LP-BM5 Retrovirus Research Articles

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.

HIF-1α is essential for T cell suppression by murine LP-BM5 retrovirus infection-augmented Monocytic Myeloid Derived Suppressor Cells

Abstract Compared to naïve mice, monocytic myeloid-derived suppressor cells (M-MDSCs) increase in number, and suppressive efficiency, after infection with the immunodeficiency causing mouse retrovirus, LP-BM5. In ex vivo suppression assays, M-MDSCs, enriched from spleens of LP-BM5 infected mice inhibited both T- and as a novel finding, B-cell responsiveness. For M-MDSC suppression of T-cell proliferation and IFN-gamma production, we previously reported the mechanism to be almost completely iNOS/NO dependent. In contrast, the suppression of B-cell proliferation and IL-10 secretion by predominantly T2-B cells was only ~50% iNOS/NO dependent, with V-domain Ig suppressor of T cell activation (VISTA), superoxide and peroxynitrite also playing substantial effector roles. In light of recent reports on the Hypoxia-inducible transcription factor 1, α subunit (HIF-1α) regulating the function and differentiation of MDSCs, and reports of its role as a principal regulator of NO production, we describe here the characterization of M-MDSCs from myeloid-specific HIF-1α deficient, vs. w.t., B6 mice. As early as 5 weeks post infection, enriched M-MDSC preparations from HIF-1α deficient mice were significantly impaired functionally, in some cases with a total inability, to suppress in vitro T cell responsiveness. However, these same M-MDSC preparations suppressed B-cell responsiveness at a level very similar to that of control w.t. M-MDSCs. Experiments are in progress to further understand the role HIF-1α plays in the impairment of M-MDSC ex vivo suppression of T cell proliferation and function, including a possibility that HIF-1α regulates iNOS/NO production in M-MDSCs only when in the process of suppressing T- but not B-cell targets.

5-Fluorouracil depletion of Myeloid Derived Suppressor Cells in mice infected with LP-BM5 retrovirus.

Abstract Tumor associated MDSC inhibit tumor-directed T-cell responses. We have shown the induction of monocytic MDSCs (M-MDSC) during infection of B6 mice by LP-BM5 immunodeficiency inducing retrovirus. These M-MDSCs suppressed proliferation and function of T cells, ~100% via iNOS/NO; and that of B cells, ~50% via iNOS/NO along with the negative checkpoint regulator VISTA, NOS, ROS, and other soluble mediators. Because of overlapping cell-surface phenotypes, our attempts to selectively deplete M-MDSCs in vivo have been challenging. However, 5-Fluorouracil (5-FU), a pyrimidine analog widely used in tumor chemotherapy, has recently been reported to selectively eliminate MDSCs by apoptosis (Biomed. J. 2015:38:111–116). Here, we treated LP-BM5 infected mice with an extended course of 5-FU: at 6–7 weeks post infection achieving ~50% depletion of splenic M-MDSCs, and spleen cells from depleted mice exhibited greatly decreased suppression in ex vivo assays. LP-BM5 infected mice with this level of M-MDSC depletion, had significantly less disease, including: splenomegaly and immunodeficiency – i.e. impaired B and T-cell ex vivo polyclonal responses. Also, regarding other disease parameters, the loss of LP-BM5 induced splenic: CD4 Thy1.2 and B-cell CD23 expression was greatly reduced in M-MDSC depleted mice. As compared to sham treated mice, via flow cytometry, the splenic CD8+ T cell subset from LP-BM5 infected/M-MDSC depleted mice was greatly increased in cell number and MFI. Future directions include extending these initial experiments to further characterize the effects on B and T-cells resulting from in-vivo 5-FU-mediated M-MDSC depletion during LP-BM5 retrovirus-induced immunodeficiency.

Nitrosative damage during retrovirus infection-induced neuropathic pain

BackgroundPeripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35–50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.MethodsUsing the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism.ResultsHind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4+) and large (NF200+) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4+ and CD8+ T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals.ConclusionsResults reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain.

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.

MiR-155 and LP-BM5 retrovirus-augmented Monocytic myeloid derived suppressor cells, are players in a profound murine immunodeficiency-causing syndrome

Abstract We have shown augmentation of monocytic MDSCs (M-MDSCs) during a profound immunodeficiency causing LP-BM5 retrovirus infection of B6 mice. Enriched splenic M-MDSCs preparations in suppression assays, inhibited T-, and the much less reported B-, cell responsiveness. This inhibition of T-cell proliferation and IFN-gamma production was almost completely iNOS/NO dependent, whereas suppression of B-cell responses was only ~50% iNOS/NO dependent. To explore additional mechanism(s) in this inhibition of B-cell responsiveness, we reported that VISTA, a negative checkpoint regulator, is involved in LP-BM5 M-MDSC suppression of ex vivo B-cell responses. In light of the reported involvement of miR-155 in B-cell and myeloid cell function, we are now also evaluating M-MDSCs from 5 week LP-BM5 infected miR-155 k.o.s. Our results show miR-155 k.o. M-MDSCs are substantially less (30–50%) suppressive compared to M-MDSCs from infected Wt. mice, and at 10 w.p.i. the k.o.s exhibit a different kind of immunodeficiency: 1) spleen cells are significantly more responsive to B-cell polyclonal activators, 2) significantly less splenomegaly and 3) have fewer resident splenic M-MDSCs. In an initial experiment, involving adoptive transfer of M-MDSCs from donor LP-BM5 infected Wt. B6 to infected miR-155 k.o.s, we were able to significantly augment miR-155 k.o.: 1) splenomegaly, 2) splenic B-and T-cell immunodeficiency, and 3) the number of residing splenic M-MDSCs, to levels almost comparable to those of Wt. infected mice. These results highlight involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B-cell responses, and were compatible with a role for M-MDSC in LP-BM5-induced immunodeficiency.

Calcitonin gene related protein antiviral role in LP-BM5 infection of astrocytes and microglia

Abstract CGRP (calcitonin gene related protein) is a neuropeptide expressed by primary sensory neurons upon infection. LP-BM5 retrovirus infection induces profound immunodeficiency in B6 mice and has been used as a murine model of HIV infection. Previously, we showed that LP-BM5 induced increased CGRP expression in the spinal cords of infected mice. In vitro studies indicated a microglia dependent, CGRP-induced reduction of LP-BM5 viral loads in primary mixed glial cells. To further delineate CGRP’s effects on glial cells, we treated specific murine cell lines (C8D1A astrocyte cell line and C8B4 microglia cell line) with LP-BM5 and CGRP. We found that LP-BM5 was capable of infecting both microglia and astrocytes individually and PCR detection for selected CGRP receptor components indicated the presence of CGRP receptor in both cell lines. CGRP’s antiviral effect was observed in both astrocytes and microglia, however a lower dose of CGRP was required for microglia. Western blot analysis suggested differential changes in CGRP downstream signaling pathways in astrocyte vs. microglia upon CGRP treatment. In addition, type I interferons known for their antiviral properties were elevated in astrocytes following CGRP treatment. We propose that CGRP mediated signaling pathways may mediate type I interferon production thus further promote antiviral responses.

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.

MDSC suppression of B cell responses in murine retrovirus-induced immunodeficiency: a role for VISTA (IRC4P.603)

Abstract Inhibition by myeloid derived suppressor cells (MDSC) of T cell responses is well established in tumor microenvironments. We demonstrated (Green et al., 2013) induction of monocytic MDSCs during infection of B6 mice by LP-BM5 retrovirus, which causes profound immunodeficiency. These MDSCs suppressed not only T, but also B cell, responsiveness ex vivo. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was ~100%, iNOS/NO dependent, MDSC suppression of B-cell responses was only ~50% dependent on iNOS/NO - as shown by using iNOS inhibitors and iNOS k.o. mice as a source of MDSCs. We then discovered an additional mechanism(s) in MDSC inhibition of (only) B cell responsiveness that involved VISTA, a new negative checkpoint regulator. Using anti-VISTA blocking mAb, or LP-BM5 infected VISTA-/- MDSCs, MDSC inhibition of B cell responses was dependent on MDSC-expressed VISTA — again accounting for ~50% of total MDSC suppression. Combining the use of reagents to block both iNOS/NO and VISTA lead to an additive, if not synergistic, abrogation of MDSC suppression of B cell responsiveness. Consistent with a direct role of VISTA, in the absence of MDSCs, a VISTA-Ig fusion protein also partially inhibited B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5-induced immunodeficiency and highlight involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.

Suppression of B cell responsiveness by LP-BM5 retrovirus-induced myeloid derived suppressor cells generated during a murine acquired immunodeficiency syndrome: a role for negative checkpoint regulator expression on the MDSCs (VIR7P.1059)

Abstract The inhibitory activities of myeloid derived suppressor cells (MDSC) against T cell responses are well established in tumor microenvironments. We published (2013, J. Virol. 87:2058-2071) on the induction of monocytic MDSCs during infection of susceptible B6 mice by LP-BM5 retrovirus, which causes a profound immunodeficiency. These MDSCs inhibited not only T, but also B cell, responsiveness to polyclonal stimulation in ex vivo suppression assays. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was largely iNOS/NO dependent, MDSC suppression of B cell responses was only partially (~50%) due to iNOS/NO - as shown by using iNOS inhibitors and LP-BM5 infected iNOS k.o. mice as a source of MDSCs. Here, we further study additional suppressive mechanism(s) in the MDSC inhibition of B cell responsiveness. Using MDSCs from LP-BM5 infected mice of negative checkpoint regulator knock-out origin, and specific blocking antibody, MDSC suppression of B cell responses was partly dependent on MDSC expression of certain checkpoint regulators. Combining the use of reagents to interrupt both iNOS/NO and checkpoint regulation lead to a synergistic blocking of MDSC suppression of B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5 induced immunodeficiency and highlight the involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.

