Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.     

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).     

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.     

Generation of antigen-specific, Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function

We tested the hypothesis that immature APC, whose NF-kappaB-signaling pathway and thus maturation was blocked by the proteosome inhibitor benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI), could be a source of Ag-specific regulatory T (Treg) cells. DO11.10 CD4(+) T cells that were incubated with Ag- and PSI-pulsed APC proliferated poorly, produced less IL-2, IFN-gamma, and IL-10 in secondary cultures, and inhibited the response of both naive and memory CD4(+) T cells stimulated by Ag-pulsed APC. The generation of PSI-APC Treg cells required IL-10 production by APC. PSI-APC Treg cell inhibition required cell-cell contact but not IL-10 or TGF-beta. Addition of IL-2 did not reverse, but Ab to CTLA-4 did reverse partially the inhibitory effect. Depletion of CD25(+) T cells before initial culture with PSI-APC did not affect Treg generation. PSI-APC Treg cells expressed high levels of Foxp3, inhibited proliferation of naive DO11.10 T cells in vivo, and abrogated colitis driven by a memory Th1 response to bacterial-associated Ag. We conclude that NF-kappaB-blocked, immature APC are able to induce the differentiation of Treg cells that can function in vitro and in vivo in an Ag-specific manner.     

In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring

BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.     

A bone marrow-derived APC in the gut-associated lymphoid tissue captures oral antigens and presents them to both CD4+ and CD8+ T cells

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer's patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.     

Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.     

Antigen-specific T cell repertoire modification of CD4+CD25+ regulatory T cells

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).     

Resting B lymphocytes as APC for naive T lymphocytes: dependence on CD40 ligand/CD40

Although resting B cells as APC are tolerogenic for naive T cells in vivo, we show here that they can provide all the costimulatory signals necessary for naive T cell proliferation in vivo and in vitro. In the absence of an activating signal through the B cell Ag receptor, T cell proliferation after Ag recognition on resting B cells depends on CD40 expression on the B cells, implying that naive T cells use the membrane-bound cytokine, CD40 ligand (CD154), to induce the costimulatory signals that they need. Induction of B7-1 (CD80) and increased or sustained expression of CD44H, ICAM-1 (CD54), and B7-2 (CD86) are dependent on the interaction of CD40 ligand with CD40. Transient expression (12 h) of B7-2 is T cell- and peptide Ag-dependent, but CD40-independent. Only sustained (>/=24 h) expression of B7-2 and perhaps increased expression of ICAM-1 could be shown to be functionally important in this system. T cells cultured with CD40-deficient B cells and peptide remain about as responsive as fresh naive cells upon secondary culture with whole splenic APC. Therefore, B cells, and perhaps other APC, may be tolerogenic not because they fail to provide sufficient costimulation for T cell proliferation, but because they are deficient in some later functions necessary for a productive T cell response.     

Induction of negative signal through CD2 during antigen-specific T cell activation.

We have investigated the role of CD2 molecules in Ag-specific T cell activation by using a mouse model system in which the function of CD2 can be analyzed without the apparent influence of major accessory molecules, such as CD4 or LFA-1. Transfection of the CD2 gene into a CD2- T cell hybridoma confers the enhancement of IL-2 production upon Ag stimulation. Anti-CD2 mAb inhibits the Ag-specific response of the CD2-transfectant, not only to the level of CD2- cells but to the background. B cells, but not MHC class II-transfected L cells, serve as APC to induce the inhibition of Ag response. The complete abrogation of the response is observed only upon the stimulation through TCR with Ag in the presence of APC but not through either TCR-CD3 or other molecules such as Thy-1. Furthermore, the inhibition can also be observed when anti-CD2 mAb is immobilized on culture plates, suggesting that the inhibition of Ag response results from transducing the negative signal through the CD2 molecule. The experiments on cytoplasmic domain-deleted CD2-transfected T cells reveal that the cytoplasmic portion is responsible for the CD2-mediated abrogation of Ag responses. These results imply that CD2 has important roles in T cell responses not only as an activation and adhesion molecule but also as a regulatory molecule of Ag-specific responses through the TCR.     

Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity

Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.     

Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8(+) T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.     

