Modulation of p38 MAPK signaling enhances dendritic cell activation of human CD4+ Th17 responses to ovarian tumor antigen

The recent finding that Th17 infiltration of ovarian tumors positively predicts patient outcomes suggests that Th17 responses play a protective role in ovarian tumor immunity. This observation has led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage by tumor vaccination. In this study, we show that treatment of ovarian tumor antigen-loaded, cytokine-matured human dendritic cells (DC) with a combination of IL-15 and a p38 MAP kinase inhibitor offers potent synergy in antagonism of CD4⁺ Treg induction and redirection toward CD4⁺ Th17 responses that correlate with strong CD8⁺ cytotoxic T lymphocyte (CTL) activation. Ovarian tumor antigen-specific CD4⁺ T cells secrete high levels of IL-17 and show reduced expression of CTLA-4, PD-1, and Foxp3 following activation with IL-15/p38 inhibitor-treated DC. We further show that modulation of p38 MAPK signaling in DC is associated with reduced expression of B7-H1 (PD-L1), loss of indoleamine 2,3-dioxygenase activity, and increased phosphorylation of ERK 1/2 MAPK. These observations may allow the development of innovative DC vaccination strategies to boost Th17 immunity in ovarian cancer patients.     

Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

Involvement of CD4⁺ helper T (Th) cells is crucial for CD8⁺ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4⁺ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4⁺ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4⁺ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K^b/OVA_257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4⁺ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4⁺ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4⁺ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

IL-21-treated naive CD45RA<Superscript>+</Superscript> CD8<Superscript>+</Superscript> T cells represent a reliable source for producing leukemia-reactive cytotoxic T lymphocytes with high proliferative potential and early differentiation phenotype

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8⁺ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA⁺ CD8⁺ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8⁺ CTLs from naive CD45RA⁺ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.     

Human Th17 cells

The discovery in mice of a new lineage of CD4⁺ effector T helper (Th) cells that selectively produce IL-17 has provided exciting new insights into immune regulation, host defence, and the pathogenesis of autoimmune and other chronic inflammatory disorders. This population of CD4⁺ Th cells, which has been termed 'Th17', indeed plays an apparently critical role in the pathogenesis of some murine models of autoimmunity. Interestingly, murine Th17 cells share a common origin with Foxp3⁺ T regulatory cells, because both populations are produced in response to transforming growth factor-β, but they develop into Th17 cells only when IL-6 is simultaneously produced. Initial studies in humans have confirmed the existence of Th17 cells, but they have shown that the origin of these cells in humans differs from that in mice, with IL-1β and IL-23 being the major cytokines responsible for their development. Moreover, the presence in the circulation and in various tissues of Th cells that can produce both IL-17 and interferon-γ, as well as the flexibility of human Th17 clones to produce interferon-γ in addition to IL-17 in response to IL-12, suggests that there may be a developmental relationship between Th17 and Th1 cells, at least in humans. Resolving this issue has great implications in tems of establishing the respective pathogenic roles of Th1 and Th17 cells in autoimmune disorders. In contrast, it is unlikely that Th17 cells contribute to the pathogenesis of human allergic IgE-mediated disorders, because IL-4 and IL-25 (a powerful inducer of IL-4) are both potent inhibitors of Th17 cell development.     

Reciprocal effects of Th1 and Treg cell inducing pathogen-associated immunomodulatory molecules on anti-tumor immunity

We have addressed the hypothesis that pathogen-associated immunomodulatory molecules may influence anti-tumor immunity through their pro- and anti-inflammatory activities and abilities to induce effector and regulatory T (Treg) cells. We found that CpG oligonucleotides (CpG) and cholera toxin (CT), which promote Th1 or Th2/Treg cell biased responses, respectively, had differential effects on tumor growth. Therapeutic peritumoral administration of CpG significantly reduced subcutaneous tumor growth and prolonged survival, whereas CT enhanced tumor growth and reduced survival. Peritumoral administration of CpG enhanced the frequency of IFN-γ-secreting and reduced IL-10-secreting CD4⁺ and CD8⁺ T cells, in the tumor and in the draining lymph nodes, whereas, CT significantly enhanced the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells, but reduced IFN-γ-secreting T cells infiltrating the tumor. In contrast to the beneficial effect of CpG in mice with subcutaneous tumors, CpG or CT had no protective effect against tumor growth in the lungs when given therapeutically by the nasal route. However, prophylactic intranasal administration of CpG significantly reduced the number of lung metastases and this was associated with an enhanced frequency of IFN-γ-secreting CD8⁺ T cells in the draining lymph node and enhanced tumor-specific CTL responses. Our findings demonstrate that pathogen-associated molecules can either inhibit or enhance anti-tumor immunity by selectively promoting the induction of effector or regulatory T cells, and that the environment of the growing tumor influences the protective effect. Joanne Lysaght and Andrew G. Jarnicki contributed equally.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.     

Tim-3 ligand galectin-9 reduces IL-17 level and accelerates Klebsiella pneumoniae infection

T cell immunoglobulin and mucin domain (Tim)-3 is expressed on activated CD4⁺ and CD8⁺ T cells. Identification of galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3/galectin-9 pathway, which results in apoptosis of effector CD4⁺ and CD8⁺ T cells. Moreover, Th17 cells are a recently discovered CD4⁺ effector T cell, which are important in antimicrobial immunity. Whether the Tim-3/galectin-9 pathway affects Th17 immunity has not been elucidated. Here, we demonstrated expression of Tim-3 on Th17 cells by flow cytometry. Th17-skewed cells were sensitive to galectin-9-induced apoptosis. In vitro administration of galectin-9 decreased stimulated Th17 cells and inhibited production of IL-17. Interestingly, Klebsiella pneumoniae (K. pneumoniae) infection led to enhanced IL-17 levels. Recombinant galectin-9 significantly decreased IL-17 in vivo, which resulted in reduced bacterial clearance and high mortality. These observations suggest that the Tim-3/galectin-9 pathway plays an important role in termination of Th17-immune responses, and could be a therapeutic target for inflammatory diseases.     

Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of human antigen-specific memory CD8<Superscript>+</Superscript> T cells

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8⁺ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8⁺ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8⁺ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8⁺ T cells. The lytic capacity of memory CTLs toward CEA⁺ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8⁺ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8⁺ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8⁺ T cells.     

Interleukin-4 is effective in restoring cytotoxic T cell activity that declines during in vivo progression of a murine B lymphoma

 We previously reported [Chakrabarti et al. (1992) Cell Immunol 142:54; 144:455] that, in a murine B lymphoma model 2C3, idiotype (Id)-specific CD8⁺ cytotoxic T lymphocytes (CTL) are generated in mice following hyperimmunization with irradiated tumor cells, and that they are effective in tumor rejection. The present study reveals that 2C3-specific CTL are also induced in spleens during tumor progression, but are not sustained. At the early stage of tumor growth, the splenic T cells following a 5-day incubation in vitro with killed 2C3 tumor targets, produce high levels of cytokines, namely interleukin-4 (IL-4), IL-10 and interferon γ (IFNγ). Their cytotoxic T lymphocyte (CTL) activity and cytokine levels, except IL-2, sharply decline at the late stage when the mice are increasingly moribund. Although the decline in cytokine level is also evident with CD4⁺ T cells, a precipitous and concurrent decrease occurs primarily in the IL-4 level with both CD4⁺ and CD8⁺ T cells of late-tumor-bearing animals (TBA). Study with the unseparated splenocytes also reveals that sevenfold less IL-4 is produced at the late stage. Furthermore, the cytotoxicity of CTL from late TBA can be effectively restored by addition of supernatants from the splenocyte culture of early TBA, or by IL-4, but not by IFNγ and IL-10. In addition, only IL-4-activated CD8⁺ T cells from the late TBA are found, by Winn assay, to be protective in vivo. Thus it appears that IL-4, required to sustain antitumor CTL activity, is consumed by T and possibly other cells at the late stage of tumor growth, thereby compromising host immunity against the tumor. We contend that induction or maintenance of protective immunity depends not only on the tumor antigen but also on the specific cytokine milieu in a tumor-bearing host. Received: 8 February 1997 / Accepted: 24 April 1997     

Human Th17 cells in infection and autoimmunity

IL-17-producing T cells (Th17) have been identified in mice as a distinct lineage of CD4⁺ T helper cells. Since their discovery, efforts have been made in characterizing human Th17 cells and the factors involved in their differentiation and in understanding the role these cells play in protective immunity and autoimmune diseases.     

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.     

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4⁺Foxp3⁺ Treg cells and Gr1⁺CD11b⁺ MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8⁺ T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.     

Rheumatoid arthritis bone marrow environment supports Th17 response

Background

Rheumatoid arthritis (RA) is a systemic, autoimmune disease leading to joint destruction and ultimately disability. Bone marrow (BM) is an important compartment in RA, where pathological processes from “outside the joint” can occur. IL-17 is a cytokine that exerts proinflammatory effects and participates in the process of bone destruction. It is believed that IL-17 is involved in pathogenesis of RA. However, little is known about the biology of this cytokine in BM. In the present study we investigated Th17-related cytokines in RA BM.

Methods

BM samples were obtained from RA and osteoarthritis (OA) patients during total hip replacement surgery. Levels of IL-17AF, IL-17AA, IL-17FF, IL-1β, IL-6, IL-23, TGF-β and CCL20 in BM plasma were determined by specific enzyme-linked immunosorbent assay tests. Percentage of IL-17-producing cells in BM was evaluated by flow cytometry. The effect of IL-15 stimulation on IL-17 production by BM mononuclear cells was examined in vitro.

Results

Increased levels of IL-17AF were observed in BM plasma of RA patients in comparison to OA patients. Increased concentrations of IL-1β, IL-6 and CCL20 were observed in RA compared to OA BM plasma. Concordant with these findings, significantly increased percentages of CD3⁺CD4⁺IL-17⁺ and CD3⁺CD4⁺IL-17⁺IFN-γ⁺ cells were present in RA BM in comparison to OA BM samples. Finally, abundant in RA BM, IL-15 increased IL-17 production by cultured BM mononuclear cells.

Conclusions

In the course of RA, the BM microenvironment can promote the development of Th17 cell responses and overproduction of IL-17AF that may lead to increased inflammation and tissue destruction in RA BM.

     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

Increase of Th17 Cell Phenotype in Kidney Transplant Recipients with Chronic Allograft Dysfunction

This study was performed to determine the association of Th17 cell phenotype with chronic allograft dysfunction in kidney transplant recipients (KTRs). We compared the expression of Th17 cell phenotype in KTRs with chronic allograft dysfunction group (CAD, n = 52) with four control groups (long-term stable KTRs (LTS, n = 67), early stable KTRs (ES, n = 28), end stage renal disease (ESRD, n = 45), and healthy control (HC, n = 26). We also performed in vitro study using human proximal renal tubular epithelial cell line (HPRTEpiC) to evaluate the effect of IL-17 on human renal tubular epithelial cells. The CAD group showed increased percentage of Th17 cells out of CD4⁺ T cells and also increased proportion of IL-17 producing cells out of effector memory T cells or out of CCR4⁺CCR6⁺/CD4⁺ T cells compared to the LTS group and other control groups. Also, the serum level of IL-17, IL-33, and RAGE, and the expression of IL-1beta, RAGE, and HMGB1 mRNA showed an increase in the CAD group compared to the LTS group. In vitro study revealed that IL-17 increased production of IL-6 and IL-8 and up-regulated profibrotic gene expression such as ACTA-2 and CTGF in HPRTEpiC in a dose-dependent manner, which suggests that IL-17 has a role in the development of renal tubular cell injury. The results of our study may suggest that increase of Th17 cell phenotype could be a marker for the chronic allograft injury; hence there is a need to develop diagnostic and therapeutic tools targeting the Th17 cells pathway.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999