In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy

The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.     

Acute myeloid leukemia impairs natural killer cells through the formation of a deficient cytotoxic immunological synapse

Acute myeloid leukemia (AML) cells are killed by allogeneic NK cells. However, autologous NK cells from AML patients express decreased levels of activating receptors, and show reduced cytotoxicity. Here, we investigated how interactions between NK and AML cells might cause loss of NK‐cell activity in patients. Our results show that AML cell lines and primary blasts alter the NK‐cell phenotype, reducing their cytotoxic potential upon prolonged contact. Downregulation of NK‐cell‐activating receptors was contact‐dependent and correlated with conjugate formation. Time‐lapse imaging of HL60 AML cell line and NK‐cell interactions showed a high proportion of noncytolytic contacts. Studies of NK‐cell immunological synapses revealed a defect in lytic synapse formation. Namely, despite correct F‐actin and LFA‐1 recruitment, polarization of lytic granules toward primary blasts or AML cell lines was reduced. The NK–AML cell line synapses showed impairment of CD3ζ recruitment. Attempts to correct these synapse defects by cytokine stimulation of NK cells improved conjugate formation, but not granule polarization. Pretreatment of AML cell lines with the immunomodulating molecule lenalidomide significantly enhanced granule polarization. We speculate that combining immunomodulatory drugs and cytokines could increase AML cell sensitivity to autologous NK cells and reinforce the activity of allogeneic NK cells in adoptive immunotherapy.     

Immunotherapy prospects for acute myeloid leukaemia

While chemotherapy is successful at inducing remission of acute myeloid leukaemia (AML), the disease has a high probability of relapse. Strategies to prevent relapse involve consolidation chemotherapy, stem cell transplantation and immunotherapy. Evidence for immunosurveillance of AML and susceptibility of leukaemia cells to both T cell and natural killer (NK) cell attack and justifies the application of immune strategies to control residual AML persisting after remission induction. Immune therapy for AML includes allogeneic stem cell transplantation, adoptive transfer of allogeneic or autologous T cells or NK cells, vaccination with leukaemia cells, dendritic cells, cell lysates, peptides and DNA vaccines and treatment with cytokines, antibodies and immunomodulatory agents. Here we describe what is known about the immunological features of AML at presentation and in remission, the current status of immunotherapy and strategies combining treatment approaches with a view to achieving leukaemia cure.     

Effector functions and specificities of normal murine T cells stimulated by syngeneic blasts

Large numbers of syngeneically stimulated T cells are easily obtained by stimulation of splenic T cell populations with irradiated lipopolysaccharide (LPS) blasts. Thus, proliferative T cell responses were obtained after stimulation with allogeneic LPS blasts, concanavalin A (Con A) blasts, normal spleen cells or peritoneal macrophages while only LPS and Con A blasts were competent syngeneic stimulators. The cytolytic activity of T cells stimulated with allogeneic LPS blasts was H-2 specific, while that of T cells stimulated with syngeneic LPS blasts was often completely nonspecific, and higher for LPS than for Con A blast target cells. In other experiments, "self" H-2K- and I-A-specific cytotoxic T cells were obtained, without apparent reasons for the development of either type of cytolytic cells. Stimulation of Lyt-2- T cells with syngeneic LPS blasts led to the generation of T helper effector cells which induced normal B cell proliferation in an H-2 restricted manner. This restriction was over-come, the proliferative responses were augmented and plaque-forming cell responses generated, through addition of Con A to the cultures. The results imply the ability of B blasts to induce stimulation of syngeneic T cells, and generation of effector cells displaying cytolytic and helper activity. This phenomenon might play a role in the regulation of the immune system.     

Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Rα in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Rα production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.     

Gene Expression in CD8 and CD4 T-Cell Populations Following Activation with the Calcium Ionophore A23187

