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[Background]: Intra-vesical instillation of Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an effective therapy for high-grade non-muscle invasive bladder cancer (NMIBC), which provokes a local immune response resulting in 70% of patients free of relapse after three years. Because non-responder patients usually have a bad prognosis, the early identification of treatment failure is crucial. We hypothesized that, if an effective immune response was taking place in the bladder, soluble factors would be released to the urine many days after BCG instillations., [Methods]: An extensive panel of cytokines and chemokines released into the urine seven days after every BCG instillation was screened in a cohort of NMIBC patients over three years., [Results]: The determinations of the urinary concentrations of cytokines, chemokines, and creatinine showed that increasing concentrations of C-X-C motif chemokine 10 (CXCL10) also known as interferon-inducible protein 10 (IP10) could be detected during the six-week induction cycle of BCG-treated patients released into the urine by CD14+ cells. In vitro, CXCL10 facilitated the recruitment of effector immune cells after the BCG-mediated upregulation of CXCR3 in both T- and natural killer (NK)-cells. Conclusions: The high concentrations of chemokine detected one week after the encounter with mycobacteria suggest that the CXCL10 axis might be related to the intensity of the immune anti-tumor response.

Immunotherapy, via intra-vesical instillations of BCG, is the therapy of choice for patients with high-risk non-muscle invasive bladder cancer. The subsequent recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, is believed to induce a local immune response that eliminates these tumors, but the detailed mechanisms of action of this therapy are not well understood. Here, we have studied the phenotype and function of the responding lymphocyte populations as well as the spectrum of cytokines and chemokines produced in an in vitro model of human peripheral blood mononuclear cells (PBMCs) co-cultured with BCG. Natural killer (NK) cell activation was a prominent feature of this immune response and we have studied the expansion of this lymphocyte population in detail. We show that, after BCG stimulation, CD56dim NK cells proliferate, upregulate CD56, but maintain the expression of CD16 and the ability to mediate ADCC. CD56bright NK cells also contribute to this expansion by increasing CD16 and KIR expression. These unconventional CD56bright cells efficiently degranulated against bladder cancer cells and the expansion of this population required the release of soluble factors by other immune cells in the context of BCG. Consistent with these in vitro data, a small, but significant increase in the intensity of CD16 expression was noted in peripheral blood CD56bright cells from bladder cancer patients undergoing BCG therapy, that was not observed in patients treated with mitomycin-C instillations. These observations suggest that activation of NK cells may be an important component of the anti-tumoral immune response triggered by BCG therapy in bladder cancer.

FUNDING This work was supported by the Medical Research Council [New Investigator grant number G0601165 (to M.V.-G.)], the Royal Society [grant number IJP2007/R3] and Fondo de Investigación Sanitaria [grant numbers PS09/00181, PI08/1701 and PI11/00298]. O.A. was partially supported by The Newton Trust and the Leukaemia Research Fund [grant number 05060 (to H.T.R. and M.V.-G.)]; S.L.-C. was partially supported by CSIC (Consejo Superior de Investigaciones Científicas) [grant number 201020E086 (to M.V.-G.)]; L.F.-M. was partially supported by CSIC [grant 201020I044 (to M.V.-G.)] and the Comunidad de Madrid [grant number S2010/BMD-2326 IMMUNOTHERCAN (to M.V.-G.)]; R.P. was an exchange student of the Leonardo da Vinci program; and S.P.-Q. was a recipient of a CSIC fellowship of Introduction to Research., The human MICA (MHC I-related chain A) gene, encoding a ligand for the NKG2D (NKG2-D type II integral membrane protein) receptor, is highly polymorphic. A group of MICA alleles, named MICA 5.1 (prototype, MICA*008), produce a truncated protein due to a nucleotide insertion in the transmembrane domain. These alleles are very frequent in all of the human populations studied and they have different biological properties, compared with full-length alleles, e.g. recruitment into exosomes, which makes them very potent for down-modulating the NKG2D receptor in effector immune cells. Moreover, MICA*008 is not affected by viral immune evasion mechanisms that target other MICA alleles. In the present study, we demonstrate that MICA*008 acquires a GPI (glycosylphosphatidylinositol) anchor and that this modification is responsible for many of the distinct biological features of the truncated MICA alleles, including recruitment of the protein to exosomes. MICA*008 processing is also unusual as it is observed in the endoplasmic reticulum as a Triton™ X-114 soluble protein, partially undergoing GPI modification while the rest is exocytosed, suggesting a new model for MICA*008 release. This is the first report of a GPI-anchored MICA allele. The finding that this modification occurs in both families of human NKG2D ligands, as well as in the murine system, suggests positive pressure to maintain this biochemical feature., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgements The authors thank Nigel Miller for assistance with cell sorting. This work was supported by grants from the Medical Research Council (New Investigator Grant to M. V. G.) and Fondo de Investigación Sanitaria (PS09/00181 and PI08/1701) and, in part, by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. O. A. was supported by the Leukaemia Research Fund and The Newton Trust. H. C. H. was supported by grants from Ellison Medical Foundation and NIH/NIGMS GM087544. S. A. was a recipient of a short-term fellowship from the European Molecular Biology Organization and supported in part by fellowships from Caja Madrid and Ibercaja., MICA and MICB (MHC‐class‐I‐related chain A/B) are transmembrane proteins expressed in pathological conditions that are ligands for NKG2D, an activating receptor found on cytotoxic lymphocytes. The recognition on target cells of NKG2D ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells. Besides being expressed at the cell surface, MICA/B can be released as soluble proteins. Soluble NKG2D ligands downmodulate expression of the NKG2D receptor on lymphocytes, leading to a diminished cytotoxic response. Prior studies suggested that recruitment of MICA/B molecules to cholesterol‐enriched microdomains was an important factor regulating the proteolytic release of these molecules. We now show that recruitment of MICA to these microdomains depends on palmitoylation of two cysteine residues that allow MICA molecules to reside in the membrane in the same domains as caveolin‐1. Compared with WT molecules, nonpalmitoylated mutant MICA molecules were shed to the supernatant with low efficiency; however, both WT and mutant MICA were able to trigger NK cell cytotoxicity. These data suggest that the presence of NKG2D ligands at the plasma membrane is sufficient to activate cytotoxicity and reflect the need of different ligands to exploit different cellular pathways to reach the cell surface upon different stress situations., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgements The authors would like to thank Drs M. E. Medof, V. L. Stevens and S. Vainauskas for GPI-deficient cell lines; C. Gross and F. Colucci for critically reading the manuscript. This work was supported by the MRC (New Investigator Grant to M.V.-G.) and FIS (PS09/00181 and PI08/1701). O.A. was supported by the Leukaemia Research Fund and The Newton Trust. S.A.-G. was supported by Fundación Caja Madrid and Ibercaja. Deposited in PMC for release after 6 months., The activating immune receptor NKG2D binds to several stress-induced ligands that are structurally different. MHC-class-I-related chain (MIC) A/B molecules have a transmembrane domain, whereas most UL16 binding proteins (ULBPs) are glycosylphosphatidylinositol (GPI)-linked molecules. The significance of this variability in membrane anchors is unclear. Here, we demonstrate that ULBP2, but not ULBP1 or ULBP3, can reach the cell surface without the GPI modification. Several proteins are expressed at the cell surface as both transmembrane and GPI-linked molecules, either via alternative splicing or by the expression of linked genes. However, to our knowledge, ULBP2 is the first single mammalian cDNA that can be expressed as either a transmembrane or a GPI-anchored protein. The rate of maturation and the levels of cell surface expression of the non-GPI-linked form were lower than those of the GPI-linked ULBP2. Nonetheless, non-GPI ULBP2 was recognised by NKG2D and triggered NK cell cytotoxicity. These data show that differences in membrane attachment by NKG2D ligands are more important for regulation of their surface expression than for cytotoxic recognition by NKG2D and emphasise that detailed characterisation of the cell biology of individual NKG2D ligands will be necessary to allow targeted modulation of this system., MRC- Studies on the cell biology of the ULBPs: implications for the activation of the immune response, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgments We thank R. Apps for the ULBP constructs; N. Miller for assistance with cell sorting and F. Colucci and his group, M. Field, J. Kaufman, A. Kelly, S. Powis, P. Roda-Navarro and J. Trowsdale for helpful discussions., Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgments We thank S.J. Powis (University of St. Andrews), P. Roda-Navarro, Dr. F. Colucci and his group, and Dr. A. Kelly for helpful discussions; Prof. J. Trowsdale for critical reading of the manuscript; and N. Miller for assistance with cell sorting. Grant Support: Medical Research Council and Leukemia Research Fund. M. Valés-Gómez is a recipient of a New Investigator Award Grant from the Medical Research Council. O. Ashiru and P. Boutet were partially supported by The Newton Trust. S. Agüera-González was supported by Fundacion Caja Madrid and Ibercaja., The MHC class I–related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised immune response and progression of disease. Recently, thiol disulphide isomerases and members of the ADAM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MICA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression. Cancer Res; 70(2); 481–9, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub