Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.     

Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.     

Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-gamma-mediated effect

Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-gamma mRNA, without apparent involvement of NF-kappaB. Moreover, expression of PPAR-gamma was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-gamma ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.     

Antigen specificity acquisition of adoptive CD4+ regulatory T cells via acquired peptide-MHC class I complexes

The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

Preferential costimulation by CD80 results in IL-10-dependent TGF-beta1(+) -adaptive regulatory T cell generation

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.     

Anti-inflammatory effects of Saccharomyces boulardii mediated by myeloid dendritic cells from patients with Crohn's disease and ulcerative colitis

Saccharomyces boulardii (Sb) is a probiotic yeast that has demonstrated efficacy in pilot studies in patients with inflammatory bowel disease (IBD). Microbial antigen handling by dendritic cells (DC) is believed to be of critical importance for immunity and tolerance in IBD. The aim was to characterize the effects of Sb on DC from IBD patients. Highly purified (>95%), lipopolysaccharide-stimulated CD1c(+)CD11c(+)CD123(-) myeloid DC (mDC) from patients with ulcerative colitis (UC; n = 36), Crohn's disease (CD; n = 26), or infectious controls (IC; n = 4) were cultured in the presence or absence of fungal supernatant from Sb (SbS). Phenotype and cytokine production and/or secretion of IBD mDC were measured by flow cytometry and cytometric bead arrays, respectively. T cell phenotype and proliferation were assessed in a mixed lymphocyte reaction (MLR) with allogenic CD4(+)CD45RA(+) na?ve T cells from healthy donors. Mucosal healing was investigated in epithelial wounding and migration assays with IEC-6 cells. SbS significantly decreased the frequency of CD40-, CD80-, and CD197 (CCR7; chemokine receptor-7)-expressing IBD mDC and reduced their secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 while increasing IL-8. In the MLR, SbS significantly inhibited T cell proliferation induced by IBD mDC. Moreover, SbS inhibited T(H)1 (TNF-α and interferon-γ) polarization induced by UC mDC and promoted IL-8 and transforming growth factor-β-dependent mucosal healing. In summary, we provide novel evidence of synergistic mechanisms how Sb controls inflammation (inhibition of T cell costimulation and inflammation-associated migration and mobilization of DC) and promotes epithelial restitution relevant in IBD.     

Hierarchical signaling thresholds determine the fates of naíve T cells: partial priming leads nai;ve T cells to unresponsiveness

Differing conditions of antigen priming varying either the concentration or affinity of T cell receptor (TCR) ligands greatly alter T cell responses. Here, we demonstrate that antigen-specific CD4(+) nai;ve T cells primed with either altered peptide ligands (APLs) or a minimal concentration of antigen peptide become anergic without observable cell divisions. Transforming growth factor-beta1 (TGF-beta1) expression was induced 24h following in these stimulation conditions producing anergic cells. Productively stimulated nai;ve T cells expressed IL-2 to differentiate into T helper 1 (Th1) cells, secreting interferon-gamma (IFN-gamma) upon secondary antigen stimulation; T cells primed with an APL did not secrete either interleukin-4 (IL-4) or IFN-gamma, but expressed TGF-beta1 and Tob, a member of the anti-proliferative gene family. Therefore, T cell responses are regulated by TCR signaling depending on the extent of TCR engagement. These results suggest that partial antigen stimulation in the periphery can induce nai;ve CD4(+)T cell unresponsiveness.     

Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFalpha, IL-10, IL-12, IFNgamma, MIP1alpha, MIP1beta, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells

Anergy and suppression are cardinal features of CD4(+)CD25(+)Foxp3(+) T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4(+) and CD4(-) conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4(+)CD25(-) T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4(+)CD25(+)Foxp3(+) T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-gamma production by CD4(+)CD25(-) T cells induced by mature CD4(+) and CD4(-) DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25(-) cells but not their proliferation or IFN-gamma production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.     

The schistosome oligosaccharide lacto-N-neotetraose expands Gr1(+) cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4(+) cells: a potential mechanism for immune polarization in helminth infections.

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1(+)/CD11b(+)/F4/80(+), as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1(+)/F4/80(+) population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-beta ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1(+) cells adoptively suppressed naive CD4(+) T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4(+) cells involved cell contact and was dependent on IFN-gamma and NO, with a discrete role played by IL-10. Coculture of naive CD4(+)T cells with Gr1(+) suppressor cells did not lead to CD4(+) T cell apoptosis, although it did imprint on naive CD4(+) T cells a response characterized by lower levels of IFN-gamma, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.     

Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells

The characteristics and functions of CD4(+)CD25(+) regulatory cells have been well defined in murine and human systems. However, the interaction between CD4(+)CD25(+) T cells and dendritic cells (DC) remains unclear. In this study, we examined the effect of human CD4(+)CD25(+) T cells on maturation and function of monocyte-derived DC. We show that regulatory T cells render the DC inefficient as APCs despite prestimulation with CD40 ligand. This effect was marginally reverted by neutralizing Abs to TGF-beta. There was an increased IL-10 secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to direct suppressor effect on CD4(+) T cells, regulatory T cells may modulate the immune response through DC.     

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.     

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.