We have hypothesized that functional maturation of T lymphocytes can be dissected into a series of discrete stages. For example, activation of T lymphocytes with the calcium ionophore A23187 drives CD8+ T cells to become dividing blasts, referred to as 'pre-effector' cells in that these blasts do not express cytolytic function but are driven by IL-2 to do so. Here we characterize via Northern blots the functional maturation of CD8+ and CD4+ T-lymphocyte populations which have been activated via A23187 followed by stimulation with IL-2. Previously we have reported that no detectable IL-2 was found in the supernatants of A23187-activated pre-effector blasts. However, these cells do express levels of IL-2 mRNA very similar to those of OKT3-activated blasts, from which IL-2 is easily detected in the supernatant. Translational control may account for these findings. A23187-activated CD8+ pre-effector blasts do not respond to stimulation with IFN-gamma nor do they express IFN-gamma mRNA following stimulation with IL-2. These observations suggest that IL-2 may be sufficient to stimulate maturation of these cells. Activation via A23187 results in lower expression of the proto-oncogene c-myb relative to that found in OKT3 activation. C-myb mRNA levels are higher in CD8+ than in CD4+ A23187-activated pre-effector blasts and the c-myb level in the CD8+ pre-effector blasts falls in response to IL-2. This decrease in c-myb mRNA coincides with an increase in proliferation, and the expression of cytolytic activity.     

Cytokine responsiveness of mitogen-activated T cells derived from acute leukemia patients with chemotherapy-induced leukopenia.

The aim of the study was to characterize effects of exogenous cytokines on T lymphocytes derived from acute leukemia patients with chemotherapy-induced leukopenia. We investigated the cytokine responsiveness of long-term expanded CD4+ and CD8+ T cell clones and the effects of exogenous cytokines on anti-CD3-stimulated polyclonal T cell responses. After mitogenic activation in the presence of acute myelogenous leukemia (AML) blasts, most CD4+ and CD8+ clones proliferated in response to interleukin-2 (IL-2). Although a majority of the IL-2-responsive clones could also proliferate in the presence of exogenous IL-4, IL-7, IL-9, IL-10, IL-12, and IL-15, only IL-15 responses were equal to or exceeded the corresponding IL-2 responses. Exogenous cytokines were also added during T cell activation with phytohemagglutinin (PHA) + accessory leukemia cells derived from different AML patients, and all the cytokines then had divergent effects that depended on both differences between clones and differences between AML patients. However, for most of these T cell clone/AML blast combinations, IL-2 and IL-15 caused enhanced T cell proliferation. IL-2 and IL-15 also enhanced anti-CD3-stimulated polyclonal responses of nonexpanded T cells derived from cytopenic patients, whereas other cytokines had only minor effects. Our results demonstrate that cytokine-responsive T cells remain in the circulation during chemotherapy-induced cytopenia, and combination therapy including intensive chemotherapy and T cell-targeting cytokine therapy should, therefore, be possible in AML.     

Platelet-lymphocyte cross-talk

Platelets and lymphocytes reciprocally regulate mutual functions, i.e., platelet-lymphocyte cross-talk. The heterotypic interactions have emerged as important regulatory mechanisms in the pathophysiological processes of thrombosis, inflammation, immunity, and atherosclerosis. Platelets influence lymphocyte function via direct cell-cell contact and/or soluble mediators. Hence, platelets enhance adhesion and cell migration of T(H), T cytolytic (T(C)), NK, and B cells. Platelets affect other functional aspects of lymphocyte subpopulations in a complex manner. They may attenuate cytokine secretion and immunosuppressive responses of T(H) cells and enhance T(C) cell proliferation and cytotoxicity. Platelets promote isotype shifting and antibody production of B cells but ameliorate cytolytic activity of NK cells. On the other hand, lymphocytes can also regulate platelet aggregation and secretion, as well as the effector cell function of platelets in immune defense. The two cell types collaborate in transcellular phospholipid metabolism, CD40-CD40 ligand-mediated intercellular signaling, and their involvements in atherogenesis. The research perspectives of platelet-lymphocyte cross-talk have also been addressed.     

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.     

Different roles of PD-L1 and FasL in immunomodulation mediated by human placenta-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from either bone marrow (BMSCs) or placenta (PMSCs) have the capacity to suppress immune responses to mitogenic and allogeneic stimulations. Both cell contact and soluble factor dependent mechanisms have been proposed to explain this immunosuppression. This study explored the roles of some of cell surface molecules expressed on human PMSCs (hPMSCs) in hPMSC mediated immunomodulation. hPMSCs strongly suppressed mitogen and allogeneic peripheral mononuclear cells (PBMCs) induced T cell activation and proliferation. hPMSCs constituently expressed programmed death-ligand 1 (PD-L1) and Fas ligand (FasL) molecules. Neutralising antibodies to-PD-L1 and FasL significantly reduced the suppressive effect of hPMSCs on T cell proliferation. However, only anti-PD-L1 antibody partially restored early T cell activation suppressed by hPMSCs. Anti-FasL antibody but not anti-PD-L1 antibody reduced apoptosis of activated T cell indicating that FasL molecule plays a role in inducing apoptosis of activated T cells, although overall hPMSCs diminished T cell apoptosis. Different effects of PD-L1 and FasL molecules on T cell activation and activated T cell apoptosis suggest that these two molecules influence T cell response at different stages. hPMSCs significantly prevented activated T cells from going into S phase. Both antibodies to PD-L1 and FasL had significant effect on reversing the effect of hPMSCs on cell cycles. hPMSCs reduced INF-γ but increased IL-10 production by mitogen activated T cells. Both antibodies partially abolished the effect of hPMSCs on INF-γ and IL-10 production. These data demonstrated that PD-L1 and FasL molecules play significant roles in immunomodulation mediated by hPMSCs. This study provides a rational basis for modulation of negative costimulators on hPMSCs to increase their immunosuppressive properties in their therapeutic applications.     

