Cytolytic T-cell Response Research Articles

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Tuning formulation of multipeptide vaccination for melanoma patients at high risk of relapse

18014 Background: Vaccination with MAGE peptides is followed by tumor regression in 10–20% of metastatic melanoma patients with detectable disease, mostly those with only SC or lymph node metastases. Anti-vaccine cytolytic T lymphocyte (CTL) responses have been observed in about 50% of the regressor patients, suggesting that these vaccines were poorly immunogenic. Recent reports have documented convincing CTL responses in patients vaccinated with peptides administered in a water-in-oil emulsion with Montanide, a clinical grade incomplete Freund’s adjuvant. After SC injection, this vaccine stimulates a local inflammation which could help to the recruitment of peptide presenting cells. Considering the absence of an effective treatment to prevent relapses of cutaneous melanoma, we applied this vaccination modality in an adjuvant setting. Methods and Results: We vaccinated 14 HLA-A2 melanoma patients with no evidence of disease but at high risk of relapse, with 4 antigenic peptides (MAGE-3.A2, NA17.A2, Tyrosinase.A2 and gp100.A2) emulsified in Montanide ISA51. Seven patients received 5 vaccinations, every 3 weeks, consisting of 4 injections of 900 μg of one peptide and 1 ml Montanide. Seven other patients received the same vaccines but with 300 μg of each peptide. Toxicity was limited to grade II, at the injection sites. Two thirds of the patients mounted a CTL response to peptide gp100.A2, and 50% to peptide NA17.A2. The lower doses of peptides were as immunogenic as the higher. Conclusions: These encouraging results indicate that this simple vaccination modality is not toxic and clearly immunogenic. We wish to formally assess the role of the immunological adjuvant Montanide in these CTL responses by vaccinating a third group of patients with the same 4 antigenic peptides without Montanide. We will also vaccinate with a mix of the 4 peptides and Montanide all injected at one site, as this will reduce the number of injections and could facilitate immunological helper effects of each peptide on the others. Finally, depending on the immunological results obtained with these two additional cohorts of patients, the best modality will be used in a larger study to evaluate clinical efficacy. No significant financial relationships to disclose.

Precision guiding of cytolytic T-lymphocyte responses in cancer immunotherapy

Precision guiding of cytolytic T-lymphocyte responses in cancer immunotherapy

Tumoral and Immunologic Response After Vaccination of Melanoma Patients With an ALVAC Virus Encoding MAGE Antigens Recognized by T Cells

To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.

Recent Developments in Lipid Drugs

Lipopeptide lipid moieties induce dendritic cell (DC) internalization and epitopes are recognized by MHC, the major histocompatibility complex. HIV-1 (human immunodeficiency virus type 1) lipopeptide vaccine candidate elicits immune responses, and sustains HIV control after highly active antiretroviral therapy (HAART). Mp- and Dp-MART (anti-melanoma lipopeptides) induce strong CTL (cytolytic T lymphocyte) response. New BGTC, BGDA, TGKC lipoplexes mediate gene delivery, e.g., into mouse pancreatic tumor nodules. Triterpene glycyrrhizic acid (GL) inhibits SARS-CoV (severe acute respiratory syndrome associated coronavirus) replication. Compared to CDV (cidofovir), CDV ether lipid esters have enhanced activity against vaccinia (VV) and cowpox (CV) viruses in vitro. Oral treatment of VV and CV infected mice with CDV ether lipid esters, as effective as i.p. CDV, may be useful against orthopoxvirus infections in humans.

Fibrocytes are potent stimulators of anti-virus cytotoxic T cells

Fibrocytes (Fb) are a population of circulating leukocytes reported to be capable of presenting antigen to CD4(+) T lymphocytes. In contrast, no information is available about their capacity to stimulate CD8(+) cytolytic T lymphocyte (CTL) responses. To this end, Fb were isolated from porcine blood to investigate their ability to stimulate CTL responses using a classical swine fever virus model. The isolated Fb (referred to as primary Fb) displayed the phenotype previously reported for mouse and human Fb, particularly in terms of the surface proteins necessary for antigen presentation, major histocompatibility complex (MHC) classes I and II, and CD80/86. These primary Fb endocytosed and degraded antigen efficiently. In absence of exogenous stimuli, endocytosis and MHC II expression were lost when the Fb were passaged and cultured. Treatment of such secondary Fb with interferon-gamma (IFN-gamma) restored the MHC II expression. The primary and secondary Fb were capable of stimulating antigen-specific CD4(+) T lymphocytes relating to previous reports. In addition, an efficient stimulation of virus-specific CD8(+)CTL was measured in terms of CD8(+) T cell proliferation, IFN-gamma production, and cytotoxic activity. This was noted even at low Fb/T lymphocyte ratios, at which dendritic cells were less efficient. Although IFN-gamma pretreatment of Fb was not necessary for this function, it could enhance the Fb activity. These results demonstrate that Fb are efficient, accessory cells for the presentation of viral antigen to specific CD8(+) CTL.

