxicity against a variety of human tumor cells when compared to either CD3⁺ CD56⁻ T cells or lymphokine activated killer (LAK) cells. While the mechanisms regulating the in vivo expansion of CD56⁺ T cells are not known, here we demonstrate the importance of CD2-mediated IL-12-dependent signals in the in vitro expansion of CD56⁺ T cells. Specifically, we show that activated monocytes provide a contact dependent factor (CD58/LFA-3) and a soluble factor (IL-12), both critical for the in vitro expansion of CD56⁺ T cells. The biological and therapeutic implications of these findings are discussed. Received: 4 May 2000 / Accepted: 25 August 2000     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Cytotoxic activity and phenotypic characteristics of lymphocyte subsets after therapy of cancer patients with interleukin-2

Summary After a 5-day period of continuous intravenous infusion of recombinant interleukin 2 (rIL-2) in seven patients with malignant melanoma or gastric or pancreatic cancer, different lymphocyte subsets were separated from patients' blood and tested ex vivo for cytotoxic activity against various tumour cell lines. Lytic activity was mediated by CD3⁺CD56⁺, CD3^–CD56⁺, CD3^–CD2⁺ and CD8⁺CD56⁺ lymphocytes. No cytotoxic activity could be observed within the CD3⁺CD56^–, CD3⁺CD2⁺ or CD4⁺ T cell subsets. To characterize CD56⁺ cytotoxic cells further, the expression of other antigens on this population was analysed before and after IL-2 therapy. CD3, CD4, CD16 and CD57 antigens were weakly expressed, and the IL-2 receptor (CD25) was not detectable on these cells either before and after treatment with IL-2. In contrast, increased expression of CD2, CD8 and HLA-DR antigens occurred following therapy. The divergence of CD3 and CD8 antigen expression after IL-2 therapy was caused by an increase in CD3^–CD8⁺ cells, detectable as a low-density CD8⁺ subset. This study shows that cytotoxic activity of in vivo IL-2-activated killer cells is predominantly, but not exclusively, mediated by CD3^–CD56⁺ lymphocytes, partially coexpressing the CD8 antigen and lacking the expression of CD 16 antigens.     

Human CD80/IL2 lentivirus transduced acute myeloid leukaemia cells enhance cytolytic activity in vitro in spite of an increase in regulatory CD4<Superscript>+</Superscript> T cells in a subset of cultures

Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4⁺ T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8⁺ T cells (P = 0.046) CD3⁻CD56⁺ NK cells (P = 0.028) and CD3⁺CD4⁺CD25^highFoxp3⁺ Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8⁺ T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8⁺ T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-γ and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.     

Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8⁺ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8⁺ T cell-dependent prolonged survival time of naïve IL-4^?/? mice. In mice immunized with SV40 TAg we observed in IL-4^?/? mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8⁺ T cells. The enhanced cytotoxicity in IL-4^?/? mice was accompanied by a significant increase in the fraction of CD8⁺ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8⁺ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.     

Low dose IL-15 induces snap arming of CD44(low) T lymphocytes in the absence of antigen

It is widely accepted that naïve T cells require two signals, antigen recognition and co-simulation, to become cytotoxic over the course of 3–5 days. However, we observed that freshly isolated murine splenocytes without exposure to antigen become cytotoxic within 24 h after culture with IL-15. IL-15 is a cytokine that promotes homeostatic proliferation, maintenance and activation of memory T cells. The induced cytotoxicity, measured by anti-CD3 redirected ⁵¹Cr release, represented the combined activity of T cells regardless of their antigen specificity, and proceeded even when CD44^hi (memory-associated phenotype) CD8⁺ T cells were depleted. Cytotoxic capacity was perforin-dependent and occurred without detectable up-regulation of granzyme B or cell division. After induction, the phenotypic markers for the memory subset and for activation remained unchanged from the expression of resting T cells. Our work suggests that T cells may gain cytotoxic potential earlier than currently thought and even without TCR stimulation.     

Chronic hepatitis C viral infection reduces NK cell frequency and suppresses cytokine secretion: Reversion by anti-viral treatment

Impaired activity of NK (natural killer) cells has been proposed as a mechanism contributing to viral persistence and chronic infection in hepatitis C (HCV) infection. We aimed to assess the impact of HCV infection on NK cells regarding frequency, subset distribution, and cytotoxic and cytokine secretion functions, as well as IFN-α and ribavirin therapeutic effects on NK cells. Significant reduction of total NK frequency and the CD56^dim16⁺ subset was observed in chronic HCV patients. IFN-γ expression upon stimulation with K562 was severely suppressed but cytotoxicity measured by CD107a expression was maintained. These adverse effects were reversed after treatment with pegylated IFN-α and ribavirin; however, these skewed functions were not recovered in treatment-resistant patients. Thus, HCV chronic infection severely affects NK functions, except for cytotoxicity. Altered NK cell frequency and cytokine secretion by HCV infection may contribute to impaired cellular immune response and virus persistence.     

