Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Nicotine Inhibits Memory CTL Programming

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.     

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B‐cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL–tumor contacts in vivo. In the therapeutic settings of anti‐CD19 CAR T cells, the majority of CAR T cell–tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti‐tumor responses in vivo.     

Studies on in vivo induction of HIV-1 envelope-specific cytotoxic T lymphocytes by synthetic peptides from the V3 loop region of HIV-1 IIIB gp120

We have previously reported the induction of MHC class I-restricted, CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human immunodeficiency virus type 1 (HIV-1) in mice by a 15-amino acid peptide (R15K) from the V3 loop in gp120. We now present evidence showing that CTL activity induced by R15K was stable for 8–10 weeks after a single injection and that as little as 20 μg peptide was sufficient for efficient CTL induction in vivo. While induction of CTLs was efficient with R15K emulsified in either complete or incomplete Freund's adjuvant, only a low-level CTL response was observed in mice immunized with R15K in either alum or saline. We analyzed a series of carrier-free synthetic peptides ranging in length from 8 to 24 amino acids from the V3 loop region and observed that peptide R10I consisting of 10 amino acids from the middle portion of R15K was more efficient for CTL induction. Additionally, lymph node cells from mice immunized with 24 and 15 amino acid peptides (N24G and R15K, respectively) when restimulated in vitro with R10I exhibited greater HIV-1 env-specific CTL activity than when either of the longer peptides was used for restimulation. A peptide consisting of only 8 amino acids (R8K) was sufficient neither for inducing primary CTLs nor for in vitro restimulation of lymph node CTL precursors. These results establish that a carrier-free 10-amino acid synthetic peptide from the V3 loop region in HIV-1 gp120 has the optimal sequence for efficient induction of HIV env-specific CTLs in mice.     

Use of an In Vivo FTA Assay to Assess the Magnitude,Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

Qualitative characteristics of cytotoxic CD8⁺ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Monophosphoryl lipid A plus IFNγ maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNγ generates mature DCs that produce IL-12 and polarize CD4⁺ T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNγ induce superior tumour antigen-specific CD8⁺ CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNγ DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNγ DCs. The high amounts of induced CTLs are functional as they produce IFNγ and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNγ DCs are very promising for the use in future cancer immunotherapy.     

No evidence for competition between cytotoxic T-lymphocyte responses in HIV-1 infection

Strong competition between cytotoxic T-lymphocytes (CTLs) specific for different epitopes in human immunodeficiency virus (HIV) infection would have important implications for the design of an HIV vaccine. To investigate evidence for this type of competition, we analysed CTL response data from 97 patients with chronic HIV infection who were frequently sampled for up to 96 weeks. For each sample, CTL responses directed against a range of known epitopes in gag, pol and nef were measured using an enzyme-linked immunospot assay. The Lotka–Volterra model of competition was used to predict patterns that would be expected from these data if competitive interactions materially affect CTL numbers. In this application, the model predicts that when hosts make responses to a larger number of epitopes, they would have diminished responses to each epitope and that if one epitope-specific response becomes dramatically smaller, others would increase in size to compensate; conversely if one response grows, others would shrink. Analysis of the experimental data reveals results that are wholly inconsistent with these predictions. In hosts who respond to more epitopes, the average epitope-specific response tends to be larger, not smaller. Furthermore, responses to different epitopes almost always increase in unison or decrease in unison. Our findings are therefore inconsistent with the hypothesis that there is competition between CTL responses directed against different epitopes in HIV infection. This suggests that vaccines that elicit broad responses would be favourable because they would direct a larger total response against the virus, in addition to being more robust to the effects of CTL escape.     

The Role of the E3 Ligase Cbl-B in Murine Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb−/− bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb−/− BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb−/− BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb−/− BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8⁺ OT-I or CD4⁺ OT-II transgenic T cells. However, cblb−/− BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb−/− peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.     

Multiple paths for activation of naive CD8+ T cells: CD4-independent help

CD8(+) CTLs play a pivotal role in immune responses against many viruses and tumors. Two models have been proposed. The "three-cell" model focuses on the role of CD4(+) T cells, proposing that help is only provided to CTLs by CD4(+) T cells that recognize Ag on the same APC. The sequential "two-cell" model proposes that CD4(+) T cells can first interact with APCs, which in turn activate naive CTLs. Although these models provide a general framework for the role of CD4(+) T cells in mediating help for CTLs, a number of issues are unresolved. We have investigated the induction of CTL responses using dendritic cells (DCs) to immunize mice against defined peptide Ags. We find that help is required for activation of naive CTLs when DCs are used as APCs, regardless of the origin or MHC class I restriction of the peptides we studied in this system. However, CD8(+) T cells can provide self-help if they are present at a sufficiently high precursor frequency. The important variable is the total number of T cells responding, because class II-knockout DCs pulsed with two noncompeting peptides are effective in priming.     

Gag-specific cytotoxic T-lymphocyte-based control of primary simian immunodeficiency virus replication in a vaccine trial

Gag-specific cytotoxic T lymphocytes (CTLs) exert strong suppressive pressure on human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. However, it has remained unclear whether they can actually contain primary viral replication. Recent trials of prophylactic vaccines inducing virus-specific T-cell responses have indicated their potential to confer resistance against primary SIV replication in rhesus macaques, while the immunological determinant for this vaccine-based viral control has not been elucidated thus far. Here we present evidence implicating Gag-specific CTLs as responsible for the vaccine-based primary SIV control. Prophylactic vaccination using a Gag-expressing Sendai virus vector resulted in containment of SIVmac239 challenge in all rhesus macaques possessing the major histocompatibility complex (MHC) haplotype 90-120-Ia. In contrast, 90-120-Ia-positive vaccinees failed to contain SIVs carrying multiple gag CTL escape mutations that had been selected, at the cost of viral fitness, in SIVmac239-infected 90-120-Ia-positive macaques. These results show that Gag-specific CTL responses do play a crucial role in the control of wild-type SIVmac239 replication in vaccinees. This study implies the possibility of Gag-specific CTL-based primary HIV containment by prophylactic vaccination, although it also suggests that CTL-based AIDS vaccine efficacy may be abrogated in viral transmission between MHC-matched individuals.     

