Search

Background Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Materials and Methods CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. Results CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. Conclusions We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications. Disclosure Information A. Ventura: None. P. Palmerini: None. A. Dalla Pieta: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None.

Background Cytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI). Materials and Methods CIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent. Results The combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone. Conclusions Here we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies. References Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311. The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato Antonio Disclosure Information A. Dalla Pieta: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None.

Background Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells, and represent an attractive approach for cell-based immunotherapy as they do not require antigen-specific priming for tumor cell recognition, and can be efficiently and rapidly expanded in vitro. We recently reported that CIK cells have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the assessment and the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires reliable therapeutic options. Materials and methods Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or MDA-MB-231 cells subcutaneously or orthotopically into the mammary fat pad, or by injecting MDA-MB-231 cells intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab, while the mAb alone or CIK cells plus Rituximab served as control treatments. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. Results CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either implanted with TNBC patient-derived tumor xenografts or injected with MDA-MB-231 TNBC cell line. Moreover, in both experimental and spontaneous metastatic models the combined approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and in particular led to an enrichment of the CD16a+ subset. Conclusions Data highlight the potentiality of a novel immunotherapy approach where a non-specific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells. Disclosure Information R. Sommaggio: None. E. Cappuzzello: None. A. Dalla Pieta: None. P. Palmerini: None. A. Tosi: None. D. Carpanese: None. L. Nicole: None. A. Rosato: None.

Background & Aim Cytokine-Induced Killer (CIK) cells are a population of effector cells that represent a promising tool for adoptive cell therapy. From a technical and methodological point of view, they are easily expandable ex-vivo and safe, and demonstrated efficacy against hematological malignancies. CIK cells exert cytotoxicity against a broad range of tumor histotypes but not against normal tissues and hematopoietic precursors. We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity. Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells could be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® and Obinutuzumab®. Methods, Results & Conclusion CIK cells were obtained from peripheral blood mononucleated cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb (OKT3) and IL-2 for 14 days; fresh IL-2 was provided every 3-4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against EHEB (CLL), Raji (Burkitt lymphoma), RCK-8, TMB-8, Karpas 422 (DLBCL) tumor cell lines, and primary samples obtained from CLL and DLBCL patients after written informed consent. The combination with either the mAbs significantly increased CIK cell cytotoxicity not only against lymphoma cell lines, but also against the primary tumor samples. Depletion of NK cells from CIK cell bulk cultures did not affect target cell lysis, thus confirming that the antibody-mediated increase of cytotoxicity was mainly accountable to the CIK cell fraction. Taken together, these data indicate that the combination treatment with CIK cells and anti-CD20 mAbs could represent a novel approach for the treatment of hematological malignancies, alternative to the use of chimeric antigen receptor (CAR)-T cells.

Research in xenotransplantation implies a high experimental complexity comprising molecular, cellular, and in vivo studies to investigate the mechanisms of xenograft immune rejection and functional failure, as well as the strategies to counteract them. After major advances associated with the identification of the carbohydrate xenoantigens and their elimination through genomic edition of the source pigs, the study of the cellular immune response against the xenograft is gaining particular attention. Xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells are relevant to address this hurdle. Thus, we describe here coculture, co-stimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms of xenograft rejection. These techniques allow elucidating the key pathways that take place during the xenogeneic immune response in a simplified setting. Treatment with either pro-inflammatory or anti-inflammatory cytokines can be used for studying the regulation of adhesion, co-stimulatory molecules, and receptors involved in triggering the immune response under various conditions. Furthermore, these assays can be used for the follow-up of the immune response of in vivo studies as well as for the development of tolerogenic approaches that promote xenograft survival.

In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53–proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP–microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53. Walerych et al. show that p53 missense mutants upregulate the proteasome and increase breast cancer cell resistance to proteasome inhibitors. Combined inhibition of p53 mutants and the proteasome leads to increased therapeutic efficacy.

Background & Aim Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Methods, Results & Conclusion CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3-4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks (38.6±14.9%, 35.0±15.5%, 24.0±17.8%, respectively). Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications.

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Summary Objective Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. Design To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). Results Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. Conclusions Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies.

The aim of the study was to identify immunogenic HLA-A*0201-binding epitopes derived from a number of classical midgut carcinoid-associated proteins. CD8(+) T cells recognizing tumor-associated antigen (TAA) epitopes are of great interest for the establishment of immunotherapy as a novel treatment for this type of malignancy.Midgut carcinoid tumor specimens were microdissected and expression levels of potential TAAs were investigated by quantitative real time PCR. HLA-A*0201-binding motifs were selected using HLA peptide binding prediction algorithms and stabilization of HLA-A*0201 was verified using TAP-deficient T2 cells. Peripheral blood of midgut carcinoid patients was analyzed for peptide epitope recognition and the feasibility of generating peptide-reactive CD8(+) T cells in healthy blood donors was examined by an in vitro stimulation protocol using mature DCs. Activation of patient and healthy donor CD8(+) T cells was analyzed by intracellular flow cytometry staining of interferon gamma.Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), and islet autoantigen 2 (IA-2) are properly expressed by midgut carcinoid tumor cells, with CGA mRNA expressed to greatest level. Midgut carcinoid patients have increased frequencies of peripheral blood CD8(+) T cells recognizing a pool of HLA-A*0201 peptides derived from these proteins compared to healthy age-matched individuals. Activated peptide-specific CD8(+) T cells could also be generated in healthy blood donors by in vitro stimulation.We have identified a number of immunogenic midgut carcinoid-associated peptide epitopes recognized by CD8(+) T cells. We show that midgut carcinoid patients display immune recognition of their tumors. Memory CD8(+) T cells in patient blood are of great interest when pursuing an immunotherapeutic treatment strategy.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

// Silvia Pegoraro 1 , Gloria Ros 1 , Silvano Piazza 2 , Roberta Sommaggio 3 , Yari Ciani 1,2 , Antonio Rosato 3,4 , Riccardo Sgarra 1 , Giannino Del Sal 1,2 , and Guidalberto Manfioletti 1,* 1 Dipartimento di Scienze della Vita, Universita degli Studi di Trieste, Trieste, Italy 2 Laboratorio Nazionale CIB, (LNCIB), Area Science Park, Trieste, Italy 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy 4 Istituto Oncologico Veneto IRCCS, Padova, Italy Correspondence: Guidalberto Manfioletti, email: // Keywords : Epithelial to mesenchymal transition, stemness, HMGA1, breast cancer, invasion, metastasis, gene-signature Received : June 28, 2013 Accepted : July 7, 2013 Published : July 9, 2013 Abstract Breast cancer is a heterogeneous disease that progresses to the critical hallmark of metastasis. In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in this process in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo . Mechanistically, HMGA1 activated stemness and key migration-associated genes which were linked to the Wnt/beta-catenin, Notch and Pin1/mutant p53 signalling pathways. Moreover, we identified a specific HMGA1 gene expression signature that was activated in a large subset of human primary breast tumours and was associated with poor prognosis. Taken together, these data provide new insights into the role of HMGA1 in the acquisition of aggressive features in breast cancer.

Cellular studies are essential in the xenotransplantation field in order to investigate the cellular immune responses triggered by xenogeneic cells and identify the key molecules involved. A series of functional studies can be conducted with this purpose that include treatment with proinflammatory cytokines and xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells. The choice of the pig cell type is critical to appropriately model the transplant setting of interest. Thus, pig endothelial cells are commonly used for studying the rejection process of vascularized organs. Treatment with cytokines allows studying the regulation of adhesion, costimulatory molecules, and receptors involved in triggering the immune response in an attempt to reproduce the more complex in vivo situation. The adhesion assays are used to determine the capacity of human leukocytes to adhere to porcine cells under various conditions. Furthermore, we describe coculture, costimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms that take place during the xenogeneic immune response.

Cellular studies are essential in the xenotransplantation field in order to investigate the cellular immune responses triggered by xenogeneic cells and identify the key molecules involved. A series of functional studies can be conducted with this purpose that include treatment with proinflammatory cytokines and xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells. The choice of the pig cell type is critical to appropriately model the transplant setting of interest. Thus, pig endothelial cells are commonly used for studying the rejection process of vascularized organs. Treatment with cytokines allows studying the regulation of adhesion, costimulatory molecules, and receptors involved in triggering the immune response in an attempt to reproduce the more complex in vivo situation. The adhesion assays are used to determine the capacity of human leukocytes to adhere to porcine cells under various conditions. Furthermore, we describe coculture, costimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms that take place during the xenogeneic immune response.

Transplantation may be the best option for the repair of many cartilage lesions including early osteoarthritis. Currently, autologous and allogeneic chondrocytes are grafted into cartilage defects to treat selected patients with moderate clinical success. However, their limited use justifies exploring novel therapies for cartilage repair. Xenotransplantation could become a solution by offering high cell availability, quality and genetic engineering capabilities. The rejection process of xenogeneic cartilage is thus being elucidated in order to develop counteractive strategies. Initial studies determined that pig cartilage xenografts are rejected by a slow process comprising humoral and cellular responses in which the galactose α1,3-galactose antigen participates. Since then, our group has identified key mechanisms of the human response to pig chondrocytes (PCs). In particular, human antibody and complement contribute to PC rejection by inducing a pro-inflammatory milieu. Furthermore, PCs express and up-regulate molecules which are functionally relevant for a variety of cellular immune responses (SLA-I, the potent co-stimulatory molecule CD86, and adhesion molecules VCAM-1 and ICAM-1). These participate by triggering a T cell response, as well as supporting a prominent role of the innate immune responses led by natural killer (NK) cells and monocytes/macrophages. Human NK cells lyse PCs by using selected NK activating receptors, whereas human monocytes are activated by PCs to secrete cytokines and chemokines. All this knowledge sets the bases for the development of genetic engineering approaches designed to avert rejection of xenogeneic chondrocytes and leads the way to developing new clinical applications for cartilage repair.