Reciprocal relationship of T regulatory cells and monocytic myeloid-derived suppressor cells in LP-BM5 murine retrovirus-induced immunodeficiency.

Immunomodulatory cellular subsets, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumour microenvironment and are targets of immunotherapy, but their role in retroviral-associated immunosuppression is less well understood. Due to known crosstalk between Tregs and MDSCs in the tumour microenvironment, and also their hypothesized involvement during human immunodeficiency virus/simian immunodeficiency virus infection, studying the interplay between these immune cells during LP-BM5 retrovirus-induced murine AIDS is of interest. IL-10-producing FoxP3+ Tregs expanded after LP-BM5 infection. Following in vivo adoptive transfer of natural Treg (nTreg)-depleted CD4+T-cells, and subsequent LP-BM5 retroviral infection, enriched monocytic MDSCs (M-MDSCs) from these nTreg-depleted mice displayed altered phenotypic subsets. In addition, M-MDSCs from LP-BM5-infected nTreg-depleted mice exhibited increased suppression of T-cell, but not B-cell, responses, compared with M-MDSCs derived from non-depleted LP-BM5-infected controls. Additionally, LP-BM5-induced M-MDSCs modulated the production of IL-10 by FoxP3+ Tregs in vitro. These collective data highlight in vitro and for the first time, to the best of our knowledge, in vivo reciprocal modulation between retroviral-induced M-MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of such regulatory cells during retroviral infection.

Selective Involvement of the Checkpoint Regulator VISTA in Suppression of B-Cell, but Not T-Cell, Responsiveness by Monocytic Myeloid-Derived Suppressor Cells from Mice Infected with an Immunodeficiency-Causing Retrovirus.

Inhibition of T-cell responses in tumor microenvironments by myeloid-derived suppressor cells (MDSCs) is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-cell, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T cells by inducible nitric oxide synthase (iNOS)/nitric oxide (NO), but uniquely, inhibition of B cells was ~50% dependent each on iNOS/NO and the MDSC-expressed negative-checkpoint regulator VISTA. Blockade with a combination of iNOS/NO and VISTA caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness.

Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

Use of IRF-3 and/or IRF-7 knockout mice to study viral pathogenesis: lessons from a murine retrovirus-induced AIDS model.

Interferon regulatory factor (IRF) regulation of the type I interferon response has not been extensively explored in murine retroviral infections. IRF-3(-/-) and select IRF-3/7(-/-) mice were resistant to LP-BM5-induced pathogenesis. However, further analyses strongly suggested that resistance could be attributed to strain 129-specific contamination of the known retrovirus resistance gene Fv1. Therefore, caution should be taken when interpreting phenotypes observed in these knockout mice, as strain 129-derived genetic polymorphisms may explain observed differences.

Myeloid-derived suppressor cells in murine retrovirus-induced AIDS inhibit T- and B-cell responses in vitro that are used to define the immunodeficiency.

Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b(+) Gr-1(+) Ly6C(+)) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1-PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G(low/±) Ly6C(+) CD11b(+)-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.

Upregulated selenoprotein I during lipopolysaccharide-induced B cell activation promotes lipidomic changes and is required for effective differentiation into IgM-secreting plasma B cells.

The mechanisms driving metabolic reprogramming during B cell activation are unclear, particularly roles for enzymatic pathways involved in lipid remodeling. We found that murine B cell activation with lipopolysaccharide (LPS) led to a 1.6-fold increase in total lipids that included higher levels of phosphatidylethanolamine (PE) and plasmenyl PE. Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase involved in the synthesis of both PE and plasmenyl PE, and SELENOI expression was also upregulated during activation. Selenoi knockout (KO) B cells exhibited decreased levels of plasmenyl PE, which plays an important antioxidant role. Lipid peroxidation was measured and found to increase ∼2-fold in KO vs. wild-type (WT) B cells. Cell death was not impacted by KO in LPS-treated B cells and proliferation was only slightly reduced, but differentiation into CD138 + Blimp-1+ plasma B cells was decreased ∼2-fold. This led to examination of B cell receptors important for differentiation that recognize the ligand B cell activating factor, and levels of TACI (transmembrane activator, calcium-modulator, and cytophilin ligand interactor) (CD267) were significantly decreased on KO B cells compared with WT control cells. Vaccination with ovalbumin/adjuvant led to decreased ovalbumin-specific immunoglobulin M (IgM) levels in sera of KO mice compared with WT mice. Real-time polymerase chain reaction analyses revealed a decreased switch from surface to secreted IgM in spleens of KO mice induced by vaccination or LP-BM5 retrovirus infection. Overall, these findings detail the lipidomic response of B cells to LPS activation and reveal the importance of upregulated SELENOI for promoting differentiation into IgM-secreting plasma B cells.

The role of indoleamine 2,3-dioxygenase in LP-BPM5 murine retroviral disease progression.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory intracellular enzyme involved in tryptophan degradation. IDO is induced during cancer and microbial infections by cytokines, ligation of co-stimulatory molecules and/or activation of pattern recognition receptors, ultimately leading to modulation of the immune response. LP-BM5 murine retroviral infection induces murine AIDS (MAIDS), which is characterized by profound and broad immunosuppression of T- and B-cell responses. Our lab has previously described multiple mechanisms regulating the development of immunodeficiency of LP-BM5-induced disease, including Programmed Death 1 (PD-1), IL-10, and T-regulatory (Treg) cells. Immunosuppressive roles of IDO have been demonstrated in other retroviral models, suggesting a possible role for IDO during LP-BM5-induced retroviral disease progression and/or development of viral load. METHODS: Mice deficient in IDO (B6.IDO-/-) and wildtype C57BL/6 (B6) mice were infected with LP-BM5 murine retrovirus. MAIDS and LP-BM5 viral load were assessed at termination. RESULTS: As expected, IDO was un-inducible in B6.IDO-/- during LP-BM5 infection. B6.IDO-/- mice infected with LP-BM5 retrovirus succumbed to MAIDS as indicated by splenomegaly, serum hyper IgG2a and IgM, decreased responsiveness to B- and T-cell mitogens, conversion of a proportion of CD4+ T cells from Thy1.2+ to Thy1.2-, and increased percentages of CD11b+Gr-1+ cells. LP-BM5 infected B6.IDO-/- mice also demonstrated the development of roughly equivalent disease kinetics as compared to infected B6 mice. Splenic viral loads of B6 and B6.IDO-/- mice were also equivalent after infection as measured by LP-BM5-specific Def Gag and Eco Gag viral mRNA, determined by qRT-PCR. CONCLUSIONS: Collectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load.

Inhibition of myeloid-derived suppressor cell (MDSC) activity by redox-modulating agents restores T and B cell proliferative responses in murine AIDS.

The mechanisms by which myeloid-derived suppressor cells (MDSCs) mediate inhibition prominently include the production of reactive nitrogen species, in particular those generated by inducible nitric oxide synthase (iNOS), and reactive oxygen species. LP-BM5 murine retroviral infection results in a profound immunodeficiency, known as murine AIDS, as well as in increased numbers and activity of monocytic-type MDSCs (M-MDSCs) that suppress both T and B cell responses. While M-MDSCs suppress T cells ex vivo in a fully iNOS/NO-dependent manner, M-MDSC suppression of B cell responses is only partially due to iNOS/NO. This study preliminarily explored the role of two redox-modulating compounds in inhibiting the M-MDSC suppressive activity in LP-BM5 infection. The tested molecules were: I-152 consisting in a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-cysteamine (SMEA) and C4-GSH that is the n-butanoyl glutathione (GSH) derivative. The results show that both molecules, tested in a concentration range between 3 and 20 mM, blocked the M-MDSC suppression of activated B and T cells ex vivo and restored their proliferative capacity in vivo. Ex vivo I-152 blockade of M-MDSC suppressiveness was more significant for T cell (about 70%) while M-MDSC blockade by C4-GSH was preferential for B cell responsiveness (about 60%), which was also confirmed by in vivo investigation. Beyond insights into redox-dependent suppressive effector mechanism(s) of M-MDSCs in LP-BM5 infection, these findings may ultimately be important to identify new immunotherapeutics against infectious diseases.

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade.

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

The programmed death-1 and interleukin-10 pathways play a down-modulatory role in LP-BM5 retrovirus-induced murine immunodeficiency syndrome.

Pathology due to the immune system's response to viral infections often represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In susceptible C57BL/6 (B6) mice, the murine retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, profound B- and T-cell immunodeficiency, and increased susceptibility to opportunistic pathogens and terminal B-cell lymphomas. Here, we report that B6.PD-1 (programmed death-1) and B6.IL-10 knockout mice are substantially more susceptible to LP-BM5-induced disease than wild-type B6 mice. LP-BM5-infected B6.PD-1(-/-) mice developed more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice: PD-1(-/-) mice are more susceptible to lower doses of LP-BM5 and show more exaggerated disease early postinfection. LP-BM5-infected B6.IL-10(-/-) mice also develop exaggerated LP-BM5-induced disease, compared to B6 mice, without a significant change in the retroviral load. By reciprocal reconstitution experiments, comparing wild-type versus PD-1(-/-) sources of the requisite cells for LP-BM5 pathogenesis-CD4 T and B cells, PD-1(+) B cells appear to be crucial in the normal limitation of LP-BM5-induced disease in B6 mice. Also, infected B6 mice have increased CD11b(+) spleen cells that express interleukin-10 (IL-10). However, PD-1(-/-) mice, though showing an even greater expansion of CD11b(+) cells after LP-BM5 inoculation, did not show an equivalent increase in IL-10-producing cells. Thus, it appears that PD-1/PD-L interactions and IL-10 are primarily important in moderating the effects of LP-BM5-induced disease in B6 mice.

H. pylori-induced apoptosis in human gastric cancer cells mediated via the release of apoptosis-inducing factor from mitochondria.

BACKGROUND AND AIM: Our previous study of Helicobacter pylori-induced apoptosis showed the involvement of Bcl-2 family proteins and cytochrome c release from mitochondria. Here, we examine the release of other factors from mitochondria, such as apoptosis-inducing factor (AIF), and upstream events involving caspase-8 and Bid. METHODS: Human gastric adenocarcinoma (AGS) cells were incubated with a cagA-positive H. pylori strain for 0, 3, 6, and 24 hours and either total protein or cytoplasmic, nuclear, and mitochondrial membrane fractions were collected. RESULTS: Proteins were immunoblotted for AIF, Bid, polyadenosine ribose polymerase (PARP), caspase-8, and beta-catenin. H. pylori activated caspase-8, caused PARP cleavage, and attenuated mitochondrial membrane potential. A time-dependent decrease in beta-catenin protein expression was detected in cytoplasmic and nuclear extracts, coupled with a decrease in beta-actin. An increase in the cytoplasmic pool of AIF was seen as early as 3 hours after H. pylori exposure, and a concomitant increase was seen in nuclear AIF levels up to 6 hours. A band corresponding to full-length Bid was seen in both the cytoplasmic and the nuclear fractions of controls, but not after H. pylori exposure. Active AIF staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls. CONCLUSION: H. pylori might trigger apoptosis in AGS cells via interaction with death receptors in the plasma membrane, leading to the cleavage of procaspase-8, release of cytochrome c and AIF from mitochondria, and activation of subsequent downstream apoptotic events, as reported previously for chlorophyllin. This is consistent with AIF activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.

LP-BM5 Retrovirus-Expanded Monocytic Myeloid-Derived Suppressor Cells Alter B Cell Phenotype and Function.

Our laboratory demonstrated that infection with the murine retrovirus LP-BM5 results in increased numbers of monocytic myeloid-derived suppressor cells (M-MDSCs) and that these M-MDSCs suppress not only T but also B cell responses. Because of the paucity of studies regarding the effects of MDSCs in general on B cells, we focused on these understudied B cell targets for M-MDSC effects on B cell phenotypic and functional parameters. M-MDSCs specifically decreased the proliferation of transitional type 2 (T2) B cells in response to polyclonal stimulation but increased germinal center and Ab-secreting B cell proportions and class-switched Ig production. Additionally, M-MDSCs inhibited the expression of CD40 and MHC class II on stimulated B cells and suppressed Ag presentation to Ag-specific CD4+ T cells. These alterations of the B cell compartment coincided with decreases in aerobic glycolysis, mitochondrial respiration, and glucose consumption; the latter specifically decreased in the T2 subset. To compare B cell targets of ex vivo M-MDSC suppression with the status of B cells during the course of LP-BM5-induced pathogenesis, including immunodeficiency in vivo, B cells from LP-BM5-infected mice were collected and analyzed. LP-BM5 infection resulted in several analogous alterations of B cells, as were observed with retrovirally expanded M-MDSC suppression in vitro, including decreased proliferation of T2 B cells, an increased proportion of germinal center and Ab-secreting B cells, increased production of class-switched Abs, decreased expression of CD40, and decreased metabolic activity upon stimulation.

