Biomedical subjects

Walter J Storkus

Publications and source records attributed to Walter J Storkus.

At least 37 records · Page 2Linked to original sources

PubMed2004

alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity.

Using the principle of functional polarization of dendritic cells (DCs), we have developed a novel protocol to generate human DCs combining the three features critical for the induction of type-1 immunity: (a) fully mature status; (b) responsiveness to secondary lymphoid organ chemokines; and (c) high interleukin-12p70 (IL-12p70)-producing ability. We show that IFN-alpha and polyinosinic:polycytidylic acid (p-I:C) synergize with the "classical" type-1-polarizing cytokine cocktail [tumor necrosis factor alpha (TNFalpha)/IL-1beta/IFNgamma], allowing for serum-free generation of fully mature type-1-polarized DCs (DC1). Such "alpha-type-1-polarized DC(s)" (alphaDC1) show high migratory responses to the CCR7 ligand, 6C-kine but produce much higher levels of IL-12p70 as compared to TNFalpha/IL-1beta/IL-6/prostaglandin E2 (PGE2)-matured DCs (sDC), the current "gold standard" in DC-based cancer vaccination. A single round of in vitro sensitization with alphaDC1 (versus sDCs) induces up to 40-fold higher numbers of long-lived CTLs against melanoma-associated antigens: MART-1, gp100, and tyrosinase. Serum-free generation of alphaDC1 allows, for the first time, the clinical application of DCs that combine the key three features important for their efficacy as anticancer vaccines.

Cell Polarity↗

PubMed2004

Delivery of interferon-alpha transfected dendritic cells into central nervous system tumors enhances the antitumor efficacy of peripheral peptide-based vaccines.

We evaluated the effects, on immunity and survival, of injection of interferon (IFN)-alpha-transfected dendritic cells (DC-IFN-alpha) into intracranial tumors in mice immunized previously with syngeneic dendritic cells (DCs) pulsed either with ovalbumin-derived CTL or T helper epitopes. These immunizations protected animals from s.c. challenge with ovalbumin-expressing M05 melanoma (class I+ and class II-negative). Notably, antiovalbumin CTL responses were observed in animals vaccinated with an ovalbumin-derived T helper epitope but only after the mice were challenged with M05 cells. This cross-priming of CTL was dependent on both CD4+ and CD8+ T cells. Because we observed that s.c., but not intracranial, tumors were infiltrated with CD11c+ DCs, and because IFN-alpha promotes the activation and survival of both DCs and T cells, we evaluated the combinational antitumor effects of injecting adenoviral (Ad)-IFN-alpha-engineered DCs into intracranial M05 tumors in preimmunized mice. Delivery of DC-IFN-alpha prolonged survival. This was most notable for animals prevaccinated with both the CTL and T helper ovalbumin epitopes, with 60% (6 of 10) of mice (versus 0 of 10 of control animals) surviving for > 80 days after tumor challenge. DC-IFN-alpha appeared to persist longer than mock-transfected DCs within the intracranial tumor microenvironment, and DC-IFN-alpha-treated mice exhibited enhanced levels of ovalbumin-specific CTL in draining cervical lymph nodes. On the basis of these results, we believe that local expression of IFN-alpha by DCs within the intracranial tumor site may enhance the clinical efficacy of peripheral vaccine approaches for brain tumors.

Amino Acid Sequence↗

PubMed2004

Disease-stage variance in functional CD4(+) T-cell responses against novel pan-human leukocyte antigen-D region presented human papillomavirus-16 E7 epitopes.