Interactions between human mesenchymal stem cells and natural killer cells

Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T-cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto-toxicity against HLA-class I- expressing targets. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors, including transforming growth factor-beta1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.     

α-Fetoprotein impairs activation of natural killer cells by inhibiting the function of dendritic cells

α-Fetoprotein (AFP) is a tumour-associated antigen in hepatocellular carcinoma (HCC). The biological properties of AFP have been identified in its regulatory effects on immune responses of T cells and B cells. However, AFP effects on natural killer (NK) cells are still unclear. In this study, we examined the immunoregulation of AFP on NK activity. The cytolytic activity against K562 cells and Huh7 cells of NK cells co-cultured with AFP-treated dendritic cells (DCs) (AFP-DCs) was lower than that with albumin-treated DCs (Alb-DCs). Direct addition of AFP to NK cells did not alter the cytolytic activity of NK cells. Adding AFP inhibited the interleukin (IL)-12 production of DCs after stimulation with lipopolysaccharide (LPS) [Toll-like receptor (TLR)-4 ligand], or Poly(I:C) (TLR-3 ligand), but not IL-18 production. The mRNAs of IL-12p35 and IL-12p40 were significantly inhibited in AFP-DCs compared with Alb-DCs, but those of TLR-4 or TLR-3 were not. Transwell experiments revealed that soluble factors derived from DCs played roles in inhibition of the ability of activating NK cells by AFP-DCs. Adding the neutralizing antibody of IL-12 to NK cells co-cultured with Alb-DCs resulted in a decrease of cytolytic activity to the levels of NK cells co-cultured with AFP-DCs. Adding IL-12 to NK cells co-cultured with AFP-DCs resulted in an increase of cytolytic activity to the levels of NK cells co-cultured with Alb-DCs. These demonstrated that the impairment of IL-12 production from AFP-DCs resulted in inhibition of the ability of the activation of NK cells by DCs, and thus suggests a role of AFP in HCC development.     

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.     

MUC1-specific anti-tumor responses: molecular requirements for CD4-mediated responses

MUC1 was first defined as a tumor antigen in the late 1980s, yet little is known about the types of immune responses that mediate rejection of MUC1(+) tumors in vivo. MUC1-specific antibodies, T(h) cells and cytotoxic T cells can be detected in patients with different adenocarcinomas, yet these tumors usually progress. Thus, there is a need to better understand the in vivo mechanisms of antigen-specific tumor rejection. To characterize the nature of MUC1-specific immune responses in vivo, rejection of a MUC1-expressing melanoma tumor line (B16.MUC1) was evaluated in mice lacking specific T cell subsets, cytokines, co-stimulatory molecules or molecular effectors of cytolytic pathways. Results demonstrated that rejection of the B16.MUC1 tumor cell line was primarily mediated by CD4(+) T cells, and required Fas ligand, lymphotoxin-alpha, CD40, CD40 ligand and CD28, but not perforin, gammadelta T cells, IL-4, IL-10, IL-12 or tumor necrosis factor receptor-1. Depletion of NK cells demonstrated that NK cells might also contribute to MUC1 immunity in the B16.MUC1 tumor model. These results demonstrated that the immune response generated against MUC1 does not fit the type 1 or 2 model described for many immune responses. Additionally, multiple cytolytic mechanisms are required for B16.MUC1 rejection.     

Enhancement of immunogenicity of Jeg3 cells by ectopic expression of HLA-A*0201 and CD80

PROBLEM: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. METHOD OF STUDY: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. RESULTS: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. CONCLUSION: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells.