Induction Of Cytotoxic Effector Cells Research Articles

Induction of cytotoxic effector cells towards cholangiocellular, pancreatic, and colorectal tumor cells by activation of the immune checkpoint CD40/CD40L on dendritic cells

IntroductionGastrointestinal (GI) malignancies, such as cholangiocarcinoma, pancreatic carcinoma, and metastatic colorectal carcinoma, have a poor prognosis and effective therapeutic approaches are still challenging. Checkpoint inhibition with PD-1 or PDL-1 antibodies revealed promising results in different tumor entities; however, only few patients with GI tumors can potentially benefit from PD1/PDL1 inhibiting immunotherapy. Further immunotherapeutic strategies for GI malignancies are urgently needed. The aim of this study was to demonstrate that in vitro activation of the immune checkpoint CD40/CD40L can improve DC action towards bile duct, pancreas, and colorectal carcinoma.MethodsHuman DC were isolated from buffy coats from healthy donors, pulsed with tumor lysates and then transduced with adenoviruses encoding human CD40L (Ad-hCD40L). Using transwell assays, the effects of (m)CD40L on DC immunoactivation compared to (s)CD40L were analyzed. Surface marker and cytokine/chemokine expression were measured by flow cytometry, ELISA and cytokine arrays. Capacity of Ad-hCD40L-transduced DC to induce tumor-specific effector cells was tested using MTT proliferation assay and cytotoxicity assays. Apoptosis induction on tumor cells after culturing with supernatants of Ad-hCD40L-transduced DC was analyzed by flow cytometry.ResultsAd-hCD40L transduction induced a high expression of (s)CD40L and (m)CD40L on DC and seemed to induce a strong cellular CD40/CD40L interaction among DC, leading to the formation of cell aggregates. Due to the CD40/CD40L interaction, a significant upregulation of DC maturation markers and a Th1-shift on cytokines/chemokines in the supernatant of DC were achieved. Interestingly, a pure Th1-shift was only achieved, when a cellular CD40/CD40L interaction among DC took place. (s)CD40L induced almost no upregulation of maturation markers and rather resulted in a Th2-cytokine expression, such as IL-10. Correspondingly, (m)CD40L-expressing DC led to significant proliferation and stimulation of tumor-specific effector cells with increased cytotoxicity towards pancreatic, bile duct and colorectal tumor cells. Supernatants of Ad-hCD40L-transduced DC could also induce apoptosis in the different tumor cells in vitro.ConclusionStimulation of the immune checkpoint CD40L/CD40 by endogenous expression of (m)CD40L provokes a cellular interaction, which increases the immunomodulatory capacity of DC. A Th1 cytokine/chemokine expression is induced, leading to a significant proliferation and enabling cytotoxicity of effector cells towards human bile duct, pancreatic and colorectal tumor cells. The present data point to the promising approach for DC-based immunotherapy of gastrointestinal malignances by activating the CD40/CD40L immune checkpoint.

Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1.

HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 Å(2)) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8(+) T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-β fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-β XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

Antigen Incorporated In Virus-like Particles Is Delivered to Specific Dendritic Cell Subsets That Induce An Effective Antitumor Immune Response In Vivo

Virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) can be used as a scaffold to facilitate the delivery of antigens to induce cell-mediated immune responses. In this study, we investigated the immune response to lymphocytic choriomeningitis virus-derived peptide antigen (gp33) delivered by RHDV VLP. The gp33 peptides were incorporated into the VLP in 2 different forms, either recombinantly expressed inside the VLP (VLP-gp33r) or chemically coupled to the surface of the VLP (VLP-gp33c). We showed that VLP-gp33r induced a greater level of cytotoxicity than VLP-gp33c against gp33-coated target cells in vivo. Both VLP, when delivered as prophylactic vaccines, inhibited the growth of Lewis' lung carcinoma tumors expressing gp33 (LL-LCMV) in mice to a similar degree. Studies to investigate the mechanism induced by these VLP showed that 2 CD11c DC subsets, CD8α and CD8α, acquired VLP in vivo and in vitro, and VLP-gp33r were cross-presented by both these subsets to prime CD8 T cells through a TAP-independent, endosomal recycling pathway. Depletion of Langerin DC in vivo before and after vaccination with VLP-gp33r, lead to reduced cytotoxicity implicating these cells in the induction of cytotoxic effector cells. These results suggest that recombinant VLP expressing tumor peptides targeted to Langerin DC may have clinical application. Finally we found that VLP-gp33r were more effective antitumor vaccines than VLP-gp33c when delivered therapeutically. The findings of this study suggest the potential of VLP as a platform for delivery of tumor-associate antigen and elicit protective immunity against tumors.

Antibody blockade of Thy‐1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti‐CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.

Interleukin-12-induced cytotoxicity against syngeneic B cell lymphomas of SJL/J mice

The B cell lymphomas (RCS) that develop spontaneously in 90% of aging SJL/J mice stimulate syngeneic CD4 + Vβ16 + Th2 cells to produce cytokines, such as IL-4 and IL-5, which promote lymphoma growth. Although RCS cells express a unique superantigen (vSAg) encoded by an endogenous MMTV (Mtv-29) provirus that also elicits IFN-γ production from naı̈ve syngeneic lymphoid cells, there is no development of RCS-specific cytotoxicity. However, addition of IL-12 to co-cultures of SJL spleen and irradiated (γ-)RCS cells resulted in the appearance of effector cells that killed RCS and NK-susceptible target cells. Antibody depletion studies revealed at least two types of RCS/IL-12-induced cytotoxic cells: (1) NK cells (Asialo GM1 +) and (2) CD8 + CTL. Despite high titers of IFN-γ in the SN of co-culture of SJL spleen and γ-RCS cells, cytotoxicity only developed if IL-12 was also included in the co-cultures. The results of RNAse protection assays and multi-parameter FACS analysis demonstrated an upregulation of IFN-γ and decrease in IL-4 by activated Th cells in co-cultures with IL-12. These results indicate that inclusion of IL-12 in primary co-cultures of SJL spleen and γ-RCS cells influences the qualitative nature of the response to favor use of RCS-responsive Th1 rather than Th2 cells to facilitate the production of cytotoxic effector cells. Results of in vivo experiments support this hypothesis, as judged by tumor growth assays and FACS analysis of the tumor cell content of lymphoid tissues. Inhibition of lymphoma growth was observed in mice given γ-RCS/IL-12-induced effector cells prior to injection of viable RCS cells. These results demonstrate that IL-12 can be used to alter the host immune response leading to induction of cytotoxic effector cells that inhibit the development and/or progressive growth of otherwise resistant B cell lymphomas in SJL/J mice.

Leucocyte contamination of platelet products

Despite several adverse transfusion effects attributed to leucocytes in platelet transfusions, the main reason to remove leucocytes from transfusions is to reduce HLA alloantibody formation resulting in immunological platelet refractoriness and febrile transfusion reactions due to HLA antibodies in the recipient. When present above a critical level, leucocytes always induce an immune response. This will not always result in HLA antibody formation or the induction of cytotoxic effector cells. Often the result is immune suppression and tolerance towards donor HLA antigens. The immunological effect, either antibody formation or the induction of tolerance, is the result of various distinguished mechanisms. These will be discussed separately, although in transfusion practice these factors are interacting.

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine. II. Characterization of induced effector cells and the mechanism of induction.

In a preceding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T8OG)2. In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T8OG. Removal of macrophages from 7T8OG activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T8OG induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM1 antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T8OG induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T8OG. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T8OG resulted in effectors with reduced cytotoxicity. When injected in vivo, 7T8OG induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to alpha & beta-IFN but not to gamma-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T8OG is most likely to be mediated by alpha & beta-IFN involvement of other cytokines can not be ruled out.

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.