Effective Induction of Human NK Cells with OK-432 and Further Augmentation of Their Cytolytic Function by rIL-2

Low concentrations of exogenously added recombinant interleukin 2 (rIL-2) were able to augment OK-432-induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL-2 and manifested itself only in PBMC stimulated with OK-432 (OK-MC) followed by rIL-2; augmentation did not happen in the reverse order. The existence of CD16⁺/CD25⁺ (IL-2 receptor positive; IL-2R⁺) and CD57⁺/CD25⁺ double positive cells which possess NK cell surface markers in OK-MC markedly increased in a long-term culture (12 days). A strong positive correlation was observed between the IL-2-dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day-12-OK-MC with monoclonal anti-CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL-2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK-432 could promote and upregulate the expression of IL-2 receptor (CD25) on CD56⁺ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL-2 with IL-2 receptors on OK-432-activated NK cells could augment their lytic function.     

Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

Distribution of Several Activating and Inhibitory Receptors on CD3<Superscript>−</Superscript>CD16<Superscript>+</Superscript> NK Cells and Their Correlation with NK Cell Function in Healthy Individuals

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3⁻CD16⁺ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3⁻CD16^bright NK subset constituted 78.97%, while the regulatory CD3⁻CD16^dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3⁻CD16⁺ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3⁻CD16^bright and CD3⁻CD16^dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3⁻CD16⁺NK/K562 conjugate composition, together with the stronger target binding capacity of CD16^bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3⁻CD16⁺NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3⁻CD16^bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

Long-term proliferation of functional human NK cells,with conversion of CD56<Superscript>dim</Superscript> NK cells to a CD56<Superscript>bright</Superscript> phenotype,induced by carcinoma cells co-expressing 4-1BBL and IL-12

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56^bright, and we show that isolated CD56^dimCD16⁺ NK cells can switch to a CD56^brightCD16⁻ phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required ‘help’ from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56^bright and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.     

Brucella abortus induces intracellular retention of MHC‐I molecules in human macrophages down‐modulating cytotoxic CD8+ T cell responses

Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8⁺ T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN‐γ‐induced MHC‐I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC‐I down‐modulation correlated with the development of diminished CD8⁺ cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8⁺ T lymphocytes and a diminished percentage of IFN‐γ‐producing CD8⁺ T cells. Inhibition of MHC‐I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC‐I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC‐I molecules whereby B. abortus would avoid CD8⁺ cytotoxic T cell responses, evading their immunological surveillance.     

Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow.

We analyzed the cytotoxicity and characterized the phenotype of oncolytic bone marrow (BM) lymphocyte subsets generated in vitro by interleukin-2 (IL-2) and stimulator cells (SC). Two irradiated B-lymphoblastoid cell lines (Daudi and EBV-transformed BSM) and fresh human acute myelogenous leukemia (AML) were used as SC. Stimulation with Daudi and IL-2 resulted in a substantial increase in cytotoxic activity (100- to 1000-fold) against a broad range of tumor targets, and total cellular expansion was higher compared to stimulation with IL-2 alone. The most prominent increase was observed in the CD16+ and CD56+/CD3- natural killer (NK) cell subset; however, a significant increase was also observed in CD56+/CD3+ T cells. Functional analysis of Daudi- and IL-2-generated subsets using fluorescence-activated cell sorting (FACS) revealed that most of the lytic activity was mediated by NK cells. Significant potentiation of oncolytic activity and cell growth was also seen in the cultures stimulated with BSM or fresh AML and IL-2. The highest oncolytic activity in the latter cultures was mediated primarily by CD8+, CD3+, and CD56- T cells, although NK cells also participated in cytotoxic activity. The T cell-mediated cytotoxicity was restricted by the major histocompatibility complex (MHC), since most cytotoxicity could be blocked by HLA I antibodies. Additionally, we observed that optimum stimulation of cytotoxicity required effector cell-stimulator cell contact. These data indicate that depending on the tumor used for stimulation, different lymphocyte subsets may be generated in IL-2 cultures. These different approaches may be useful in both specific and nonspecific immunotherapy.