Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein.

BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.

Decreased invariant natural killer T-cell-mediated antitumor immune response in patients with gastric cancer.

Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Invariant natural killer T (iNKT) cells are innate-like cytotoxic T lymphocytes involved in tumor immune surveillance. They can be activated either through CD1d-presented glycolipid antigens recognized by their invariant T-cell receptor, cytokines or by sensing tumor-associated stress-induced ligands through the natural killer group 2, member D (NKG2D) receptor. Although the number and functionality of iNKT cells may be decreased in several types of cancer, here we show that GC patients presented a mild increase in iNKT cell frequencies and numbers in the blood compared with healthy donors. In GC patients, iNKT cells, expanded in vitro with α-galactosyl ceramide and stimulated with phorbol 12-myristate 13-acetate and ionomycin, produced higher levels of interleukin-2 and transforming growth factor-beta, while their capacity to degranulate remained preserved. Because tumor-derived epithelial cell adhesion molecule-positive epithelial cells did not display surface CD1d, and NKG2D ligands (NKG2DLs) were detected in the gastric tumor milieu, we envisioned a role for NKG2D in iNKT cell functions. Peripheral iNKT cells from GC patients and controls presented similar levels of NKG2D; nevertheless, the percentages of interferon-γ-producing and CD107a-positive iNKT cells from patients were reduced upon challenge with CD1d-negative, NKG2DL-positive K562 cells, suggesting a compromised response by iNKT cells in GC patients, which may not result from impaired NKG2D/NKG2DL signaling. The decreased response of iNKT cells may explain the fact that higher frequencies of circulating iNKT cells did not confer a survival benefit for GC patients. Therefore, functional impairment of iNKT cells in GC may contribute to tumor immune escape and favor disease progression.

The association of HLA-G polymorphisms and the synergistic effect of sMICA and sHLA-G with chronic kidney disease and allograft acceptance.

The HLA-G and MICA genes are stimulated under inflammatory conditions and code for soluble (sMICA and sHLA-G) or membrane-bound molecules that exhibit immunomodulatory properties. It is still unclear whether they would have a synergistic or antagonistic effect on the immunomodulation of the inflammatory response, such as in chronic kidney disease (CKD), contributing to a better prognosis after the kidney transplantation. In this study, we went from genetic to plasma analysis, first evaluating the polymorphism of MICA, NKG2D and HLA-G in a cohort from Southern Brazil, subdivided in a control group of individuals (n = 75), patients with CKD (n = 94), and kidney-transplant (KT) patients (n = 64). MICA, NKG2D and HLA-G genotyping was performed by polymerase chain reaction with specific oligonucleotide probes, Taqman and Sanger sequencing, respectively. Levels of soluble forms of MICA and HLA-G were measured in plasma with ELISA. Case-control analysis showed that the individuals with haplotype HLA-G*01:01/UTR-4 have a lower susceptibility to develop chronic kidney disease (OR = 0.480; p = 0.032). Concerning the group of kidney-transplant patients, the HLA-G genotypes +3010 GC (rs1710) and +3142 GC (rs1063320) were associated with higher risk for allograft rejection (OR = 5.357; p = 0.013 and OR = 5.357, p = 0.013, respectively). Nevertheless, the genotype +3010 GG (OR = 0.136; p = 0.041) was associated with kidney allograft acceptance, suggesting that it is a protection factor for rejection. In addition, the phenotypic analysis revealed higher levels of sHLA-G (p = 0.003) and sMICA (p < 0.001) in plasma were associated with the development of CKD. For patients who were already under chronic pathological stress and underwent a kidney transplant, a high sMICA (p = 0.001) in pre-transplant proved to favor immunomodulation and allograft acceptance. Even so, the association of genetic factors with differential levels of soluble molecules were not evidenced, we displayed a synergistic effect of sMICA and sHLA-G in response to inflammation. This increase was observed in CKD patients, that when undergo transplantation, had this previous amount of immunoregulatory molecules as a positive factor for the allograft acceptance.

Viral inhibitors of NKG2D ligands for tumor surveillance.

INTRODUCTION: Natural Killer cells (NK) are key for the innate immune response against tumors and viral infections. Several viral proteins evade host immune response and target the NK cell receptor NKG2D and its ligands. Areas covered: This review aimed to describe the viruses and their proteins that interfere with the NKG2D receptor and their ligands, and how these interactions lead to immune evasion, host protection, and tissue damage from acute and chronic viral infections. Expert opinion: The study of viral proteins has already impacted the field of oncology. A prime example is the HBV vaccine and the development of antiviral drugs for HIV, Hepatitis C, and the family of Herpesviridae viruses. The NKG2D system seems to be a rational therapeutic target. Nevertheless, an effective cytotoxic response by NK cells is mediated by a network of activating and inhibitory receptors, the integration of which determines if the NK cell becomes cytotoxic or permissive. Immunotherapeutic agents that increase the antitumor lytic activity of NK cells through modulating activation and inhibitory signaling of NK cells are being developed. Nevertheless, more research is needed to dissect the integrative mechanism of NK cells function to fully exploit their antitumor and antiviral effector mechanisms.

Clinical significance of tumor expression of major histocompatibility complex class I-related chains A and B (MICA/B) in gastric cancer patients.

Gastric cancer (GC) is the third most common cause of cancer death worldwide. Natural killer cells play an important role in the immune defense against transformed cells. They express the activating receptor NKG2D, whose ligands belong to the MIC and ULBP/RAET family. Although it is well established that these ligands are generally expressed in tumors, the association between their expression in the tumor and gastric mucosa and clinical parameters and prognosis of GC remains to be addressed. In the present study, MICA and MICB expression was analyzed, by flow cytometry, in 23 and 20 pairs of gastric tumor and adjacent non-neoplasic gastric mucosa, respectively. Additionally, ligands expression in 13 tumors and 7 gastric mucosa samples from GC patients were evaluated by immunohistochemistry. The mRNA levels of MICA in 9 pairs of tumor and mucosa were determined by quantitative PCR. Data were associated with the clinicopathological characteristics and the patient outcome. MICA expression was observed in 57% of tumors (13/23) and 44% of mucosal samples (10/23), while MICB was detected in 50% of tumors (10/20) and 45% of mucosal tissues (9/20). At the protein level, ligand expression was significantly higher in the tumor than in the gastric mucosa. MICA mRNA levels were also increased in the tumor as compared to the mucosa. However, clinicopathological analysis indicated that, in patients with tumors >5 cm, the expression of MICA and MICB in the tumor did not differ from that of the mucosa, and tumors >5 cm showed significantly higher MICA and MICB expression than tumors ≤5 cm. Patients presenting tumors >5 cm that expressed MICA and MICB had substantially shorter survival than those with large tumors that did not express these ligands. Our results suggest that locally sustained expression of MICA and MICB in the tumor may contribute to the malignant progression of GC and that expression of these ligands predicts an unfavorable prognosis in GC patients presenting large tumors.

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications.

BACKGROUND: Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. RESULTS: We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. CONCLUSIONS: Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Re-organization of mitochondria at the NK cell immune synapse.

As part of the innate immune response NK cells destroy infected, transformed, or otherwise stressed cells within hours of activation. In contrast, CD4(+) T lymphocytes require a sustained increase in their metabolism in order to cope with the biogenesis of cell components, in a process of proliferation and differentiation into effector cells. Recently, mitochondria have been implied in T lymphocyte immune synapse function but little is known on the role of mitochondria in the NK cell interaction with tumour cells. Here we analysed NK cells mitochondrial membrane potential (Deltapsi(m)) as an indicator of mitochondrial energy status and cellular homeostasis. Upon contact with K562 tumour cells, NK cells undergo Deltapsi(m) depolarization, indicating a rapid consumption of their metabolic energy. Furthermore, pharmacological inhibition of ATP synthesis down-regulates NK cell cytotoxic activity. Confocal- and electron-microscopy analyses showed re-organization of NK cells mitochondria towards the site of interaction with K562 tumour cell (NK cell immune synapse), perhaps as a way to compensate for local energy consumption. Interestingly, mitochondrial re-organization also takes place following NK stimulation with anti-NKGD2 antibodies but not with anti-KIR2DL1 antibodies, suggesting that activating rather than inhibiting cell signalling, triggered by NK cell receptors, is involved in NK cell mitochondria dynamics.

Major histocompatibility complex class I chain-related gene A polymorphism and linkage disequilibrium with HLA-B alleles in Euro-Brazilians.

Major histocompatibility complex class I chain-related gene A (MICA) was identified within the human leukocyte antigen (HLA) class I region and was located 46 kb centromeric from HLA-B locus. It functions as a ligand for human gammadelta T, CD8 T and natural killer (NK) cells by binding the NKG2D receptor. The aims of the present study were to determine the distribution of MICA alleles and MICA-HLA-B haplotypes in a sample of Euro-Brazilians. Through the combination of three typing methods, polymerase chain reaction (PCR)-sequence-specific oligonucleotide probe, PCR-sequence-specific primer and PCR-restriction fragment length polymorphism, 19 alleles were detected besides a MICA gene deletion in a sample composed by 204 unrelated Euro-Brazilians. The most commonly observed alleles were: MICA*00801 (25.3%), MICA*00201 (17.7%) and MICA*00901 (13.7%). The GCT repeat polymorphism variant A6 was the most commonly found. The most frequent haplotype found in this study was MICA*00901-B*51 (8.1%), followed by haplotypes MICA*00201-B*35 (6.1%) and MICA*00801-B*07 (6.1%). MICA*00801 truncated product, and its low affinity for NKG2D receptor may work as an inhibitor in its putative soluble form. It may also be that selective forces may favor MICA*00801 heterozygosity with NKG2D high affinity MICA alleles enabling activation and inhibition of cytotoxic activity of cells expressing the NKG2D receptor. The possibility of selective neutrality or of balancing selection still provides no explanation for MICA gene polymorphisms. Is it maintained by genetic drift or by the influence of migratory waves? Are there favored alleles while others present the same adaptive value?