Enhanced anti-retroviral cytotoxic T cell response by PD-1 blockade (VIR4P.1015)

Abstract 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). Our lab has previously shown that a cytotoxicT lymphocyte (CTL) response to MAIDS-associated epitopes that are shared by MAIDS-associated terminal B-cell lymphomas can be detected in uninfected, MAIDS-associated tumor cell primed B6 mice. We further confirmed that B6 mice infected with LP-BM5 retrovirus can generate equally robust anti-MAIDS tumor CTLs compared to the B6 mice primed with tumor cells. To determine if the anti-tumor CD8 T-cell responses can be enhanced by interrupting the down-regulatory PD-1 pathway specifically in the CD8 T-cell compartment, thus to treat MAIDS-associated B-cell lymphomas, we determined whether adoptive transfer of PD-1KO CD8 T cells with or without total CD4 T cells, was able to protect Rag-1KO mice from B6-1710 tumor challenge. Rag-1KO recipients that received B6.PD-1KO.CD8 T cells showed delayed tumor growth following B6-1710 tumor challenge compared to Rag-1KO mice that received equal numbers of w.t B6.CD8 T cells. This enhanced anti-tumor activity was similar to what was observed in mice that received combined PD-1 blockade in CD8 T cell compartment and depletion of CD4 T regulatory cells. Our data indicate that anti-tumor CD8 T cell response can be increased by interrupting the down-regulatory, CD8 T-cell mediated PD-1 pathway.

Suppression of T and B cell responses by myeloid derived suppressor cells after natural CD4 T regulatory cell depletion and LP-BM5 retrovirus infection (VIR7P.1057)

Abstract Immunomodulatory cellular subsets, including myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumor microenvironment and have been targets of immunotherapy, but their role in retrovirus-associated immunosuppression is less understood. Tregs are associated with HIV-infected patients, however it remains unclear whether Tregs suppress the anti-viral immune response or dampen HIV viral spread; and also unclear is the stage(s) of infection of Treg involvement. MDSCs have only recently been identified in HIV-infected patients, and thus the phenotype and mechanism(s) of suppression are still being defined. Due to known crosstalk between Tregs and MDSCs in the tumor microenvironment, it is of interest to look at the interplay between these cellular types during LP-BM5-induced murine AIDS (MAIDS). Following in vivo nTreg depletion, using adoptive transfer approaches, and subsequent LP-BM5 retroviral infection, enriched MDSCs from infected nTreg-depleted mice exhibit increased suppression of T cell, but not B cell, responses, as compared to MDSCs derived from non-depleted LP-BM5 infected controls. In addition, MDSCs from LP-BM5-infected nTreg-depleted mice have altered phenotypic subsets, which may correlate with functional differences. These collective data may provide insight into the possible immunotherapeutic targeting of Tregs and MDSCs during retroviral infection. (NIH AI007363, CA50157)

The role of indoleamine 2,3-dioxygenase in LP-BM5 murine retroviral disease progression

BackgroundIndoleamine 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.MethodsMice 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.ResultsAs 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.ConclusionsCollectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load.

Myeloid-derived suppressor cell subsets inhibit T- and B-cell responses in murine Retrovirus-induced immunodeficiency (P6179)

Abstract Compared to tumor microenvironments, myeloid-derived suppressor cells (MDSCs) have been less well studied in infectious diseases, particularly for retroviruses inducing immunodeficiency. We have found the development of a large population of immunosuppressive MDSCs after LP-BM5 retrovirus infection which causes murine acquired immunodeficiency. These MDSCs express a cell-surface marker signature (CD11b+Gr-1+Ly6C+) and inhibit not only T-cell, but B-cell, responsiveness by a substantially iNOS-dependent mechanism. MDSC immunosuppressiveness parallels the extent of disease in LP-BM5-infected w.t. vs. k.o. mice, differentially susceptible to disease. Retroviral load indicate that the suppressive Ly6C+CD11b+MDSC subset is LP-BM5 positive, although significantly lower than non-fractionated splenocytes from infected mice. Further fractionation of the MDSCs by FACS indicated, whereas all four subsets defined by Ly6C versus CD11b expression suppressed B-cell responses, only the Ly6Cbright CD11b+ subset suppressed T-cells, indicating distinct versus overlapping targeting of the two adaptive immune arms. These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression, and, as the total MDSC suppressive function mirrors that of LP-BM5-induced disease, support a pathogenic effector role for these retrovirus-induced MDSCs.

