Genetics: Anticipating chemotherapy resistance

Genetics: Anticipating chemotherapy resistance
Owing to the complex processes required for anthracycline-induced cytotoxicity, a prospectively defined multifactorial Consensus Signature (ConSig) might improve prediction of anthracycline response in triple-negative breast cancer (TNBC) patients, whose only standard systemic treatment option is chemotherapy. In this study, researchers led by Angelo Di Leo of the Istituto Toscano Tumori in Italy set out to construct and evaluate a multifactorial signature, comprising measures of each function required for anthracycline sensitivity in TNBC.

Mutations of the human interferon alpha-2b gene in brain tumor patients exposed to different environmental conditions

S Shahid, M Nawaz Chaudhry, N Mahmood and S Sheikh. Mutations of the human interferon alpha-2b gene in brain tumor patients exposed to different environmental conditions. Cancer Gene Therapy (2015) 22, 246–261; doi:10.1038/cgt.2015.12; published online 3 April 2015.

This study was aimed at finding out mutations of an anticancerous, antiviral and immunomodulatory gene (human interferon alpha-2b) in low- and high-grade brain tumor patients and correlate from hematological profiles. A molecular analysis was performed in which DNAs were extracted from brain biopsy samples of brain tumor patients. The gene was amplified through the PCR technique, and genetic data from sequencing were analyzed by bioinformatics to determine how mutations will lead to changes in human interferon alpha 2b protein in patients. A total of 38%gene mutations were identified among brain tumor patients. The highest percentage of (36%) frameshift mutations was identified. Hematological analysis shows modulations in the ‘lymphocytes’ parameter in a majority (64%) of the brain tumor patients. Environmental factors have been reported as risks of brain cancer. Patients were found to be under environmental stress from contaminated drinking water and from local gamma radiations. Brain tumor patients were found to have various mutations in an immunomodulating human interferon alpha-2b gene. These patients had immunosuppression that was further affirmed from their hematological profiles. This analysis may be helpful to develop certain biomarkers that may be used to develop novel immunotherapeutical drugs, which enhance a better immune response.

Activated NKT Cells Can Condition Different Splenic Dendritic Cell Subsets To Respond More Effectively to TLR Engagement and Enhance Cross-Priming.

Osmond TL, Farrand KJ, Painter GF, Ruedl C, Petersen TR, Hermans IF.
Activated NKT Cells Can Condition Different Splenic Dendritic Cell Subsets To Respond More Effectively to TLR Engagement and Enhance Cross-Priming. J Immunol. 2015 Jun 15. pii: 1401751. [Epub ahead of print]

The function of dendritic cells (DCs) can be modulated through multiple signals, including recognition of pathogen-associated molecular patterns, as well as signals provided by rapidly activated leukocytes in the local environment, such as innate-like T cells. In this article, we addressed the possibility that the roles of different murine DC subsets in cross-priming CD8+ T cells can change with the nature and timing of activatory stimuli. We show that CD8α+ DCs play a critical role in cross-priming CD8+ T cell responses to circulating proteins that enter the spleen in close temporal association with ligands for TLRs and/or compounds that activate NKT cells. However, if NKT cells are activated first, then CD8α- DCs become conditioned to respond more vigorously to TLR ligation, and if triggered directly, these cells can also contribute to priming of CD8+ T cell responses. In fact, the initial activation of NKT cells can condition multiple DC subsets to respond more effectively to TLR ligation, with plasmacytoid DCs making more IFN-α and both CD8α+ and CD8α- DCs manufacturing more IL-12. These results suggest that different DC subsets can contribute to T cell priming if provided appropriately phased activatory stimuli, an observation that could be factored into the design of more effective vaccines.

A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production.

Birkholz AM, Girardi E, Wingender G, Khurana A, Wang J, Zhao M, Zahner S, Illarionov PA, Wen X, Li M, Yuan W, Porcelli SA, Besra GS, Zajonc DM, Kronenberg M. A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production. J Immunol. 2015 Jun 15. pii: 1500070. [Epub ahead of print]

In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.

Reconstitution models to evaluate natural killer T cell function in tumor control.

