Dissection of the NKG2C NK cell response against Puumala Orthohantavirus

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C⁺ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C⁺ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C⁺ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2C^wt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2C^del/del) and heterozygous (NKG2C^wt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C⁺ cells. We therefore analyzed the PUUV-mediated NKG2C⁺ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C⁺ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C⁺ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2C^del and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2C^wt/del allele was significantly overrepresented, compared to the NKG2C^wt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2C^wt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2C^wt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C⁺ NK cell response, as reflected by NKG2C^wt/del variant, may be associated with a higher risk for a severe hantavirus infections.     

慢性活动型EB病毒感染的致病机理研究进展

慢性活动型EB病毒(Epstein Barr virus,EBV)感染(chronic active EBV infection,CAEBV)是一类EBV相关的T/NK淋巴细胞增殖性疾病(Epstein Barr virus-associated T/NK-cell lymphoproliferative diseases,EBV~+T/NK-cell LPD),以持续反复的类似传染性单核细胞增多症(infectious mononucleosis,IM)临床病征和EBV感染细胞的克隆性增殖为主要特征,在临床上具有较高的发病率和致死率.目前对于CAEBV与其他各类EBV相关的T/NK淋巴细胞增殖性疾病之间的界定以及致病机理的研究仍处于发展阶段,临床上对于该类疾病的治疗也无完全有效的手段.本文主要从EBV如何感染T/NK细胞、EBV相关的病毒学研究、机体自身遗传及免疫背景几方面,综述了目前对于CAEBV致病机理的研究进展,旨在为进一步研究提供思路和线索.     

Effect of the simultaneous administration of glucocorticoids and IL-15 on human NK cell phenotype,proliferation and function

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56⁺ cells induces increased expansion of CD56⁺CD3⁻ cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16^low/neg NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.     

Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens

Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO⁺ or CD45RO^- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO⁺ but not CD45RO^- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45RO^highNKG2D^highgranzyme B^high. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO⁺ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.     

MAGT1 messenger RNA-corrected autologous T and natural killer cells for potential cell therapy in X-linked immunodeficiency with magnesium defect,Epstein-Barr virus infection and neoplasia disease

Background aimX-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect' (XMEN) disease is caused by mutations in the magnesium transporter 1 (MAGT1) gene. Loss of MAGT1 function results in a glycosylation defect that abrogates expression of key immune proteins such as the NKG2D receptor on CD8⁺ T and NK cells, which is critical for the recognition and killing of virus-infected and transformed cells, a biomarker for MAGT1 function. Patients with XMEN disease frequently have increased susceptibility to EBV infections and EBV-associated B cell malignancies, for which no specific treatment options are currently available. Experimental transfer of donor EBV-specific cytotoxic T cells may be beneficial but carries the risks of eliciting alloimmune responses. An approach for cell therapy to address viral infections and associated complications that avoids the risks of alloimmunity is needed.MethodsHere the authors assess the feasibility and efficiency of correcting autologous lymphocytes from XMEN patients by MAGT1 mRNA electroporation (EP) that avoids genomic integration and can be scaled for clinical application.Results and conclusionsRestoration of NKG2D expression was demonstrated in XMEN patient lymphocytes after MAGT1 mRNA electroporation that reach healthy donor levels in CD8⁺ T and NK cells at 1-2 days after EP. NKG2D expression persisted at ～50% for 2 weeks after EP. Functionally, mRNA-correction of XMEN NK cells rescued cytotoxic activity also to healthy donor NK cell level. The restored NKG2D receptor expression and function were unaffected by cryopreservation, which will make feasible repeat infusions of MAGT1 mRNA-corrected autologous XMEN CD8⁺ T and NK cells for potential short term therapy for XMEN patients without the risks of alloimmunization.     

Tumour‐experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)‐γ secretion on engagement of the natural‐killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell‐mediated anti‐tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co‐culture with some melanomas, activated CD4⁺ T cells promoted degranulation, and NKG2D‐ and NKp46‐dependent IFN‐γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour‐cell surface (trogocytosis). This effect was observed in CD4⁺, CD8⁺ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain‐related protein A on co‐culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.     