Liposomal delivery of CTL epitopes to dendritic cells

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.     

Dendritic Cell Cross-Priming Is Essential for Immune Responses to Listeria monocytogenes

Cross-presentation is now recognized as a major mechanism for initiating CD8 T cell responses to virus and tumor antigens in vivo. It provides an elegant mechanism that allows relatively few Dendritic cells (DCs) to initiate primary immune responses while avoiding the consumptive nature of pathogenic infection. CD8 T cells play a major role in anti-bacterial immune responses; however, the contribution of cross-presentation for priming CD8 T cell responses to bacteria, in vivo, is not well established. Listeria monocytogenes (Listeria) is the causative agent of Listeriosis, an opportunistic food-borne bacterial infection that poses a significant public health risk. Here, we employ a transgenic mouse model in which cross-presentation is uniquely inactivated, to investigate cross-priming during primary Listeria infection. We show that cross-priming deficient mice are severely compromised in their ability to generate antigen-specific T cells to stimulate MHC I-restricted CTL responses following Listeria infection. The defect in generation of Listeria-elicited CD8 T cell responses is also apparent in vitro. However, in this setting, the endogenous route of processing Listeria-derived antigens is predominant. This reveals a new experimental dichotomy whereby functional sampling of Listeria-derived antigens in vivo but not in vitro is dependent on cross-presentation of exogenously derived antigen. Thus, under normal physiological circumstances, cross-presentation is demonstrated to play an essential role in priming CD8 T cell responses to bacteria.     

Biologically-Directed Modeling Reflects Cytolytic Clearance of SIV-Infected Cells In Vivo in Macaques

The disappointing outcomes of cellular immune-based vaccines against HIV-1 despite strong evidence for the protective role of CD8⁺ T lymphocytes (CTLs) has prompted revisiting the mechanisms of cellular immunity. Prior data from experiments examining the kinetics of Simian Immunodeficiency Virus (SIV) clearance in infected macaques with or without in vivo CD8 depletion were interpreted as refuting the concept that CTLs suppress SIV/HIV by direct killing of infected cells. Here we briefly review the biological evidence for CTL cytolytic activity in viral infections, and utilize biologically-directed modeling to assess the possibility of a killing mechanism for the antiviral effect of CTLs, taking into account the generation, proliferation, and survival of activated CD4⁺ and CD8⁺ T lymphocytes, as well as the life cycle of the virus. Our analyses of the published macaque data using these models support a killing mechanism, when one considers T lymphocyte and HIV-1 lifecycles, and factors such as the eclipse period before release of virions by infected cells, an exponential pattern of virion production by infected cells, and a variable lifespan for acutely infected cells. We conclude that for SIV/HIV pathogenesis, CTLs deserve their reputation as being cytolytic.     

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of T_h1 and T_h17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher T_h17 and T_h1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c⁺ DCs in the vagina were the predominant APC population responsible for priming these T_h17 responses, and a potent source of IL-6 and IL-1β, important factors for T_h17 differentiation. T_h17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for T_h17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored T_h17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient T_h1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4⁺ T cell anti-viral immunity by priming vaginal DCs to induce T_h17 responses through an IL-1-dependent pathway.     

An engineered novel lentivector specifically transducing dendritic cells and eliciting robust HBV-specific CTL response by upregulating autophagy in T cells

Dendritic cells (DCs) play a predominant role in initiating cell immune responses. Here we generated a DC-targeting lentiviral vector (LVDC-UbHBcAg-LIGHT) and evaluated its capacity to elicit HBV-specific cytotoxic T lymphocyte (CTL) responses. DC-SIGN-mediated specific transduction using this construct was confirmed in DC-SIGN-expressing 293T cells and ex vivo-cultured bone marrow cells. LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in inducing HBV-specific CTLs. Mechanistic studies demonstrated autophagy blocking led to a significant increase in apoptosis and obvious inhibition of CD8 + T cells entry into S-phase, correspondingly attenuated LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses. This observation was supported by accumulation of pro-apoptotic proteins and the main negative cell cycle regulator-CDKN1B that otherwise would be degraded in activated T cells where autophagy preferentially occured. Our findings revealed an important role of autophagy in the activation of T cells and suggested LVDC-UbHBcAg-LIGHT may potentially be used as a therapeutic strategy to combat persistent HBV infection with higher security.     

IL-6 and IFN-α from dsRNA-stimulated dendritic cells control expansion of regulatory T cells

Foxp3⁺CD4⁺ regulatory T cells (Treg) control not only autoimmunity but also the effective immune response against RNA virus infections, which produces virus-derived double-stranded RNA (dsRNA). To induce effective anti-viral immunity, it is a key issue to learn how Treg respond to dsRNA in vitro and in vivo. We here showed that synthetic dsRNA, polyI:C, caused peripheral expansion of functional Treg in a TICAM-1- and IL-6-dependent manner in vivo. PolyI:C did not expand Treg directly, but promoted the expansion of naturally occurring Treg indirectly through IL-6 produced from dendritic cells (DCs). In addition, the expansion of Treg by IL-6 was inhibited by IFN-α from polyI:C-stimulated DCs. These data suggest that the balance of IL-6 and IFN-α in the region of RNA virus infection may determine the number of peripheral Treg, which affects the effective immune responses against viruses.