Paul Zajac

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class‐I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class‐I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV‐US12) or together with endoplasmic reticulum (ER)‐targeted Melan‐A/MART‐1_27–35 model tumor epitope (rVV‐MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV‐US12, display a decreased ability to present TAP dependent MHC class‐I restricted viral antigens to CD8+ T‐cells. While HLA class‐I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class‐II are unaffected. Characterization of rVV‐MUS12 infected cells demonstrates that over‐expression of a TAP‐independent peptide, partially compensates for ICP47 induced surface MHC class‐I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus‐specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV‐MUS12, as compared to those stimulated by rVV‐MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector‐specific responsiveness.

Background: Lymphocytic infiltration is known to be associated with a favourable prognosis in human colorectal cancer (CRC). In particular, the presence of CD8+ T cells and, unexpectedly, of Foxp3+ regulatory T cells, has been found to be associated with improved patient survival. Recent evidence suggests that IL-17 and T helper (Th) 17 cells might also have an impact on anti-tumour immune responses. We have investigated prevalence, phenotype and prognostic significance of IL-17-producing cells in human CRC. Material and Methods: IL-17 expression was evaluated by immunohistochemistry on a tissue micro-array (TMA) including 1420 cases of primary CRC with full clinico-pathological data. Furthermore, gene expression levels were assessed on CRC tissues by quantitative PCR. Finally, in order to characterize the phenotype of IL-17-positive cells, expression of IL-17, in combination with that of specific surface molecules, was analyzed on freshly excised CRC specimens by flow cytometry. Results: Frequencies of IL-17-producing cells, as well as IL-17 gene expression levels were significantly increased in tumour tissues as compared to autologous normal mucosa. IL-17-producing cells isolated from clinical specimens were exclusively comprised within the lymphocyte population and expressed CD4, but not CD8, and surprisingly, Foxp3 molecules. Accordingly, mRNA levels of genes encoding for cytokines favouring IL-17 acquisition by Foxp3+ T cells, including IL-6, IL-1beta, TGF-beta and IL-23, were found more elevated in CRC tissues as comparing to corresponding healthy mucosa. High infiltration by IL-17 producing cells significantly correlated with low T and N stages, and, most importantly, with prolonged survival time in mismatch repair (MMR)- proficient, but not-deficient CRC. Moreover, the simultaneous CRC-infiltration by IL-17+ and Foxp3+ cells was significantly associated with improved survival in both MMR-proficient and -deficient tumours. Conclusions: Our data suggest that IL-17 produced by tumour-infiltrating either CD4+ or Foxp3+ cells may promote a benign clinical outcome in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2011-784

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM‐MSC) or adipose tissue (AT‐MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM‐MSC with bovine AC, (ii) BM‐MSC from HLA‐A2+ with AC from HLA‐A2− donors, or (iii) human green fluorescent protein transduced BM‐MSC with AC. Human BM‐MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT‐PCR. Coculture of NC or AC (25%) with BM‐MSC or AT‐MSC (75%) in pellets resulted in up to 1.6‐fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM‐MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2‐fold) if cocultured with BM‐MSC and maintained a differentiated phenotype. Chondro‐induction in MSC–chondrocyte coculture is a robust process mediated by two concomitant effects: MSC‐induced chondrocyte proliferation and chondrocyte‐enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair. J. Cell. Physiol. 227: 88–97, 2012.

MAGE‐A10 is a highly immunogenic member of the MAGE‐A family of cancer/testis tumor‐associated antigens (C/T TAAs). Studies performed with broadly reactive antibodies have helped to initially characterize this TAA. However, no specific reagents have been developed so far, thus preventing a thorough analysis of its expression in healthy and tumoral tissues. We have produced MAGE‐A10 gene product in soluble recombinant form, and we have used it to generate specific monoclonal antibodies (mAbs). One of these reagents, recognizing an epitope located at the COOH terminus of the MAGE‐A10 gene product, was used to stain a multitumor tissue microarray comprising more than 2,500 paraffin‐embedded specimens including healthy tissues, benign tumors and malignancies of different histological origin. MAGE‐A10 protein was identified as an intranuclear protein of an apparent molecular weight of 70 kDa, expressed in normal spermatogonia and spermatocytes but in no other healthy tissue. Most importantly, this C/T TAA appears to be expressed in high (>50%) percentages of cancer cells from a number of malignancies, including lung, skin and urothelial tumors. Unexpectedly, high expression of MAGE‐A10 TAA at the protein level was also detectable in gynecological malignancies and stomach and gall bladder cancers. The characterization of MAGE‐A10‐specific reagents might set the stage for the development of targeted active immunotherapy by clarifying potential indications and by allowing the selection of patients eligible for treatment and the monitoring of its effectiveness.

Objective: To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications.Methods: Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF‐2), platelet‐derived growth factor BB (PDGF‐BB), human platelet lysate, or interferon‐γ (IFNγ). The roles of cell proliferation and growth factor–induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity.Results: FGF‐2 and, to a lesser extent, PDGF‐BB induced in adult human MSCs the expression of HLA–DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA–DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA–DR expression in human MSCs. HLA–DR expression occurred following FGF‐specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK‐1/2 and phosphatidylinositol 3‐kinase/Akt controlled cell proliferation and HLA–DR expression, but only MAPK/ERK‐1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA–DR transcription.Conclusion: The induction of functional HLA–DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

Common receptor gamma chain (c-gamma) cytokines (CKs) support proliferation of CD8+ T cells in presence or absence of antigen triggering and help maintaining the immunologic memory. We addressed the effects of low (< or = 5 ng/mL)-dose interleukin (IL)-2, IL-7, or IL-15 on human naive and memory antigen-specific CD8+ T cells. Peripheral blood CD8+ lymphocytes proliferated with decreasing efficiency in response to IL-15, IL-7, and IL-2. Of note, IL-15 preferentially promoted expansion of CD45RA/CD8+ T-cell memory subset. Accordingly, cytotoxic T lymphocytes specific for cytomegalovirus-derived antigens from seropositive donors proliferated in response to IL-15 and, to lesser extent to IL-7, but poorly to IL-2. CD8+ T cells were then pretreated with CK before antigen stimulation using, as read out, specific cytotoxic activity. After the pretreatment with IL-15, but not IL-2, previously experienced viral antigens induced vigorous cytotoxic responses. Minor effects of IL-7 were also detectable. In contrast, IL-2 best supported the cytotoxic T lymphocyte generation from prevailingly naive CD8 T cells from HLA-A*0201 healthy donors, specific for L27Melan-A/MART-126-35 melanoma-associated antigen. Cells from melanoma patients were tested before and after Melan-A/MART-1-targeted antigen-specific immunotherapy. Untreated patients showed heterogeneous patterns of responsiveness to c-gamma CK. However, when naive patients whose CD8+ T cells best responded to IL-2 were vaccinated, a modified responsiveness pattern was detectable. After immunization, cells displayed a significantly higher response to IL-15 than to IL-2 pretreatment. Thus, responsiveness to c-gamma CK is critically influenced by naive or memory status of peripheral blood CD8+ T cells.

The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1). The procedure first requires the cloning, by classical methods not described here, of the gene of interest, e.g. CD80, into a vaccinia shuttle plasmid under the control of a virus-specific promoter enabling a transcription during the early phase of infection. Flanking the insert, the plasmid contains viral sequences and a selection maker needed for the insertion into the viral genome. The successive plaque isolation of recombinant virus on cell monolayer described here is based on the transient “gpt” selection system which enables other insertions in different loci of the same virus. Finally, after verification amplification and titration of the recombinant vector, replication will be impaired by a psoralen-UV treatment in order to produce a non-replicating virus. Expression and function of inserts, following infection of cells, are verified by specific phenotypic and functional assays.

Background: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals.Methods: WB (300 μl) was incubated at 37°C with specific antigens, in the form of peptides or commercial vaccines for 5–16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4°C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes.Results: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 μl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1β (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-γ gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination.Conclusion: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities.

Dendritic cells (DC) can be activated by proinflammatory cytokines or upon toll-like receptor (TLR) triggering. These stimuli induce specific patterns of phenotypic modulation and gene expression profiles. We investigated whether TLR triggering represents an indispensable requirement for the induction of T cell responses by human DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interferon-alpha (IFN-DC). As model stimulator we chose imidazoquinolone (3M-001), a synthetic TLR7 agonist used in the treatment of skin infections and tumors and as experimental adjuvant. At difference with DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL-4) (IL-4-DC), IFN-DC display a semimature phenotype. Furthermore, IFN-DC, but not IL-4-DC are able to induce CD4+ and CD8+ T cell responses, in steady state, for example, in the absence of TLR triggering. 3M-001 treatment induces up-regulation of the surface expression of costimulatory molecules and "de novo" production of IL-12 and IL-6 in IFN-DC. However, TLR7 triggering fails to significantly enhance the capacity of IFN-DC to induce antigen-specific cytotoxic T lymphocytes and to stimulate allogeneic CD4+ T cells. These data indicate that TLR engagement and IL-12 production do not represent indispensable prerequisites for optimal antigen-presenting cell function in IFN-DC, qualifying these cells as powerful cellular reagents of potential use in active specific immunotherapy.