CD4+ T cell-mediated killing of MHC class II-positive antigen-presenting cells. I. Characterization of target cell recognition by in vivo or in vitro activated CD4+ killer T cells

Ag-specific as well as Ia-restricted killing of certain APC by CD4+ T cells was investigated. The CD4-mediated killing is not only a characteristic of in vitro long term cultured T cell lines or clones, but is also manifest after in vivo priming. Thus, CD4+ killer T cells are generated in vivo as well. CD4+ killer T cells are detected in the Th1, but not in the Th2 subset, and they do not appear to lyse Ia+ APC or bystander cells by a pathway mediated by secreted T cell factors. The latter observation is demonstrated by cold target inhibition experiments as well as by the failure of puromycin to inhibit killing, if applied in doses which completely block lymphokine secretion. Ia+ APC differ in their susceptibility to lysis. Transformed APC are usually better lysed than nontransformed APC. Unstimulated B cells are not killed, while LPS-stimulated B cell blasts are killed. The results of cold target inhibition and bystander killing experiments suggest that CD4+ killer T cells are activated by the common pathway, i.e., by Ag presented in the context of Ia, but killing requires the recognition of additional determinant(s) on APC. It is proposed that these killing-inducing determinants are continuously expressed on most transformed Ia+ cells and on nontransformed but stimulated APC.     

Expansion of functional endogenous antigen-specific CD4+CD25+ regulatory T cells from nonobese diabetic mice

CD4+CD25+Foxp3+ regulatory T cells (T(reg)) are critical for controlling autoimmunity. Evidence suggests that T(reg) development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the T(reg) responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific T(reg) are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific T(reg) in NOD mice. Ag-specific T(reg) from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific T(reg) expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded T(reg) were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific T(reg) were more efficient than polyclonal T(reg) in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific T(reg) in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.     

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.     

Mature dendritic cells prime functionally superior melan-A-specific CD8+ lymphocytes as compared with nonprofessional APC

Priming of melan-A(26/27-35)-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-gamma secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-gamma-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45RO(+) melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL.     

License to heal: bidirectional interaction of antigen-specific regulatory T cells and tolerogenic APC

Naturally occurring CD4(+)CD25(+) regulatory T (T(R)) cells, a component of the innate immune response, which play a key role in the maintenance of self-tolerance, have become the focus of numerous studies over the last decade. These cells inhibit the immune response in an Ag-nonspecific manner, interacting with other T cells. Much less is known about adaptive T(R) cells, which develop in response to chronic antigenic stimulation, and act directly on professional and nonprofessional APC, rendering them tolerogenic and able to elicit the differentiation of CD8(+) and CD4(+) T cells with suppressive activity. In this review, we will discuss data pertaining to the bidirectional interaction between Ag-specific T(R) with APC and their clinical relevance.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

CD4+ T cells can protect APC from CTL-mediated elimination

Professional APC play a central role in generating antiviral CD8(+) CTL immunity. However, the fate of such APC following interaction with these same CTL remains poorly understood. We have shown previously that prolonged Ag presentation persists in the presence of a strong CTL response following HSV infection. In this study, we examined the mechanism of survival of APC in vivo when presenting an immunodominant determinant from HSV. We show that transferred peptide-labeled dendritic cells were eliminated from draining lymph nodes in the presence of HSV-specific CTL. Maturation of dendritic cells with LPS or anti-CD40 before injection protected against CTL lysis in vivo. Furthermore, endogenous APC could be eliminated from draining lymph nodes early after HSV infection by adoptive transfer of HSV-specific CTL, yet the cotransfer of significant virus-specific CD4(+) T cell help promoted prolonged Ag presentation. This suggests that Th cells may assist in prolonging class I-restricted Ag presentation, potentially enhancing CTL recruitment and allowing more efficient T cell priming.     

Alteration of cell surface sialylation regulates antigen-induced naive CD8+ T cell responses

The strength of interactions with APC instructs naive T cells to undergo programmed expansion and differentiation, which is largely determined by the peptide affinity and dose as well as the duration of TCR ligation. Although, most ligands mediating these interactions are terminally sialylated, the impact of the T cell sialylation status on Ag-dependent response remains poorly understood. In this study, by monitoring TCR transgenic CD8+ T cells, OT-I, we show that biochemical desialylation of naive OT-I T cells increases their sensitivity for agonist as well as partial agonist peptides. Desialylation enhances early activation and shortens the duration of TCR stimulation required for proliferation and differentiation, without increasing apoptosis. Moreover, desialylation of naive OT-I T cells augments their response to tumor-presented Ag. These results provide direct evidence for a regulatory role for sialylation in Ag-dependent CD8+ T cell responses and offer a new approach to sensitize or dampen Ag-specific CD8+ T cell responses.