Optic nerve hydropic axonal degeneration and blocked retrograde axoplasmic transport: histopathologic features in human high-pressure secondary glaucoma.

OBJECTIVE: To describe histopathologic features of anterior optic nerves of 12 eyes enucleated for sustained high ocular pressure from iris-ciliary body melanomas in 10 and choroidal melanomas with chronic angle closure in 2. METHODS: In this retrospective study, we analyzed cases indexed in 2 eye pathology laboratories and reviewed the pertinent literature. Cases were identified from diagnostic indexes; microscopic study of slides stained with hematoxylin-eosin and Verhoeff-van Gieson, Mallory trichrome, periodic acid-Schiff, alcian blue, or colloidal iron for acid mucopolysaccharide; review of available clinical documentation; and analysis of features and photography. The main outcome measures were description of optic nerve heads, prelaminar atrophy, laminar posterior bowing, locations and density of hydropic axonal degeneration, blocked retrograde axoplasmic transport, posterior atrophy, and optic nerve disorganization with glial proliferation. RESULTS: Hydropic axonal degeneration was present in front of, within, and posterior to the lamina cribrosa in all 12 eyes. This degeneration extended diffusely and posteriorly from the peripheral lamina and was most dense centrally in 10 eyes. Retrolaminar changes compatible with blockage of retrograde axoplasmic transport were seen in 9 eyes. Posterior atrophy with disorganization and glial proliferation was seen in 10 eyes. No eye had classic glaucomatous atrophic cupping. CONCLUSIONS: Diffuse and centrally intense hydropic axonal degeneration and central blocked retrograde axoplasmic transport explain loss of central acuity, generalized contraction of visual field, and generalized optic atrophy without glaucomatous cupping in eyes with prolonged high-pressure secondary glaucoma.

The Role of Myeloid-Derived Suppressor Cells in Viral Infection.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that are well described as potent immune regulatory cells during human cancer and murine tumor models. Reports of MDSCs during viral infections remain limited, and their association with immunomodulation of viral diseases is still being defined. Here, we provide an overview of MDSCs or MDSC-like cells identified during viral infections, including murine viral models and human viral diseases. Understanding the similarities and/or differences of virally induced versus tumor-derived MDSCs will be important for designing future immunotherapeutic approaches.

Apoptosis of epitope-specific antiretroviral cytotoxic T lymphocytes via Fas ligand-Fas interactions.

C57BL/6 (B6; H-2b) mice are capable of mounting a vigorous AKR/Gross Murine Leukemia Virus (MuLV)-specific cytotoxic T lymphocyte (CTL) response to AKR/Gross MuLVs whereas AKR.H- 2b congenic mice, although carrying the responder H-2b major histocompatibility haplotype, are specifically nonresponsive. Furthermore, when viable AKR.H-2b spleen cells are cocultured with primed responder B6 antiviral precursor CTLs, the AKR.H-2b cells function as "veto" cells that actively mediate the inhibition by apoptosis of B6 antiviral CTL generation in a contact-dependent, MHC-restricted, and veto cell Fas ligand (FasL)/responder T cell Fas-dependent manner. In the present study we show that antigen-specific, antiviral CTLs that survive apoptotic inhibition by AKR.H-2b veto cells display a less activated cell surface phenotype, and are less able to bind specific MHC-peptide tetramers, including on a per-T cell receptor (TcR) basis. In addition, surviving antiviral CTLs also appeared to be functionally deficient, based on both their reduced ability to lyse specific target cells and to produce interferon (IFN)-gamma. Carboxyfluorescein diacetate succinimidyl ester staining confirmed that AKR/Gross MuLV-specific CTLs proliferated less extensively when AKR.H-2b veto cells were included in cocultures. AKR/Gross MuLV-specific effector CTLs as well as memory CTLs were each efficiently targeted for inhibition by AKR.H-2b veto cells. Attempts to enhance the quality of the priming by multiple in vivo immunizations did not alter the capacity of the AKR.H-2b cells to inhibit the antiviral CTL response. These results further characterize the nature of the interaction between veto cells and antiviral CTLs, and underscore the efficiency of veto cell-mediated inhibition of the CTL response.

Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-ß.

Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-ß, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression.

Subpopulations of M-MDSCs from mice infected by an immunodeficiency-causing retrovirus and their differential suppression of T- vs B-cell responses.

Monocytic (CD11b(+)Ly6G(±/Lo)Ly6C(+)) myeloid derived suppressor cells (M-MDSCs) expand following murine retroviral LP-BM5 infection and suppress ex vivo polyclonal T-cell and B-cell responses. M-MDSCs 3 weeks post LP-BM5 infection have decreased suppression of T-cell, but not B-cell, responses and alterations in the degree of iNOS/NO dependence of suppression. M-MDSCs from LP-BM5 infected mice were sorted into four quadrant populations (Ly6C/CD11b density): all quadrants suppressed B-cell responses, but only M-MDSCs expressing the highest levels of Ly6C and CD11b (Q2) significantly suppressed T-cell responses. Further subdivision of this Q2 population revealed the Ly6C(+/Hi) M-MDSC subpopulation as the most suppressive, inhibiting T- and B-cell responses in a full, or partially, iNOS/NO-dependent manner, respectively. In contrast, the lower/moderate levels of suppression by the Ly6C(+/Lo) and Ly6C(+/Mid) M-MDSC Q2 subpopulations, whether versus T- and/or B-cells, displayed little/no iNOS dependency for suppression. These results highlight differential phenotypic and functional immunosuppressive M-MDSC subsets in a retroviral immunodeficiency model.

Analysis of the helper virus in murine retrovirus-induced immunodeficiency syndrome: evidence for immunoselection of the dominant and subdominant CTL epitopes of the BM5 ecotropic virus.

In genetically susceptible strains, such as C57BL/6 (B6) mice, LP-BM5 causes murine AIDS (MAIDS). LP-BM5 is a complex mixture of murine leukemia viruses (MuLV) that includes replication competent ecotropic (BM5eco) and mink cell focus inducing (MCF), and replication defective (BM5d) MuLV. At present, for the BM5eco virus, sequence information on only the gag region is available. In this paper, we describe for the first time the sequencing of the entire BM5eco viral genome as well as analysis of homology with two other previously sequenced and well-characterized MuLVs, Emv-11 and Emv-2, the latter constituting the parental virus for BM5eco. We propose that the detailed sequence comparisons herein provide cogent evidence that BM5eco utilizes variations in cytotoxic T lymphocytes (CTL) epitopes as an immune escape mechanism. This CTL evasion mechanism may contribute substantially to the underlying prototypic susceptibility of B6 mice to LP-BM5-induced MAIDS.

Quantitative analysis of LP-BM5 murine leukemia retrovirus RNA using real-time RT-PCR.

Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus (MuLV) complex that contains a mixture of defective (BM5def) and replication-competent viruses. While the BM5def virus is the causative agent in MAIDS, the replication-competent viruses in LP-BM5, including ecotropic MuLV (BM5eco), are required for BM5def propagation and thus function as helper viruses. We describe quantitative real-time RT-PCR assays for RNA encoded by the BM5def and BM5eco components of LP-BM5. The assays were used to standardize better the input doses of LP-BM5 viruses across viral preparations and to quantify BM5def and BM5eco gag RNA levels in spleen and blood cells from MAIDS-susceptible and -insusceptible infected mice. Spleens of MAIDS-susceptible infected mice harbored approximately similar levels of BM5def gag RNA as infected spleens of mice that are insusceptible to MAIDS due to lack of CD40. In contrast, the same infected spleens of CD40-deficient mice contained substantially higher (up to 10-fold) levels of BM5eco gag RNA compared with susceptible controls. Similar to that seen in spleen, infected blood of CD40-deficient mice contained similar levels of BM5def gag as susceptible strains, but increased levels (up to threefold) of BM5eco gag RNA. The assays described below can be used to characterize better the contributions of different functional viral components of the LP-BM5 mixture to the development of MAIDS.