Gebremeskel S, Slauenwhite D, Johnston B. Reconstitution models to evaluate natural killer T cell function in tumor control. Immunol Cell Biol. 2015 Jun 22. doi: 10.1038/icb.2015.67. [Epub ahead of print]

Natural killer T (NKT) cells are glycolipid-reactive T lymphocytes that function in immunosurveillance and immune regulation. However, reduced tumor control in NKT cell deficient Jα18-/- mice may be confounded by an overall reduction in TCR repertoire diversity in these animals. Mechanistic studies are also hindered by a lack of tools to target molecules specifically in NKT cells. To address these issues, we developed protocols to expand functional NKT cells and stably reconstitute them in Jα18-/- mice. In vivo delivery of α-galactosylceramide (α-GalCer)-loaded dendritic cells expandedNKT cells in wild-type mice without skewing CD4 or TCR Vβ expression profiles. Expanded NKT cells exhibited enhanced cytokine responses upon re-stimulation with glycolipid or CD3 ligation. Adoptive transfer of recently expanded wild-type or IFN-γ-/- NKT cells protected recipient Jα18-/- mice from B16 melanoma metastasis without the need for additional glycolipid stimulation. However, NKT cell reconstitution in recipient Jα18-/- mice was short lived. Long term reconstitution was only achieved when expanded NKT cells were transferred into sublethally irradiated recipients. Thirty days after transfer, NKT cell numbers, phenotype, and α-GalCer-induced cytokine responses were equivalent to naïve wild-type mice. Jα18-/- recipients reconstituted with wild-type or IFN-γ-/- NKT cells were both protected from B16 melanoma metastasis following α-GalCer treatment, and NK cell transactivation was intact in mice reconstituted with IFN-γ-/- NKT cells. These studies validate the use of reconstitution protocols to investigate mechanisms of NKT cell immune function, demonstrating that NKT cell-derived IFN-γ and the altered TCR repertoire in Jα18-/- mice do not impactNKT cell-mediated anti-tumor responses.

CD20; CD73; Co-stimulatory molecules; Cytokine; IL-10; α-galactosylceramide

Almishri W, Deans J, Swain MG. Rapid activation and hepatic recruitment of innate-like regulatory B cells after invariant NKT cell stimulation in mice. J Hepatol. 2015 Jun 18. pii: S0168-8278(15)00398-0. doi: 10.1016/j.jhep.2015.06.007. [Epub ahead of print]

BACKGROUND & AIMS:
Invariant natural killer T (iNKT) cells are present within the liver and have been implicated in the development of many liver diseases. Upon activation by glycolipid ligands (including α-galactosylceramide; αGalCer), hepatic iNKT cells produce numerous cytokines and recruit both pro-inflammatory and regulatory immune cells. However, the involvement of B cells in this process is poorly defined.
METHODS:
Wildtype (male, C57BL/6), B cell deficient, or B cell depleted mice were injected with αGalCer or vehicle and hepatic B cell phenotype and liver injury subsequently determined.
RESULTS:
iNKT cell activation resulted in liver injury and the rapid activation and hepatic recruitment of B cells (mainly innate-like B1 and MZ-like B cells) from the spleen and peritoneal cavity. B cells recruited to the liver produce IL-10 and TGFβ, and express cell surface CD73 (ectoenzyme which generates adenosine). B cell deficient mice developed augmented αGalCer-induced hepatitis, enhanced neutrophil recruitment and striking alterations in the hepatic cytokine milieu. αGalCer-induced hepatitis was unaltered in IL-10 -/- mice, or after TGFβ neutralization, but was significantly worsened in mice treated with a CD73 inhibitor.
CONCLUSIONS:
iNKT cell stimulation recruits innate-like regulatory B cells to the liver which suppress hepatic inflammation through IL-10 and TGFβ1 independent mechanisms, but involve CD73 activity. These findings highlight an important inflammation-suppressing role for B cells at early time points during the development of an innate immune response within the liver, and represent a potential therapeutic target for the treatment of liver disease.

Dysregulated osteoclastogenesis is related to natural killer T cell dysfunction in rheumatoid arthritis.