Myeloid-Derived Suppressor Cells in Murine Retrovirus-Induced AIDS Inhibit T- and B-Cell ResponsesIn VitroThat 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.

Isothiocyanates Ameliorate the Symptom of Heart Dysfunction and Mortality in a Murine AIDS Model by Inhibiting Apoptosis in the Left Ventricle

Cardiac involvement has been reported in as many as 45-55% of patients with human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS), and significant cardiac morbidity is reported in 6-7% of HIV patients. We investigated the inhibitory effects of isothiocyanates (ITCs) on heart dysfunction and mortality by regulating apoptosis in the left ventricle of the heart in a murine AIDS model. Mice were divided into six groups: an uninfected group, an untreated LP-BM5 retrovirus-infected group, and four LP-BM5 retrovirus-infected groups treated with one of four ITCs (sulforaphane [SUL], indolo[3,2-b]carbazole, benzyl isothiocyanate [BITC], or phenethyl isothiocyanate [PEITC]). After 16 weeks, the median survival time of the LP-BM5 retrovirus-infected mice was 87 days, whereas that of the uninfected control group and all ITC treatment groups was over 112 days. SUL, PEITC, and BITC significantly inhibited apoptosis in the left ventricle by increasing the Bcl-2/Bax ratio compared with LP-BM5-infected mice. In addition, SUL and PEITC suppressed inducible nitric oxide synthase (iNOS) expression at both the mRNA and protein levels in the left ventricle of heart tissue infected with the LP-BM5 retrovirus by inactivating cytoplasmic nuclear factor κB (NF-κB). In conclusion, LP-BM5 retrovirus infection was related to survival of murine AIDS mice, and NF-κB-mediated iNOS expression may be an important mediator of left ventricle dysfunction of the heart. Furthermore, certain ITCs may have the potential to improve AIDS-related heart dysfunction due to their inhibition of apoptosis by decreasing iNOS and Bax expression through suppression of NF-κB.

Myeloid-Derived suppressor cells in murine retrovirus-induced AIDS: inhibition of the T-cell and B-cell responsiveness that define the immunodeficiency (170.7)

Abstract Myeloid-derived suppressor cells (MDSCs) have been characterized in several tumor settings however, they have been less well studied in response to infectious disease processes, in particular against retroviruses that induce immunodeficiency. Here we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on LP-BM5 retrovirus infection, which causes murine acquired immunodeficiency. This MDSC population is CD11b+, Gr-1+, Ly6C+ , exhibits profound inhibition of immune responsiveness by a cell-dose and substantially iNOS-dependent mechanism that is independent of arginase activity, PD-1/PD-L1 expression, and IL-10; and the degree of immunosuppression parallels the extent of disease in differentially LP-BM5 susceptible w.t. vs. knock-out mouse strains. Not only T-cell, but also interestingly B-cell host responses, were suppressed by these MDSCs. The direct immunosuppression of B cell responses was confirmed by use of: purified B responder cells, multiple B-cell specific stimuli, and independent assays measuring B-cell expansion. This suppressive MDSC subset is positive for proviral LP-BM5 RNA, albeit at a significantly lower level than LP-BM5 infected non-fractionated spleen cells. These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression, and support a possible pathogenic effector role for these retrovirus-induced MDSCs.

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.

Inhibition of Premature Death by Isothiocyanates through Immune Restoration in LP-BM5 Leukemia Retrovirus-Infected C57BL/6 Mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.

Impaired memory CD8 T cell responses against an immunodominant retroviral cryptic epitope

The immunodominant cryptic epitope SYNTGRFPPL, encoded within open reading frame 2 of the LP-BM5 retroviral gag gene, is critical for protection against retroviral-induced pathogenesis. The goal of this study was to dissect the memory response against this unique immunodominant cryptic epitope. Unlike the protective acute effector population of SYNTGRFPPL-specific CD8 T cells, long-lived SYNTGRFPPL-specific CD8 T cells lacked the ability to protect susceptible mice infected with LP-BM5 retrovirus. Compared to memory CD8 T cells against a conventional epitope with similar MHC-I specificity, primed and restimulated using similar conditions, long-lived SYNTGRFPPL-specific CD8 T cells were impaired in their ability to recall against antigen, with reduced cytolytic capabilities and cytokine production. Since similar priming and restimulation regimes were utilized to generate each effector CD8 T cell population, this study has potentially broad implications with regard to the selection criteria of potent, highly conserved cryptic epitopes for use in epitope-based vaccines.