Modulation of murine in vitro immune response by verapamil (V)

Functionally distinct lymphocyte subsets differ with regard to necessary activation signals. In selected circumstances lymphocyte activation has been shown to be critically dependent upon transcellular calcium influx. Whether calcium plays a central role in the activation of all lymphocytes remains to be determined. The effect of the calcium channel blocker verapamil on the induction of murine cytotoxic T lymphocytes (CTL), suppressor cells, T helper cells, and B cells was investigated. Verapamil (V) was found to inhibit the induction of cytotoxic effector cells. V acted primarily on the afferent limb of this immune response, was synergistic with cyclosporin A (CsA), and its effects could be largely reversed by the addition of exogenous helper factors. V also inhibited B cell proliferation in response to anti-mouse IgM in the presence of 2-mercaptoethanol, but in the absence of cognate or non-cognate T cell help. In contrast to this, V did not inhibit the activation of cells capable of inducing B cell proliferation nor did it inhibit the induction of suppressor cells. The selective suppression of V is discussed in terms of activation requirements of CTL, suppressor cells and helper cell subsets.

Plant lectin, ATF1011, on the tumor cell surface augments tumor-specific immunity through activation of T cells specific for the lectin.

The possibility that a plant lectin as a carrier protein would specifically activate T cells, resulting in the augmentation of antitumor immunity was investigated. ATF1011, a nonmitogenic lectin for T cells purified from Aloe arborescens Mill, bound equally to normal and tumor cells. ATF1011 binding on the MM102 tumor cell surfaces augmented anti-trinitrophenyl (TNP) antibody production of murine splenocytes when the mice were primarily immunized with TNP-conjugated MM102 tumor cells. The alloreactive cytotoxic T cell response was also augmented by allostimulator cells binding ATF1011 on the cell surfaces. These augmented responses may be assumed to be mediated by the activation of helper T cells recognizing ATF1011 as a carrier protein. Killer T cells were induced against ATF1011 antigen in the H-2 restricted manner using syngeneic stimulator cells bearing ATF1011 on the cell surfaces. When this lectin was administered intralesionally into the tumors, induction of cytotoxic effector cells was demonstrated. These results suggest that intralesionally administered ATF1011 binds to the tumor cell membrane and activates T cells specific for this carrier lectin in situ, which results in the augmented induction of systemic antitumor immunity.

Cellular origin of interleukin 2 (IL 2) in man: evidence for stimulus-restricted IL 2 production by T4+ and T8+ T lymphocytes.

The human T cell subset(s) responsible for production of interleukin 2 (IL 2) was investigated in the present study. For this purpose, highly purified T4+ and T8+ T lymphocytes were stimulated with mitogens and alloantigens. Subsequently, culture supernatants were analyzed for IL 2 activity in each of two assay systems: 1) proliferation of long-term T cell lines and 2) induction of cytotoxic effector cells from a resting T8+ population incubated in MLC. Mitogen stimulation led to secretion of equivalent amounts of IL 2 from both the major T cell subsets; in contrast, after allogeneic activation, IL 2 was produced predominantly from the T4+ subset. The stimulus dependency of IL 2 production suggests that the individual functional repertoires of T4+ and T8+ T cell subsets may be linked to unique surface receptors and/or interaction molecules involved in cell triggering rather than a restricted capacity to produce lymphokine.

Cell-mediated immune response of marmosets to herpesvirus-associated antigens in vitro

Immune responses in vitro of some species of marmosets to herpesvirus-associated antigens expressed on virus-transformed lymphoblastoid cell lines (LCL) were studied by determining lymphocyte proliferation, interferon production, and the induction of cytotoxic effector cells in mixed lymphocyte-LCL cultures (MLLC). Autologous Epstein-Barr virus (EBV)-transformed B-cell lines induced neither lymphocyte proliferation nor interferon production in MLLC, while autologous Herpesvirus ateles (HVA)-transformed T-cell lines stimulated responder cell DNA synthesis and interferon production. Both EBV-LCL and HVA-LCL failed to induce cytotoxic effector cells in autologous MLLC responder cells. These findings differ markedly from the human immune response to autologous EBV-LCL in vitro and may have implications for the unusual susceptibility of marmosets to the induction of lymphoproliferative disease following inoculation with oncogenic herpesviruses.

In vitro induction of cytotoxic effector cells against human neoplasms. I. Sensitization conditions and effect of cryopreservation on the induction and expression of cytotoxic responses to allogeneic leukemia cells

Peripheral blood lymphocytes (PBL) from normal human donors were sensitized in vitro against allogeneic human acute myelocytic leukemia (AML) cells by means of an unidirectional mixed lymphocyte-tumor cell culture (MLTC) technique. The cytotoxic responsiveness of the sensitized lymphocytes, as determined in vitro by the 51Cr-release assay, varied among individual lymphocyte donors and was greatly dependent on the sensitization culture conditions. Induction of cytotoxic effector cells was augmented appreciably by adding to the cultures minute amounts of the immunopotentiating agent MER-BCG. Responding lymphocytes and stimulating leukemia cells cryopreserved for several weeks in liquid nitrogen were as effective as fresh cells in generating effector lymphocytes; the cytotoxic capacity of already sensitized lymphocytes was fully retained by cryopreservation. The implications of these findings for possible clinical employment of in vitro sensitized lymphocytes in adoptive immunotherapy of cancer are discussed.

In vitro induction of cytotoxic effector cells with spontaneous killer cell specificity.

The present work shows that stimulation of peripheral blood lymphocytes with autologous or allogeneic B-cell lines leads to a strong induction of cytotoxic effector cells with spontaneous killer (SK) cell specificity, apart from the specific response directed against the particular stimulating cell. To demonstrate this we have determined a relative target cell specificity in the SK system, defined by the short-term 51Cr release assay, and established a relative specificity index (RSI). Using this approach we have been able to show that killer cells induced during a 5-day cocultivation period with B-cell lines have a similar PSI to that of unstimulated SK cells. In addition, we have shown that such killer cells can be induced from several different lymphocyte subpopulations and that they, in contrast to SK cells, do not express Fc receptors. The implications of these findings in relation to the nature, mechanism, and biological significance of the SK cell system is discussed.

Effects of α-Fetoprotein on Murine Immune Responses

Abstract Because of the current interest in the possible “immunosuppressive” effects of α-fetoprotein a systematic study of the effect of normal sera, amniotic fluid (AmF), sera from hepatoma-bearing animals (Tu sera), and AFP isolated by immunoabsorption from AmF or Tu sera on various models of the immune response of the mouse was carried out. Whereas some in vitro reactions were inhibited by AFP, other in vitro systems were only weakly inhibited, unaffected, or even enhanced by the addition of AFP. AFP by itself was mitogenic for murine lymphoid cells, inhibited the LPS mitogenic response 3-fold, but did not affect the response to Con A. Very high concentrations of AFP partially inhibited primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC). The secondary in vitro PFC response to keyhole limpet hemocyanin (KLH) conjugated to trinitrophenol (TNP) was inhibited 2- to 4-fold. The induction of cytotoxic effector cells directed against P815 mastocytoma cells was increased 9-fold in the presence of AFP. There was no significant difference between the effect of AmFAFP and TuAFP on any of the in vitro reactions studied. Normal and tumor sera produced a similar reduction in secondary PFC responses in spite of a 6000-fold difference in AFP concentration. The in vivo PFC response to SRBC in mice bearing a transplantable hepatoma was greater than that of normal mice even though the serum AFP concentration of the tumor-bearing mice was 1000 times normal. In addition, no AFP synthesis or coating of lymphocytes was observed during the course of a graft-vs-host or mixed lymphocyte reactions. Therefore, although some effects on immune phenomena occur upon addition of serum proteins and AFP, these data do not support a general immunosuppressive role for AFP in the mouse.