CD8+ T-Cell Tolerance Induced by Delivery of Antigen to the Anterior Chamber is not the Result of de Facto Intravenous or Mucosal Administration of Antigen

Purpose: We tested whether antigen administration via the anterior chamber (a.c.) was equivalent to intravenous (i.v.) or mucosal administration of antigen. Methods: Ovalbumin (OVA)-specific CD8+ T cells (OT-I) were enumerated in lymphoid tissues of C57Bl/6 (B6) mice via adoptive transfer after the same amount of antigen was administered via a.c., i.v., or mucosal routes. Lytic activity was measured in B6 and γδT cell-deficient B6 mice given OVA via a.c., i.v, or mucosal routes after injection with OVA in adjuvant. Results: OVA a.c. induced a pattern of T-cell proliferation distinct from i.v. or mucosal administration. A.c. and i.v., but not mucosal, OVA induced cytolytic T lymphocyte (CTL) tolerance. The inhibition of CTL responses was significantly greater in mice given OVA a.c. rather than i.v. γδT cells contributed to a.c.–, but not i.v.-, induced CTL tolerance. Conclusions: A.c. administration of antigen is not de-facto i.v. or mucosal administration of antigen.

Lack of Increased Clinical Efficacy When Interleukin-12 Is Added to Rituximab in B-Cell Lymphoma Patients Is Related to Inadequate Delivery of the Cytokine to the Sites of Lymphoma.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Binding of the Fab domain to B-cells directly induces apoptosis, while the Fc domain recruits immune effector functions to mediate cell lysis. Because rituximab therapy alone does not result in durable responses for all patients, combination therapies have been explored. We have previously shown in a phase I study that interleukin-12 (IL-12), which facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells, can be safely combined with rituximab and that IL-12 significantly upregulated gamma interferon, CXCL10 (inducible protein-10) and NK cell activity in the peripheral blood. To confirm whether IL-12 could augment the immune mediated cell lysis induced by rituximab, a subsequent randomized phase II study of the combination was performed in patients with B-cell lymphoma. While the combination of IL-12 and rituximab was well tolerated with acceptable toxicity, only moderate disease activity was seen and the response rate to the combination was similar to that seen with rituximab alone. Additionally, the sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. This study was therefore performed to determine potential biologic reasons for the lack of increased clinical efficacy when IL-12 was added to rituximab therapy in patients with B-cell non-Hodgkin lymphoma.Of the 52 patients treated on the phase II study, 8 patients had matched tumor biopsies and peripheral blood specimens obtained prior to therapy and again 2 weeks after treatment was started. Six of the patients were receiving IL-12 plus rituximab at the time the specimens were obtained while 2 were receiving rituximab alone. Gene expression array analysis using the Affymetrix U133 plus chip was performed on RNA isolated from cells from involved lymph nodes and from peripheral blood mononuclear cells. Specimens from the peripheral blood of patients who received IL-12 in combination with rituximab showed a greater than 5-fold increase in the expression of multiple genes known to be upregulated by IL-12 signaling including interferon gamma, CXCL10, IFIT2 and 4 (interferon-induced protein with tetratricopeptide repeats 2 and 4), IL-8 and CXCL2 (macrophage inflammatory protein-2). These increases in gene expression were not seen in the peripheral blood of patients who received rituximab alone. Furthermore, the significant changes seen in cells obtained from the peripheral blood were not seen in cells obtained from lymph nodes involved by lymphoma, despite the samples being obtained from the same patient on the same day. In the tumor specimens, the changes in gene expression involved none of the genes downstream of IL-12 signaling. Instead, many of the upregulated genes were associated with cell cycle and spindle checkpoint proteins suggesting ongoing tumor cell proliferation. In conclusion, while IL-12 significantly upregulated gene expression in the peripheral blood, the same changes were not seen in the tumor. This would suggest that systemically administered IL-12 may not be effectively delivered to the site of tumor involvement. This finding may explain the lack of additional clinical benefit when IL-12 was added to rituximab as therapy for patients with non-Hodgkin lymphoma.

A Combination of flk1-Based DNA Vaccine and an Immunomodulatory Gene (IL-12) in the Treatment of Murine Cancer

The aim of this study was to investigate the antivasculature effects and the antitumor effects of combining attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) with plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. Mouse models of Gl261 glioblastoma were treated with combining orally given attenuated Salmonella typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. The volumes of tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by a 4-hour 51Cr release assay, vessle density and tumor cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. Compared to mice receiving single agent therapy, received either oral immunization flk1-based vaccine only or the therapeutic gene-IL-12 plasmid DNA only or those in the control group, the combination therapy groups developed a strong CTL response and showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization and cell proliferation in these mice. The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.

Peptides complexed with the protein HSP70 generate efficient human cytolytic T-lymphocyte responses

Microbial HSPs (heat-shock proteins) are implicated in the induction of the innate and adaptive arms of the immune response. We set out to determine whether peptides complexed with HSP70 generate efficient CTL (cytolytic T-lymphocyte) responses. Human dendritic cells pulsed with peptide-loaded microbial HSP70 complexes generate potent antigen-specific CTL responses. Using fluorescence anisotropy, we have calculated the peptide-binding affinity of mycobacterial HSP70 (K(D)=14 microM) and show that 120 pM HSP70-bound peptide is sufficient to generate a peptide-specific CTL response that is four orders of magnitude more efficient than the peptide alone. Through the generation of mycobacterial HSP70 truncations, we find that the minimal 136 amino acid, mycobacterial HSP70 peptide-binding domain is sufficient to generate CTL responses. The design of an HSP70 mutant, in which the peptide-binding site of HSP70 is filled with a bulky hydrophobic residue, leads to a large decrease in the peptide-binding affinity. This mutant HSP70 retains stimulatory capacity but is unable to generate CTL and has separated antigen delivery from immunostimulation of dendritic cells.

Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma

Abstract A role of CD4 + cells in the regulation of immune responses has steadily gained renewed recognition. The understanding of these T-regulatory (T-reg) cells in the generation of antitumor cytolytic T lymphocyte (CTL) response is therefore important. It has been shown that immunization with specific peptides, DNA, or tumor lysate-based vaccines can induce CTL responses in vivo. We have immunized melanoma patients with major histocompatibility complex (MHC) class I restricted peptide- or melanoma tumor lysate-loaded antigen-presenting cell (APC)-based vaccines and have monitored the generation of CTL responses and T-reg cell responses, if any. Using tetramer staining and limiting dilution analyses as monitors of CTL responses, we found significant increases in the number of antigen-specific CTL in circulation after vaccination with the MART-1 27–35 peptide (AAGIGILTV)-pulsed autologous APC, the MAGE-1 161–169 peptide (EADPTGHSY)-pulsed APC, or with autologous tumor lysate-pulsed APC. The antigen-specific CTL reached the peak expansion by day 7 and then declined to the prevaccine levels by day 28. The decline in the CTL response was associated by a concomitant expansion of CD4 + CD25 +T cells. Analysis of postvaccine peripheral blood lymphocytes (PBL) from patients showed an increased amount of interleukin (IL)-10 secretion on in vitro stimulation with IL-2 after successive vaccination. Triple color flow cytometric analyses revealed cytoplasmic IL-10 in the CD4 +CD25 + T-cell fraction and the number of CD4 +CD25 + IL-10 + T cells were found to increase significantly in postvaccine PBL. These observations have implications in tumor antigen and APC/dendritic cell (DC)-based cancer vaccine strategies.

