Inmunología del cáncer I: bases moleculares y celulares de la respuesta inmune antitumoral / Cancer immunology I: molecular and cellular bases of the antitumor immune response

Significativos recursos materiales, financieros y humanos se dedican a la investigación de la biología del cáncer. La validación de biomarcadores, el desarrollo de novedosos métodos para el diagnóstico y la terapia, la implementación de programas de pesquisa a nivel poblacional y la promoción de estilos de vida saludables han impactado positivamente en la prevención y control de este grupo de enfermedades. Sin embargo, el cáncer sigue siendo un problema de salud mundial, entre otros factores, por la compleja relación que se establece entre el sistema inmune del hospedero y las células neoplásicas. Está demostrado que los mecanismos efectores que posee el sistema inmune permiten detectar y eliminar las células transformadas. Sin embargo, estos mismos mecanismos promueven la evolución somática de los tumores, al seleccionar variantes celulares resistentes a la acción de la inmunidad. Esta interacción ocurre fundamentalmente en el microambiente tumoral y ha sido conceptualizada como inmunoedición tumoral. Lo anterior sustenta la racionalidad de la inmunoterapia, la que buscar reforzar la inmunidad antitumoral, a la vez que bloquea los mecanismos de evasión a la inmunovigilancia. Con este trabajo de revisión iniciamos una serie de tres artículos que, en este orden, recorrerán las bases moleculares y celulares de la respuesta inmune antitumoral, presentarán los fundamentos de la biología tumoral y, finalmente, abordarán las implicaciones de la compleja relación entre el sistema inmune y las neoplasias para la inmunoterapia en cáncer

Substantial material, financial and human resources are dedicated to research on tumor biology. The validation of biomarkers, the development of novel diagnostic and therapeutic methods, the implementation of early detection programs in communities, and the promotion of healthy lifestyles have positively impacted on the prevention and control of this group of diseases. However, cancer remains a global health problem, due to the complex relationship between the host immune system and the cancer cells. It is a fact that the immune system´s effector mechanisms enable the detection and elimination of cancer cells. However, these immune mechanisms also promote the somatic evolution of tumors by selecting cellular clones resistant to immunity. This complex relationship conceptualized as cancer immunoediting develops mainly at the tumor microenvironment. Immunotherapy approaches are designed to reinforce antitumor immunity, while blocking immune escape mechanisms. This review starts a series of three articles that are going to review the molecular and cellular bases of the antitumor immune response, present the bases of tumor biology and, finally, address the implications of the complex relationship between the immune system and neoplasias for cancer immunotherapy

Técnica de reconstrucción en roturas recidivantes del tendón patelar / Reconstruction technique in recurrent patellar tendon ruptures

La rotura recidivante del tendón patelar es infrecuente y generalmente asociada a comorbilidad sistémica que altera la interfaz tendón-hueso o a consecuencia de un retorno inadecuado a actividades, previo a completar el proceso de rehabilitación. La reconstrucción quirúrgica se basa en el aumento tendíneo con autoinjertos o aloinjertos. El uso de autoinjertos evita el riesgo de transmisión de enfermedades infecciosas y costos en relación a los aloinjertos y no requiere un segundo tiempo quirúrgico para remover material utilizado en la protección del tendón reconstruido, en el caso de utilizar refuerzos con alambres de cerclaje o cables de acero. Se presenta la reconstrucción del tendón patelar con autoinjerto semitendinoso-gracilis, por ser una técnica validada en la literatura, que restablece la fuerza y la estabilidad del mecanismo extensor de forma costo-efectiva y permite el retorno adecuado a las actividades de la vida diaria.

Recurrent rupture of the patellar tendon is infrequent and usually associated with systemic comorbidity that alters the tendon-bone interface or as a result of an inadequate return to activities, prior to completing the rehabilitation process. Surgical reconstruction is based on tendon augmentation with autografts or allografts. The use of autografts avoids the risk of infectious diseases and costs in relation to allografts and does not require a second surgical time to remove material used in the protection of the reconstructed tendon, in case of using cerclage wires or steel cables reinforcements. Reconstruction of the patellar tendon with semitendinosus-gracilis autograft is presented as a technique validated in the literature, which restores the strength and stability of the extensor mechanism in a cost-effective way and allows adequate return to activities of daily living.

Oncogenic Transformation Can Orchestrate Immune Evasion and Inflammation in Human Mesenchymal Stem Cells Independently of Extrinsic Immune-Selective Pressure.

Immune escape is a hallmark of cancer, but whether it relies upon extrinsic immune-selective pressure or is inherently orchestrated by oncogenic pathways is unresolved. To address this question, we took advantage of an in vitro model of sequentially transformed human mesenchymal stem cells (hMSC). Neoplastic transformation in this model increased the natural immune-evasive properties of hMSC, both by reducing their immunogenicity and by increasing their capacity to inhibit mitogen-driven T-cell proliferation. We also found that IFNγ signaling was globally affected in transformed hMSC. As a consequence, the natural inhibitory effect of hMSC on T-cell proliferation switched from an inducible mechanism depending on IFNγ signaling and mediated by indoleamine 2,3-dioxygenase to a constitutive mechanism that relied upon IL1ß involving both secreted and membrane-expressed molecules. After transformation, increased IL1ß expression both sustained the immunosuppressive properties of hMSC and increased their tumorigenicity. Thus, in this model system, IL1ß acted as intrinsic inflammatory mediator that exerted an autocrine influence on tumor growth by coordinately linking immune escape and tumorigenicity. Collectively, our findings show how oncogenes directly orchestrate inflammation and immune escape to drive the multistep process of cancer progression, independently of any need for immunoediting in the tumor microenvironment.

Oncogenic transformation tunes the cross-talk between mesenchymal stem cells and T lymphocytes.

Stem cells from mesenchymal origin (MSC) exert a plethora of immunomodulatory effects. We created a neoplastic model based on in vitro step-wise transformation to assess whether oncogenic pathways have the capacity to mould the cross-talk of MSC and lymphocytes. Neoplastic MSC exhibit an increased inhibitory effect on T cell proliferation, either directly or mediated by myeloid derived suppressor cells. Additionally, transformation of MSC enhances T cell apoptosis without reducing either the percentage of CD25 expressing cells or the level of this protein expression. Malignant transformation drives MSC to lose dependency on nitric oxide for immunosuppression whilst increasing the constitutive production of PGE2. Our results indicate that oncogenesis tunes the interplay between MSC and immune cells, favoring cancer immune evasion.

A view on EGFR-targeted therapies from the oncogene-addiction perspective.

Tumor cell growth and survival can often be impaired by inactivating a single oncogen- a phenomenon that has been called as "oncogene addiction." It is in such scenarios that molecular targeted therapies may succeed. among known oncogenes, the epidermal growth factor receptor (EGFR) has become the target of different cancer therapies. So far, however, the clinical benefit from EGFR-targeted therapies has been rather limited. a critical review of the large amount of clinical data obtained with anti-EGFR agents, carried out from the perspective of the oncogene addiction concept, may help to understand the causes of the unsatisfactory results. In this article we intend to do such an exercise taking as basis for the analysis a few case studies of anti-EGFR agents that are currently in the clinic. There, the "EGFR addiction" phenomenon becomes apparent in high-responder patients. We further discuss how the concept of oncogene addiction needs to be interpreted on the light of emerging experimental evidences and ideas; in particular, that EGFR addiction may reflect the interconnection of several cellular pathways. In this regard we set forth several hypotheses; namely, that requirement of higher glucose uptake by hypoxic tumor cells may reinforce EGFR addiction; and that chronic use of EGFR-targeted antibodies in EGFR-addicted tumors would induce stable disease by reversing the malignant phenotype of cancer stem cells and also by sustaining an anti-tumor T cell response. Finally, we discuss possible reasons for the failure of certain combinatorial therapies involving anti-EGFR agents, arguing that some of these agents might produce either a negative or a positive trans-modulation effect on other oncogenes. It becomes evident that we need operational definitions of EGFR addiction in order to determine which patient populations may benefit from treatment with anti-EGFR drugs, and to improve the design of these therapies.

Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes.

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.

Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles.

We investigated the contribution of lipopolysaccharide (LPS) to adjuvant properties of native outer membrane vesicles (NOMV), a vaccine candidate for meningococcal B disease. NOMV induce the maturation of and cytokine production by murine bone marrow-derived dendritic cells through both toll-like receptors (TLR) 2 and 4 which are mostly dependent on the signalling adaptor MyD88. NOMV are also able to induce B cell proliferation in splenocytes from LPS-hyporesponsive mice. However, induction of IL-10 and type I interferon-dependent, antigen-specific and IFN(gamma)-secreting CD8(+) cytotoxic T lymphocyte responses in vivo by NOMV requires LPS. The importance of LPS in the induction of IL-10 and functional cross-priming has implications for NOMV-based vaccine and adjuvant development.

Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity.

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid-containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4(+) T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab')(2) but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.

Differential influence of the tumour-specific non-human sialic acid containing GM3 ganglioside on CD4+CD25- effector and naturally occurring CD4+CD25+ regulatory T cells function.

Increasing evidences suggest that the aberrant expression of certain gangliosides on malignant cells could affect host's anti-tumour-specific immune responses. We have recently documented the relevance of the N-glycolylated variant of GM3 ganglioside (NGcGM3), a tumour-specific non-human sialic acid containing ganglioside, for tumour progression. However, evidences about the implication of host's immunity in NGcGM3-promoted cancer progression had not been obtained previously. In this work, we compared tumour growth of X63 myeloma cells pre-treated or not with an inhibitor of the glucosylceramide synthase enzyme, in wild or CD4+ T cell-depleted BALB/c mice. Results clearly showed a relationship between the agonistic effect of NGcGM3 in tumour growth and the presence of CD4+ T lymphocytes. For the first time, a description of a ganglioside-differential effect over purified CD4+CD25- and naturally occurring regulatory CD4+CD25+ T cells is provided. While NGcGM3 similarly down-modulated the CD4 expression in both cell populations, the inhibitory capacity of the CD4+CD25+ lymphocytes and their proliferation, induced by an anti-CD3 mAb and IL2, were not modified. In a different fashion, a reduction in proliferative capacity and a noteworthy secretion of anti-inflammatory cytokines were detected when CD4+CD25- T cells were cultured in the presence of NGcGM3. Considering the relevance of dendritic cells (DC) on primary activation of T cells, the effect of NGcGM3 over DC differentiation and TLR4-mediated maturation was also assessed. Our results indicate that NGcGM3 contributes to cancer progression mainly by influencing DC and CD4+CD25- T lymphocyte functions, rather than increasing the inhibitory capacity of naturally occurring regulatory T cells.

Gangliosides as immunomodulators.

Gangliosides are glycosphingolipids expressed at the outer leaflet of the plasmatic membrane of cells from vertebrate organisms. These molecules exert diverse biological functions including modulation of the immune system responses. Aberrant expression of gangliosides has been demonstrated on malignant cells. Besides expression on tumor cell membranes, gangliosides are also shed in the tumor microenvironment and eventually circulate in patients blood. Gangliosides derived from tumors posses the capability to affect the immune system responses by altering the function of lymphocytes and antigen-presenting cells and promoting tumor growth. These molecules can be considered as tumor weapons directed to attack and destroy immunosurveillance mechanisms devoted to control cancer progression.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for dendritic cell activation.

Recent findings in the interactions between pathogens and the innate immune system, particularly with dendritic cells (DC), have opened new opportunities for adjuvants designs. We have elaborated a new approach, in which gangliosides are incorporated into the outer membrane complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). VSSP demonstrated a unique ability to render highly tolerated gangliosides immunogenic. These results drove our attention to the immunopotentiating properties of VSSP. Here we examined the VSSP adjuvant effect on dendritic cell activation. Also the role of LPS on this effect was dissected. This study reveals that VSSP is a potent adjuvant for DC activation.

Very small size proteoliposomes derived from Neisseria meningitidis: An effective adjuvant for generation of CTL responses to peptide and protein antigens.

The development of potent adjuvants, conditioning innate and adaptative immunity, particularly CTL responses, has become currently a hot point in the rational design of vaccines for cancer immunotherapy. We have described a new approach, in which gangliosides are incorporated into vesicles from Neisseria meningitidis to form Very Small Size Proteoliposomes (VSSP). VSSP is a good alternative to the existing adjuvants for use in whole cells vaccines since it promotes 80% tumour rejection and growing delay in the CT26 and F3II tumour models respectively. Also VSSP induces activation of CTL responses to co-injected trimmed peptides and soluble proteins. This phenomena is facilitated by the cross-presentation of exogenous antigen and do not need cooperation of CD4 T cells for primary CD8 T cells expansion.

Role of tumour-associated N-glycolylated variant of GM3 ganglioside in cancer progression: effect over CD4 expression on T cells.

Gangliosides have diverse biological functions including modulation of immune system response. These molecules are differentially expressed on malignant cells compared with the corresponding normal ones and are involved in cancer progression affecting, in different ways, the host's anti-tumour specific immune responses. Although in humans the N-glycolylated variant of GM3 ganglioside is almost exclusively expressed in tumour tissues, the significance of this glycolipid for malignant cell biology remains obscure, while for NAcGM3 strong immune suppressive effects have been reported. The present work demonstrates, for the first time, the capacity of NGcGM3 ganglioside to down-modulate CD4 expression in murine and human T lymphocytes, especially in non-activated T cells. Thirty and tenfold reductions in CD4 expression were induced by purified NGcGM3 ganglioside in murine and human T lymphocytes, respectively. The CD4 complete recovery in these cells occurred after 48 h of ganglioside removal, due to neo-synthesis. Restored T cells kept similar sensitivity to ganglioside-induced CD4 down-modulation after a new challenge. In addition, a clear association between NGcGM3 insertion in lymphocyte plasma membranes and the CD4 down-modulation effect was documented. Notably, a possible role of this ganglioside in tumour progression, taking advantage of the X63 myeloma model, was also outlined. The relevance of these findings, characterizing NGcGM3 as a possible tumour immunesurveillance inhibitor and supporting the reason for its neo-expression in certain human cancers, is contributing to this unique heterophilic ganglioside validation as target for cancer immunotherapy.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for Th1 induction and dendritic cell activation.

Recent findings about pathogens and innate immune system interactions have opened new opportunities for adjuvants designs. We have elaborated a new approach, in which gangliosides are incorporated into the outer membrane complex of Neisseria meningitidis (Nm) to form very small size proteoliposomes (VSSP). VSSP, used as monotherapy, demonstrated a unique ability to render immunogenic highly tolerated gangliosides. These results drove our attention to the immunopotentiating properties of VSSP. Here, we examined the VSSP adjuvant effect on the humoral and cellular responses, dendritic cell (DC) activation, and differentiation of Th cells. Also, the role of LPS in VSSP effect was dissected. This study reveals that VSSP is a potent adjuvant for dendritic cells activation and Th1 differentiation.