Acute Cytomegalovirus Infection Is Associated with Increased Frequencies of Activated and Apoptosis-Vulnerable T Cells in HIV-1-Infected Infants

Cytomegalovirus (CMV) coinfection is associated with infant HIV-1 disease progression and mortality. In a cohort of Kenyan HIV-infected infants, the frequencies of activated (CD38⁺ HLA-DR⁺) and apoptosis-vulnerable (CD95⁺ Bcl-2⁻) CD4⁺ and CD8⁺ T cells increased substantially during acute CMV infection. The frequency of activated CD4⁺ T cells was strongly associated with both concurrent CMV coinfection (P = 0.001) and HIV-1 viral load (P = 0.05). The frequency of apoptosis-vulnerable cells was also associated with CMV coinfection in the CD4 (P = 0.02) and CD8 (P < 0.001) T cell subsets. Similar observations were made in HIV-exposed uninfected infants. CMV-induced increases in T cell activation and apoptosis may contribute to the rapid disease progression in coinfected infants.     

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells.MethodsPC3^PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells.ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen.ConclusionsCollectively, the results demonstrate the feasibility of PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.     

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94⁺ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94⁺ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL⁺ CD56^dim NK cells, in contrast to the efficient responses by CD56^bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94⁺ KIR2DL^- NK cells may be uniquely beneficial.     

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

Background

The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8⁺ cytotoxic T cells and is classically considered absent in CD4⁺ T cells. However, recent studies have identified a distinctive population of CD4⁺ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4⁺ T cell-immunocompromised individuals suggests that CD4⁺ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4⁺ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4⁺NKG2D⁺ T cell expansion were also measured.

Results

Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4⁺ T cells and CD8⁺ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4⁺ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4⁺NKG2D⁺ T cells was seen in patients with CIN 1, despite the overall levels of CD4⁺ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4⁺NKG2D⁺ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group.

Conclusions

Taken together, our results suggest that the significant increase within the CD4⁺NKG2D⁺ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.     

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-β, IL-10 and IFN-γ by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-γ is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-β only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-β induced IL-10 production. IL-10 produced in naïve subjects when TGF-β is neutralized or in LCL patients did not modify the IFN-γ production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-β is neutralized clearly showed that they are memory CD45RA⁻ CD8⁺ T cells. In LCL patients, IL-10 producing cells are both CD45RA⁻ CD4 and CD8⁺ T cells. The role of these IL-10 producing CD8⁺ T cells in the development of the diseases should be carefully evaluated.     

Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70⁺/HLA-E⁻) CX⁺ and K562 cells. However, it had no effect on the responsiveness to Hsp70⁻/HLA-E⁻ CX⁻ cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70⁺/HLA-E⁺ leukemic blasts was weaker than that against Hsp70⁺/HLA-E⁻ K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E^R or HLA-E^G alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.     

Long-term proliferation of functional human NK cells,with conversion of CD56<Superscript>dim</Superscript> NK cells to a CD56<Superscript>bright</Superscript> phenotype,induced by carcinoma cells co-expressing 4-1BBL and IL-12

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56^bright, and we show that isolated CD56^dimCD16⁺ NK cells can switch to a CD56^brightCD16⁻ phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required ‘help’ from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56^bright and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.     

Establishment of Novel Monoclonal Fabs Specific for Epstein-Barr Virus Encoded Latent Membrane Protein 1

<正>Dear Editor,Epstein-Barr virus (EBV, also termed human herpesvirus-4) was the first identified human tumor virus. Since its discovery in 1964, studies have shown that EBV infects over 90%of all people by the time they are adults(Williams and Crawford 2006). EBV infection can result in     

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A⁺ and KIR3DL1⁺ cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D⁻NKG2A⁺, and NKG2D⁻KIR3DL1⁺ on the NK CD56^dim cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D⁻NKG2A⁺ expression on the CD56^bright subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.     

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis¹. LCL have been used to present antigens in a variety of immunologic assays^{2, 3}. In addition, LCL can be used to generate human monoclonal antibodies^{4, 5} and provide a potentially unlimited source when access to primary biologic materials is limited^{6, 7}.A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide⁸, and pokeweed mitogen⁹ to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells^{7, 10-12}.The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23^hiCD58⁺ cells observed as early as three days post-infection indicates a successful outcome.     

Expansion of CD11b+Ly6G+Ly6Cint cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b⁺Gr-1⁺, mostly CD11b⁺Ly6G⁺Ly6C^int and CD11b⁺Ly6G^?Ly6C^high cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b⁺Ly6G⁺Ly6C^int cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b⁺Ly6G⁺Ly6C^int cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b⁺Gr-1⁺ cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b⁺Gr-1⁺ cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b⁺Ly6G⁺Ly6C^int cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.