Given the anticipated clinical importance of helper and regulatory CD4(+) T cells reactive against human papillomavirus-16 E7 in the cervical carcinoma setting, we performed this study to identify novel E7-derived T helper (Th) epitopes and to characterize functional anti-E7 Th responses in normal donors and patients with cervical intraepithelial neoplasia I-III or cervical cancer. Candidate pan-HLA-DR (D region) binding peptides were identified and synthesized based on results obtained using a predictive computer algorithm, then applied in short-term in vitro T-cell sensitization assays. Using IFN-gamma/IL-5 (interleukin 5) enzyme-linked immunospot assays as readouts for Th1-type and Th2-type CD4(+) T-cell responses, respectively, we identified three E7-derived T helper epitopes (E7(1-12), E7(48-62), and E7(62-75)), two of which are novel. Normal donor CD4(+) T cells failed to react against these E7 peptides, whereas patients with premalignant cervical intraepithelial neoplasia I-III lesions displayed preferential Th1-type responses against all three E7 epitopes. Th1-type responses were still observed to the E7(48-62) but not to the E7(1-12) and E7(62-75) peptides in cancer patients, where these latter two epitopes evoked Th2-type responses. Notably all responders to the E7(1-12) and E7(62-75) peptides expressed the HLA-DR4 or -DR15 alleles, whereas all responders to the E7(48-62) peptide failed to express the HLA-DR4 allele. Our results are consistent with a model in which cervical cancer progression is linked to an undesirable Th1- to Th2-type shift in functional CD4(+) T cell responses to two novel E7-derived epitopes. These peptides may prove important in vaccines to promote and maintain protective Th1-type antihuman papillomavirus immunity and in the immune monitoring of treated patients harboring HPV-16(+) malignancies.

Adult↗

PubMed2003

Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity.

Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when compared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralateral, untreated tumors. Both CD4+ and CD8+ T cells were required for tumor rejection. CD8+ splenic T cells from mice treated with AdIL12/IL18DC produced the highest levels of IFN-gamma in response to tumor rechallenge in vitro and displayed the broadest repertoire of Tc1-type reactivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immunogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor alpha, with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors.

Adenoviridae↗

PubMed2003

Dendritic cells mediate NK cell help for Th1 and CTL responses: two-signal requirement for the induction of NK cell helper function.

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other's functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized "effector/memory" DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-gamma, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, "helper" function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.

CD8-Positive T-Lymphocytes↗

PubMed2003

Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma.

We have evaluated CD8+ and CD4+ T-cell responses against a new tumor-associated antigen, the receptor tyrosine kinase EphA2, which is broadly expressed in diverse cancer histologies and is frequently overexpressed in advanced stage/metastatic disease. We report herein that EphA2 is overexpressed in renal cell carcinoma (RCC) cell lines and clinical specimens of RCC, and find that the highest levels of EphA2 are consistently found in the most advanced stages of the disease. We identified and synthesized five putative HLA class I-binding and three class II-binding peptides derived from EphA2 that might serve as targets for immune reactivity. Each peptide induced specific, tumor-reactive CD8+ or CD4+T-cell responses as measured using IFN-gamma enzyme-linked immunospot assays. The EphA2 peptides elicited relatively weak responses from CD8+ T cells derived from normal healthy volunteers or from RCC patients with active disease. In marked contrast, immune reactivity to EphA2-derived epitopes was greatly enhanced in CD8+ T cells that had been isolated from patients who were rendered disease-free, after surgery. Furthermore, enzyme-linked immunospot analyses demonstrated prominent EphA2-restricted T-helper 1-type CD4+ T cell activity in patients with early stage disease, whereas T-helper 2-type and T regulatory-type responses predominated in patients with more advanced forms of RCC. These data suggest that the immune system of cancer patients actively monitors EphA2-derived epitopes, and that the magnitude and character of T-cell responses to EphA2 epitopes may convey much-needed predictive information about disease stage and outcome.

Adult↗

PubMed2003

Melanocyte-specific immune response in melanoma and vitiligo: two faces of the same coin?

The appearance of depigmentation during the course of malignant melanoma has been considered a good prognostic sign. Is it only a side-effect, informative of the immune system's response to the treatment, or does it act as a necessary amplifier of these clinically important anti-tumor responses? The current review will attempt to tackle this question by reviewing the current literature, as well as by posing some novel hypotheses. Understanding the nature of humoral and cellular immune responses directed against normal melanocytes and their malignant counterparts may lead to the design of improved therapeutic strategies relevant to both vitiligo and melanoma.