Jin HM, Kee SJ, Cho YN, Kang JH, Kim MJ, Jung HJ, Park KJ, Kim TJ, Lee SI, Choi H, Koh JT, Kim N, Park YW. Dysregulated osteoclastogenesis is related to natural killer T cell dysfunction in rheumatoid arthritis. Arthritis Rheumatol. 2015 Jun 19. doi: 10.1002/art.39244. [Epub ahead of print]

OBJECTIVE:
To investigate the role played by NKT cells in osteoclastogenesis and their effects on inflammatory bone destruction.
METHODS:
Patients with RA (n=25) and healthy controls (n=12) were enrolled in this study. In vitro osteoclastogenesis experiments were performed using peripheral blood mononuclear cells (PBMCs) in the presence of M-CSF and receptor activator of nuclear factor κB ligand (RANKL). PBMCs were cultured in vitro with α-galactosylceramide (αGalCer), and proliferation indices of NKT cells were estimated by flow cytometry. In vivo effects of αGalCer-stimulated NKT cells on inflammation and bone destruction were determined in collagen-induced arthritis (CIA) mice.
RESULTS:
In vitro osteoclastogenesis was found to be significantly inhibited by αGalCer in healthy controls, but not in RA patients. Proliferative responses of NKT cells and STAT-1 phosphorylation in monocytes in response to αGalCer were impaired in RA patients. Notably, αGalCer-stimulatedNKT cells inhibited osteoclastogenesis mainly via interferon-γ production, in a cytokine-dependent manner (not by cell-cell contact), and down-regulated osteoclast-associated genes. αGalCer-treated mice showed less severe arthritis and reduced bone destruction. Moreover, proinflammatory cytokine expression in arthritic joints was found to be reduced by αGalCer treatment.
CONCLUSIONS:
This study primarily demonstrates that αGalCer-stimulated NKT cells have a regulatory effect on osteoclastogenesis and a protective effect on inflammatory bone destruction. However, it also shows that these effects of αGalCer are diminished in RA patients, and that this is related to NKT cell dysfunction. These findings provide important information for those searching for novel therapeutic strategies to prevent bone destruction in RA. This article is protected by copyright. All rights reserved.

Impact of Pre-Transplant Anti-T Cell Globulin (ATG) on Immune Recovery after Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation.

Servais S, Menten-Dedoyart C, Beguin Y, Seidel L, Gothot A, Daulne C, Willems E, Delens L, Humblet-Baron S, Hannon M, Baron F. Impact of Pre-Transplant Anti-T Cell Globulin (ATG) on Immune Recovery after Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation. PLoS One. 2015 Jun 22;10(6):e0130026. doi: 10.1371/journal.pone.0130026. eCollection 2015.

BACKGROUND:
Pre-transplant infusion of rabbit anti-T cell globulin (ATG) is increasingly used as prevention of graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (PBSCT). However, the precise impact of pre-transplant ATG on immune recovery after PBSCT is still poorly documented.
METHODS:
In the current study, we compared immune recovery after myeloablative PBSCT in 65 patients who either received (n = 37) or did not (n = 28) pre-transplant ATG-Fresenius (ATG-F). Detailed phenotypes of circulating T, B, natural killer (NK) and invariant NKT (iNKT) cells were analyzed by multicolor flow cytometry at serial time-points from day 40 to day 365 after transplantation. Thymic function was also assessed by sjTREC quantification. Serious infectious events were collected up to 2 years post-transplantation.
RESULTS:
Pre-transplant ATG-F had a prolonged (for at least up to 1-year) and selective negative impact on the T-cell pool, while it did not impair the recovery of B, NK nor iNKT cells. Among T cells, ATG-F selectively compromised the recovery of naïve CD4+, central memory CD4+ and naïve CD8+ cells, while it spared effector memory T and regulatory T cells. Levels of sjTRECs were similar in both cohorts at 1-year after PBSCT, suggesting that ATG-F unlikely impaired thymopoiesis at long-term after PBSCT. Finally, the incidence and rate of serious infections were similar in both groups, while ATG-F patients had a lower incidence of grade II-IV acute graft-versus-host disease.
CONCLUSIONS:
Pre-transplant ATG-F induces long-lasting modulation of the circulating T-cell pool after myeloablative PBSCT, that may participate in preventing graft-versus-host disease without deeply compromising anti-pathogen defenses.

Candida albicans up-regulates the Fas-L expression in liver Natural Killer and Natural Killer T cells.

Candida albicans, Fas-Fas-L, Liver, NK cells, NKT cells, TLR2, TNF-alpha

Renna MS, Figueredo CM, Rodríguez-Galán MC, Icely PA, Cejas H, Cano R, Correa SG, Sotomayor CE. Candida albicans up-regulates the Fas-L expression in liver Natural Killer and Natural Killer T cells. Immunobiology. 2015 Jun 10. pii: S0171-2985(15)30013-9. doi: 10.1016/j.imbio.2015.06.014. [Epub ahead of print]

After Candida albicans arrival to the liver, the local production of proinflammatory cytokines and the expanded intrahepatic lymphocytes (IHL) can be either beneficial or detrimental to the host. Herein we explored the balance between protective inflammatory reaction and liver damage, focusing our study on the contribution of TNF-α and Fas-Fas-L pathways in the hepatocellular apoptosis associated to C. albicans infection. A robust tissue reaction and a progressive increase of IL-1β, IL-6 and TNF-α were observed in infected animals. Blocking the biological activity of TNF-α did not modify the number of apoptotic cells observed in C. albicans infected animals. Fas-L molecule was up regulated on purified hepatic mononuclear cells and its expression progressed with the infection. In the IHL compartment, the absolute number of Fas-L+ NK and NKT cells increased on days 1 and 3 of the infection. C. albicans was also able to up regulate Fas-L expression in normal liver NK and NKT cells after in vitro contact. The innate receptor TLR2 was involved in this phenomenon. In the interplay between host factors and evasion strategies exploited by pathogens, the mechanism supported here could represent an additional way that allows this fungus to circumvent protective immune responses in the liver.

T, B, and NKT Cells in Systemic Inflammation in Obstructive Sleep Apnoea.

Domagała-Kulawik J, Osińska I, Piechuta A, Bielicki P, Skirecki T. T, B, and NKT Cells in Systemic Inflammation in Obstructive Sleep Apnoea. Mediators Inflamm. 2015;2015:161579. doi: 10.1155/2015/161579. Epub 2015 May 25.

Background. Obstructive sleep apnoea syndrome (OSAS) brings risk of serious complications. The study objective was to assess elements of the cellular immune response in the course of OSAS. Methods. Peripheral blood (PB) lymphocytes: T, B, NK, NKT-like, Th, Tc, and HLA DR+ T cells were evaluated by flow cytometry of 48 OSA patients; the concentration of adiponectin, interleukin 1β, and TNFα was measured by ELISA method. The OSA complication score was developed and used for statistical analysis. 

Results. The proportion of B cells and Th/Tc ratio were significantly lower in the BP of OSA patients when compared with control subjects (median 7.9 versus 10.9%, 0.9 versus 1.5, p < 0.05). The proportion of Tc, NK, NKT-like, and HLADR positive T cells were elevated in OSA patients when compared with healthy subjects (36.4 versus 26.8, 15.5 versus 8.5, 5.7 versus 3.0, and 8.4 versus 4.5%, p < 0.05, resp.) and were more pronounced in patients with metabolic syndrome. The grade of OSA complication score correlated with systemic inflammation markers and the proportion of B cells. The value of adiponectin/BMI ratio correlated significantly with SpO2 (r = 0.31, p < 0.05), CRP (r = -0.35, p < 0.05), TNFα concentration (r = -0.36, p < 0.05), and proportion of B cells (r = 0.32, p < 0.05). 

Conclusion. Lymphocytes B, Tc, NK, NKT-like, and adiponectin are involved in systemic immune response in OSA patients possibly predisposing them to cardiovascular and metabolic complications.

Administration of α-Galactosylceramide improves adenine-induced renal injury.

Aguiar CF, Naffah-de-Souza C, Castoldi A, Corrêa-Costa M, Braga TT, Naka ÉL, Amano MT, Abate DT, Hiyane MI, Cenedeze MA, Filho AP, Câmara NO. Administration of α-Galactosylceramide improves adenine-induced renal injury. Mol Med. 2015 Jun 18. doi: 10.2119/molmed.2014.00090. [Epub ahead of print]

NKT cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to more than 90% of the total population of NKTs and reacts to α-Galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as IFN-γ and IL-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-week-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 days. Both adenine-fed WT and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18 KO mice presented higher expression of KIM-1, TNF-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection we observed a significantly reduction in serum creatinine, pro-inflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation.

Mycobacterium bovis; NKG2D; bladder cancer; natural cytotoxicity receptors; natural killer cells

García-Cuesta EM, López-Cobo S, Álvarez-Maestro M, Esteso G, Romera-Cárdenas G, Rey M, Cassady-Cain RL, Linares A, Valés-Gómez A, Reyburn HT, Martínez-Piñeiro L, Valés-Gómez M. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation. Front Immunol. 2015 Jun 8;6:284. doi: 10.3389/fimmu.2015.00284. eCollection 2015.

Intravesical instillation of bacillus Calmette-Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.