Immunization of melanoma patients with a MAGE-3 peptide combined with IFNa

2606 Background: Melanoma patients with lymph node metastasis at diagnosis have a less than 60% survival rate at 5 years. Interferon-a (IFNa) is used as adjuvant treatment for such patients, even though recent studies showed no increase in survival. Vaccination with tumor-specific MAGE peptides may induce tumor regression in 10–20% of metastatic melanoma patients, mostly those with only subcutaneous or lymph node metastases. However, cytolytic T lymphocyte (CTL) responses to the vaccine antigens have been observed only in half of the patients with tumor regression. We examined whether IFNa could serve as an adjuvant to induce CTL responses against tumor-specific peptides in mice and in melanoma patients without evidence of disease. Methods and Results: No detectable CTL responses can be observed in DBA/2 mice vaccinated with 50 ug of peptide P1A, which is carried on mastocytoma cells P815, injected subcutaneously without adjuvant. In the presence of immunological adjuvant AS02B (QS21/MPL from GlaxoSmithKl...

Alpha1,3-Galactosyltransferase gene-knockout miniature swine produce natural cytotoxic anti-Gal antibodies.

The expression of galactose alpha 1,3 galactose (Gal) in pigs has proved a barrier to xenotransplantation. Miniature swine lacking Gal (Gal pigs) have been produced by nuclear transfer/embryo transfer. The tissues of five Gal pigs of SLA dd haplotype (SLA) were tested for the presence of Gal epitopes by staining with the Griffonia simplicifolia IB4 lectin. Their sera were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (Gal) SLA-matched pigs; serum cytotoxicity was also assessed. The cellular responses of PBMC from Gal swine toward Gal SLA-matched PBMC were tested by mixed leukocyte reaction and cell-mediated lympholysis assays. None of the tissues tested showed Gal expression. Sera from all five Gal pigs manifested IgM binding to Gal pig PBMC, and sera from three showed IgG binding. In all five cases, cytotoxicity to Gal cells could be demonstrated, which was lost after treatment of the sera with dithiothreitol, indicating IgM antibody-mediated cytotoxicity. PBMC from Gal swine had no proliferative or cytolytic T-cell response toward Gal SLA-matched PBMC. Gal pigs do not express Gal epitopes and develop anti-Gal antibodies that are cytotoxic to Gal pig cells. The absence of an in vitro cellular immune response between Gal and Gal pigs is related to their identical SLA haplotype and indicates the absence of immunogenicity of Gal in T-cell responses. The model of Gal organ transplantation into a Gal SLA-matched recipient would be a valuable large animal model in the study of accommodation or B-cell tolerance.

NCCTG trial of concomitant or sequential IL-12 in combination with rituximab in previously treated non-Hodgkin lymphoma patients

6591 Background: Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Interleukin-12 (IL-12) facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells. This study was performed to determine the efficacy and toxicity of the combination of IL-12 and rituximab in patients with B-cell non-Hodgkin lymphoma (NHL). Methods: 52 patients with previously treated, histologically confirmed CD20+ low-grade B-cell NHL or mantle cell NHL have been enrolled in this phase 2 study. Patients were randomized to one of two arms: concurrent treatment with rituximab+IL-12 (Arm A) or rituximab with subsequent treatment with IL-12 after documented non-response, defined as less than a partial response (Arm B). Treatment consisted of rituximab 375 mg/m2 on days 1, 8, 15, and 22; and IL-12 300 ng/kg given SQ twice weekly starting on day 2 of cycle 1 for Arm A or upon progression or non-response for Arm B. We focus here on the interim analyses of the first 21 evaluable patients in each arm. Results: To date, 14 of 21 patients on Arm A and 9 of 21 patients on Arm B have discontinued treatment. 8 patients on Arm B have progressed, and have gone on to receive IL-12. On Arm A, 8 patients had grade 3 and 2 patients grade 4 toxicities vs. 2 patients on Arm B with grade 3 toxicities. Of the first 21 evaluable patients accrued to Arm A, 8 responses (38%) have been observed to date. Eight responses (38%) have also been observed on Arm B. All responses observed in Arm B were due to rituximab and none from subsequent treatment with IL-12. Based on these results, accrual to Arm B was closed. However, the subset of mantle cell NHL patients on Arm A had a higher response rate (3/5 patients). As a result, mantle cell NHL patients will continue to be accrued to Arm A. Conclusions: The combination of IL-12 and rituximab was well tolerated. The sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. The concomitant use of IL-12 and rituximab had moderate disease activity in this study; however, promising clinical activity was seen in patients with mantle cell NHL. No significant financial relationships to disclose.

NCCTG trial of concomitant or sequential IL-12 in combination with rituximab in previously treated non-Hodgkin lymphoma patients

6591 Background: Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Interleukin-12 (IL-12) facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells. This study was performed to determine the efficacy and toxicity of the combination of IL-12 and rituximab in patients with B-cell non-Hodgkin lymphoma (NHL). Methods: 52 patients with previously treated, histologically confirmed CD20+ low-grade B-cell NHL or mantle cell NHL have been enrolled in this phase 2 study. Patients were randomized to one of two arms: concurrent treatment with rituximab+IL-12 (Arm A) or rituximab with subsequent treatment with IL-12 after documented non-response, defined as less than a partial response (Arm B). Treatment consisted of rituximab 375 mg/m2 on days 1, 8, 15, and 22; and IL-12 300 ng/kg given SQ twice weekly starting on day 2 of cycle 1 for Arm A or upon progression or non-response for Arm B. We focus here on the interim analyses of the first 21 evaluable patients in each arm. Results: To date, 14 of 21 patients on Arm A and 9 of 21 patients on Arm B have discontinued treatment. 8 patients on Arm B have progressed, and have gone on to receive IL-12. On Arm A, 8 patients had grade 3 and 2 patients grade 4 toxicities vs. 2 patients on Arm B with grade 3 toxicities. Of the first 21 evaluable patients accrued to Arm A, 8 responses (38%) have been observed to date. Eight responses (38%) have also been observed on Arm B. All responses observed in Arm B were due to rituximab and none from subsequent treatment with IL-12. Based on these results, accrual to Arm B was closed. However, the subset of mantle cell NHL patients on Arm A had a higher response rate (3/5 patients). As a result, mantle cell NHL patients will continue to be accrued to Arm A. Conclusions: The combination of IL-12 and rituximab was well tolerated. The sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. The concomitant use of IL-12 and rituximab had moderate disease activity in this study; however, promising clinical activity was seen in patients with mantle cell NHL. No significant financial relationships to disclose.

Exocytosis and Fas mediated cytolytic mechanisms exert protection from West Nile virus induced encephalitis in mice.

Infection of mice with the flaviviruses West Nile virus (WNV) and Murray Valley encephalitis (MVE) induces cytolytic T-cell responses which are highly cross-reactive on target cells infected with heterologous flaviviruses. Of C57BL/6 mice infected with low doses (10(2)-10(6) PFU) of either virus, 30-40% develop encephalitis and die within 10-12 days. Mice with defects in the Fas or granule exocytosis (perforin and granzymes A and B) pathway of cellular cytotoxicity display reduced mortality and increased survival time when infected with MVE and are protected from encephalitis when deficient in both pathways. This contrasts with infection with WNV where defects in these cytolytic mechanisms increase the percentage of mice that succumb to encephalitis. Thus, no generalizations as to protective or detrimental effects of cytolytic effector functions in recovery from closely related flavivirus infections can be made. Virus-host immune interactions have to be assessed individually and cannot be generalized.

Simulating the effect of vaccine-induced immune responses on HIV infection

The need for anti-HIV-1 vaccines is universally recognized. Although several potential vaccine formulations are being tested in clinical trials, the complexity of the viral system and the length of the experimentation required and its costs makes the goal of obtaining such a vaccine still elusive. We have built a mathematical model for the simulation of HIV-1 infection spreading into the body, which allows us study in silico the effect of hypothetical anti-HIV-1 vaccines having different properties. In particular, vaccines eliciting a cytolytic T-cell response, a humoral response, or both can be simulated. The vaccines considered can be envisaged either as preventive or therapeutic and can have different strength. The kinetic parameters used for solving the model are those of HIV-1 infection obtained from experimental and clinical observations. The vaccines are instead characterized by parameters that can be varied in order to mimic different behaviors: the rate of killing of the single effector cell and the rate of neutralization of the single antibody molecule; and the level of the immune response raised. The model allows us to predict which characteristics of immunogenicity a preventive or therapeutic vaccine should possess to be efficacious, and which are the key factors that most likely will affect its ability to control the spread of the infection. We discuss here the conclusions that can be drawn from a such a model and some of its limitations.

The role of TNF-TNFR2 interactions in generation of CTL responses and clearance of hepatic adenovirus infection.

Deficiency or inhibition of tumor necrosis factor (TNF) significantly prolongs hepatic expression of recombinant adenoviral vectors. To explore mechanisms responsible for this observation, the present studies examined the effects of TNF versus TNF receptor 1 (TNFR1) or TNFR2 deficiency on the course of antiviral-immune responses to a replication-deficient, beta-galactosidase-encoding recombinant adenovirus (AdCMV-lacZ). Clearance of AdCMV-lacZ was significantly delayed in TNF-deficient mice. Less pronounced but significant delays in AdCMV-lacZ clearance were observed in TNFR2-deficient but not TNFR1-deficient mice. Numbers of interferon-gamma expressing intrahepatic lymphocytes (IHL) were similar in AdCMV-lacZ-infected, TNF-deficient, TNFR1-deficient, TNFR2-deficient, and control mice. However, IHL isolated from AdCMV-lacZ-infected, TNF-deficient or AdCMV-lacZ-infected, TNFR2-deficient mice exhibited decreased levels of FasL expression and adenovirus-specific cytolytic T lymphocyte (CTL) activity. Similar defects in allo-specific killing of Fas-sensitive hepatocyte targets by TNF-deficient or TNFR2-deficient but not TNFR1-deficient CTL were also noted. No defects in generation of allo-specific cytotoxicity directed against perforin-sensitive target cells were noted in TNF-, TNFR1-, or TNFR2-deficient lymphocytes. These findings indicate that TNF/TNFR2 interactions facilitate generation of FasL-dependent CTL effector pathways that play an important role in in vivo antiviral-immune responses in the liver.

Adding Cytokines to Monoclonal Antibody Therapy: Does the Concurrent Administration of Interleukin-12 Add to the Efficacy of Rituximab in B-cell Non-Hodgkin Lymphoma?

Interleukin-12 (IL-12) is a cytokine that facilitates cytolytic T-cell responses, enhances the lytic activity of NK cells and induces the secretion of interferon-gamma by both T and NK cells. Binding of rituximab, a chimeric murine/human monoclonal antibody, to CD20 on B-lymphocytes induces apoptosis and the Fc domain of the antibody recruits immune effector functions to mediate cell lysis. Therefore, combining IL-12 with rituximab in patients with B-cell non-Hodgkin lymphoma (NHL) may augment the immune mediated cell lysis induced by rituximab. To determine whether a synergistic effect exists when IL-12 is given in combination with rituximab, clinical trials are being done to evaluate the clinical efficacy of the combination. The two agents, when given in combination, significantly upregulate the patient's immune mechanisms. The combination upregulates gamma interferon and IP-10 expression and increases NK cell lytic activity. The combination appears to have significant clinical activity with a high clinical response rate in early phase clinical trials. However, a randomized controlled trial will be required to determine whether the addition of IL-12 adds to the efficacy of rituximab in B-cell NHL.

Effective therapeutic anticancer vaccines based on precision guiding of cytolytic T lymphocytes

In natural immune responses CD4+ T helper (Th) cells, reactive with peptide antigens presented by major histocompatibility complex (MHC) class II molecules on dendritic cells (DC), can drive the maturation of DC that is required for induction of CD8+ cytolytic T-lymphocyte (CTL) immunity. Proper induction, expansion and maintenance of CTL responses are achieved through delicate interactions between CD4+ T cells, DC and CD8+ T cells involving several ligand-receptor pairs. Th cells to a large extent operate through up-regulation of CD40L, which then interacts with CD40 on DC to cause DC maturation. Subsequent CTL induction by activated DC requires CD80/CD86 on the DC to interact with the CD28 costimulatory receptor on CD8+ T cells. For maintenance and full expansion of CTL, interaction of the DC-expressed 4-1BB ligand with its receptor 4-1BB on CTL is also important. Alternative molecular triggers of DC activation that can support induction of powerful CTL responses include agonistic anti-CD40 antibody or ligands of Toll-like receptors (TLR) such as LPS (TLR4 ligand) or oligodeoxynucleotides containing CpG-motifs (TLR9 ligand). The combination of CpG adjuvant with a 35 amino acid long synthetic peptide comprising both tumor-specific CTL and Th epitopes proved to be a highly effective vaccine formulation capable of inducing therapeutic immunity against human papillomavirus-induced mouse tumors. The recently acquired insights into antigen presentation and costimulatory signals have made possible the development of a new generation of therapeutic anticancer vaccines.