Animals↗

PubMed2003

Effective induction of antiglioma cytotoxic T cells by coadministration of interferon-beta gene vector and dendritic cells.

As type I interferons (IFNs) enhance the stimulatory activity of dendritic cells (DCs), we hypothesized that transfection of glioma cells with the IFN-beta gene in the presence of DCs would provide particularly effective antitumor activity by both facilitating apoptosis of glioma cells and presenting the resulting glioma antigens to T cell by DCs, thereby inducing specific T-cell responses against glioma cells. A mouse glioma cell line 203G was first transfected with cDNA encoding IFN-beta using cationic liposomes, then cocultured with syngeneic bone marrow-derived DCs and naïve splenic T cells. The 203G cells were almost completely killed following 96-hour coculture with DCs and T cells, and strong tumor-specific cytotoxic T-lymphocyte (CTL) activity accompanied by high level interleukin (IL)-12 and IFN-gamma production was observed in culture. In addition, omission of either IFN-beta gene delivery, DCs or T cells from the coculture completely abrogated the induction of the CTL activity, suggesting that the combination of these components was required to elicit an optimal effect. On the basis of these in vitro data, syngeneic animals bearing subcutaneous 203G tumors received intratumoral injections of IFN-beta gene and DCs. Suppression of the tumor growth by this combinational therapy was superior to treatment with DC or IFN-beta gene solely. This combination may constitute a new therapeutic strategy to induce potent antiglioma immune responses.

Animals↗

PubMed2003

Ex vivo priming of naïve T cells into EBV-specific Th1/Tc1 effector cells by mature autologous DC loaded with apoptotic/necrotic LCL.

Posttransplant lymphoproliferative disorders (PTLDs) represent life-threatening complications of bone marrow and solid organ transplantation (SOTx). These are B-cell malignancies triggered by Epstein-Barr Virus (EBV) infection in chronically immunosuppressed (IS) recipients. Immunosuppressed EBV seronegative (EBV(-)) organ recipients are at highest risk of developing PTLD owing to the lack of anti-EBV memory T cells to control subsequent EBV challenges. Our aim is to establish effective anti-EBV T-cell generation protocols for prevention or treatment of PTLD encountered in SOTx. We have used autologous dendritic cells (DCs) loaded with apoptotic/necrotic lymphoblastoid cell lines (LCLs) to evaluate the ability of such an approach to activate naïve T cells in vitro. In EBV(-) individuals, both CD8+ and CD4+ T-cell responses were amplified by this approach, as detected by IFN-gamma ELISPOT and cytotoxicity assays. The CD8+ T cells were poly-specific anti-EBNA3 A, -LMP2 and -BMLF1, with uniform reversion to a CD45RO+/RA-phenotype, decreased CD62L expression, and up-regulation of the activation markers CD28 and CD69. Addition of rhIL-12 improved anti-viral T-cell responses and reduced the functional differences observed between EBV(+) and EBV(-) responders. In conclusion, the DC/LCL method promotes cross-presentation of EBV-associated epitopes and may serve as an effective protocol for the adoptive immunotherapy of PTLD in EBV(-) SOTx patients.

Antigen Presentation↗

PubMed2003

Immunopolarization of CD4+ and CD8+ T cells to Type-1-like is associated with melanocyte loss in human vitiligo.

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. High frequencies of melanocyte-reactive cytotoxic T cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional T-cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. We isolated T cells from both perilesional and nonlesional skin biopsies obtained from five vitiligo patients, then cloned and analyzed their profile of cytokine production after short-term, nonspecific expansion in vitro. Perilesional T-cell clones (TCC) derived from patients with vitiligo exhibited a predominant Type-1-like cytokine secretion profile, whereas the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Detailed analysis of broad spectrum of cytokines produced by perilesional- and nonlesional-derived CD4+ and CD8+ TCC confirmed polarization toward Type-1-like in both CD4 and CD8 compartments, which paralleled depigmentation process observed locally in the skin. Furthermore, CD8+ TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8+ TCC isolated from perilesional biopsies of two patients with vitiligo. Finally, in two of five patients, tetramer analysis revealed presence of high frequencies of Mart-1-specific CD8 T cells in T-cell lines derived from perilesional skin. Altogether our data support the role of cellular mechanisms playing a significant part in the destruction of melanocytes in human autoimmune vitiligo.

Adult↗

PubMed2003

MAGE-6 encodes HLA-DRbeta1*0401-presented epitopes recognized by CD4+ T cells from patients with melanoma or renal cell carcinoma.

CD4+ T cells modulate the magnitude and durability of CTL responses in vivo and may serve as potent effector cells within the tumor microenvironment. The current study was undertaken to define novel epitopes from the broadly expressed tumor antigen MAGE-6 that are recognized by CD4+ T cells. We have combined the use of a HLA-DR4/peptide binding algorithm with the IFN-gamma enzyme-linked immunospot assay to identify four nonoverlapping sequences derived from the MAGE-6 protein that served as CD4+ T-cell epitopes in HLA-DR4+ donors. Strikingly, patients with active melanoma or renal cell carcinoma failed to secrete IFN-gamma in response to MAGE-6-derived epitopes, whereas both normal donors and cancer patients with no current evidence of disease were responsive, particularly after short-term in vitro stimulations with peptide-pulsed dendritic cells. Importantly, peptide-specific CD4+ T cells also recognized HLA-DRbeta1*0401+ tumor cells that constitutively expressed the MAGE-6 protein and autologous HLA-DRbeta1*0401+ dendritic cells transfected with MAGE-6 cDNA-elicited CD4+ T cells that reacted against individual peptide epitopes in vitro. These data suggest that MAGE-6-derived epitopes could serve as useful vaccine candidate components and may provide an immune-monitoring index of clinically important Th1-type immunity in patients with renal cell carcinoma or melanoma.

Adenoviridae↗

PubMed2002

Interleukin 18 gene transfer expands the repertoire of antitumor Th1-type immunity elicited by dendritic cell-based vaccines in association with enhanced therapeutic efficacy.

Dendritic cells (DCs) are potent antigen-presenting cells that can prime and boost systemic antitumor immunity. Here, we have evaluated the ability of DCs transfected to secrete the potent Th1-biasing cytokine interleukin (IL)-18 to promote enhanced antitumor immunity in a mouse sarcoma model. DCs infected with a recombinant adenovirus encoding IL-18 (AdIL18DC) expressed higher levels of MHC and costimulatory molecules and were better stimulators than control DCs in mixed leukocyte reactions in vitro. Immunization of BALB/c mice bearing established day 7 CMS4 tumors with tumor peptide-pulsed control Adpsi5-transfected DCs or nontransduced DCs significantly inhibited the growth of established tumors but did not lead to complete regression of established tumors. Importantly, immunization with antigen-loaded AdIL18DC resulted in tumor rejection or further suppression of tumor growth when compared with controls. The repertoire of naturally presented tumor peptides recognized by splenocytes (as deduced in IFN-gamma ELISA assays) from AdIL18DC-treated animals was far more diverse and of greater magnitude than that of all other groups, in association with improved therapeutic outcome. These results support the ability of IL-18 gene transfer to enhance the capacity of DCs to drive broadly reactive Th1-type therapeutic immunity prompted by single peptide epitope-based vaccines (i.e., epitope spreading).

Adenoviridae↗

PubMed2002

Disease-associated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell responses against MAGE-6 in HLA-DRB10401(+) patients with renal cell carcinoma or melanoma.

T helper type 1 (Th1)-type CD4(+) antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4(+) T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4(+) T cells from human histocompatibility leukocyte antigens (HLA)-DRbeta1*0401(+) patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-gamma and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6-derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus- or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4(+) T cell secretion of IL-10 and transforming growth factor (TGF)-beta1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4(+) subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4(+) T cell responses to provide optimal clinical benefit.

Adult↗

PubMed2002

Progenipoietin-generated dendritic cells exhibit anti-tumor efficacy in a therapeutic murine tumor model.

Progenipoietin (ProGP-4) is a chimeric molecule, exhibiting both Flt-3 and granulocyte-colony stimulating factor (G-CSF) receptor agonist activities. Subcutaneous administration of ProGP-4 to BALB/c mice at a dose of 40-100 microg/day for up to 12 consecutive days induces both CD11c(+) dendritic cells (DCs) and CD11c(-)/CD11b(+) granulocytes in spleen, blood and lymph nodes of treated animals. Peak numbers of all cell populations were observed on day 7 of treatment, with CD11c(+) DCs representing approximately 8% of total splenocytes at that time. Approximately 40-50% of these CD11c(+) cells were also able to endocytose and process the exogenous fluorescent antigen DQ-BSA. As a test of their therapeutic utility, freshly prepared CD11c(+) DCs were pulsed with a defined tumor-associated peptide epitope (murine p53(232-240)) and injected as a vaccine into BALB/c mice bearing day 7 established CMS4 sarcomas. Similarly prepared DCs were injected again 1 week later. Based on our results, we conclude that (i) both peptide-pulsed CD11c(+) DCs (harvested directly from ProGP-4 treated mice) and pulsed bone marrow-derived DCs effectively slow the growth of or mediate the regression (in 25 of 89 [28%] cases) of CMS4 tumors, and (ii) nonpulsed DCs mediated minimal or no therapeutic effect. These data support the ability of ProGP-4 to enhance the peripheral frequencies of DCs that exhibit therapeutic efficacy when applied as a vaccine to treat tumor-bearing animals.

Animals↗

PubMed2002

In vitro generated cytolytic T lymphocytes reactive against head and neck cancer recognize multiple epitopes presented by HLA-A2, including peptides derived from the p53 and MDM-2 proteins.

In previous studies, we were successful in generating HLA-A2-restricted CD8+ CTLs reactive with head and neck carcinomas (HNCs) in 4/10 cases using traditional mixed lymphocyte tumor cultures (MLTCs) employing a semi-allogeneic HLA-A2+ HNC cell line, PCI-13, as the stimulator of normal HLA-A2+ donor T lymphocytes. However, these T cell lines contained only 1-1.5% HLA-A2-restricted, tumor-reactive CD8+ CTLs, as assessed by both limiting dilution and IFN-gamma ELISPOT assays. In order to increase the success rate in generating such HNC-reactive CTL lines, we modified the procedure to allow for T cell crosspriming by autologous DCs pulsed with PCI-13 lysates. In all three attempts, HLA-A2-restricted effector T cell lines were obtained that contained PCI-13-reactive CD8+ T cells at frequencies as high as 1 in 6. These cultured bulk lines recognized at least five predominant HLA-A2-restricted epitopes based on ELISPOT fingerprinting of HPLC-fractionated, naturally presented PCI-13-derived peptides. Two of these epitopes appear to be derived from the p53 and MDM-2 proteins overexpressed by the PCI-13 cell line. Interestingly, the synthetic wild type sequence p53 (264-272) and MDM-2 (53-61) peptides were able to drive in vitro generation of tumor-specific CTLs from the PBMCs of normal HLA-A2+ donors. However, this MDM-2 peptide was not able to elicit responses from HLA-A2+ patients with HNC in short-term in vitro cultures. Overall, these data suggest that tumor lysate-loaded DCs elicit a broad repertoire of CTL responses, some of which are directed against peptides derived from cell cycle regulatory proteins that may prove to be of clinical significance in the therapy of HNC.

Amino Acid Sequence↗

PubMed2002

Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses.

To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/10(5) (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p53(22-36) peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings.

Antigens, Viral↗

PubMed2002

Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses.

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of "T helper (Th)" signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood- or peripheral blood-isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-gamma at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I-presented epitopes by antigen-specific CD8+ T cells results in the TNF-alpha- and IFN-gamma-dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I-restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I-presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.

CD40 Ligand↗

PubMed2002

Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway.

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.

Apoptosis↗