Kathy A. Green

Myeloid-Derived Suppressor Cells in Murine Retrovirus-Induced AIDS Inhibit T- and B-Cell Responses In Vitro That Are Used To Define the Immunodeficiency

ABSTRACT 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 Ly6Glow/± 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.

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.

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

Background: P. aeruginosa Cif degrades the ABC transporters CFTR and P-glycoprotein. Results: Cif increases the ubiquitination and degradation of TAP1 and decreases MHC class I antigen presentation in airway epithelial cells. Conclusion: Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. Significance: These observations suggest a mechanism whereby Pseudomonas infection increases the severity and duration of respiratory viral infections. 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.

Characterization of the CD154-Positive and CD40-Positive Cellular Subsets Required for Pathogenesis in Retrovirus-Induced Murine Immunodeficiency

ABSTRACT Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.

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

ABSTRACT 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.

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.

The Programmed Death-1 and Interleukin-10 Pathways Play a Down-Modulatory Role in LP-BM5 Retrovirus-Induced Murine Immunodeficiency Syndrome

ABSTRACT Pathology due to the immune systems 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.

The CD154/CD40 Interaction Required for Retrovirus-Induced Murine Immunodeficiency Syndrome Is Not Mediated by Upregulation of the CD80/CD86 Costimulatory Molecules

ABSTRACT C57BL/6 (B6) mice infected with LP-BM5 retroviruses develop disease, including an immunodeficiency similar to AIDS. This disease, murine AIDS (MAIDS), is inhibited by in vivo anti-CD154 monoclonal antibody treatment. The similar levels of insusceptibility of CD40−/− and CD154−/− B6 mice indicate that CD154/CD40 molecular interactions are required for MAIDS. CD4+ T and B cells, respectively, provide the CD154 and CD40 expression needed for MAIDS induction. Here, the required CD154/CD40 interaction is shown to be independent of CD80 and CD86 expression: CD80/CD86−/− B6 mice develop MAIDS after LP-BM5 infection.

CD40-associated TRAF 6 signaling is required for disease induction in a retrovirus-induced murine immunodeficiency.

ABSTRACT LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.

Alternative Translational Reading Frames as a Novel Source of Epitopes for an Expanded CD8 T-Cell Repertoire: Use of a Retroviral System to Assess the Translational Requirements for CTL Recognition and Lysis

CD8 T-cell responses constitute a key host defense mechanism against tumor cells and a variety of viral infections, including retroviral infections that lead to acquired immunodeficiency. However, both for tumor cells and for many viral infections, there can be a relative paucity of immunodominant protective CD8 T-cell responses. For retroviruses, their rapid and error-prone replication, coupled with initial CD8 T-cell immunoselection of epitope-variant, retroviral quasi-species, are major impediments to sustaining a protective CD8 T-cell response. To approach this limitation of functional CD8 T-cell epitopes, here we further characterize an underappreciated source of additional T-cell epitopes: cryptic determinants, in particular those encoded in unconventional, alternative reading frames (ARFs). By use of the CD8 T-cell epitope, SYNTGRFPPL, which we have defined as encoded by the +1NT ARF of the gag gene of the LP-BM5 retrovirus that causes murine AIDS, we further characterize the regulation of ARF-epitope expression. Specifically, we examine the translation initiation requirements for production of sufficient epitope for effector CD8 T-cell recognition. Such translation must arise from rare frame-shifting events, making it crucial to understand any other constraints on epitope production, and therefore on the ability of the anti-Kd/SYNTGRFPPL CD8 T cells to protect from LP-BM5 pathogenesis and retroviral load, as we have previously shown. The data herein demonstrate that ARF epitope production depends entirely on conventional AUG-initiated translation, and that the more proximal in-frame ARF AUG is most important. However, maximal epitope production for protective CD8 T-cell lytic function also requires synergy of this initiation codon with a counterpart conventional AUG codon upstream in the same ARF (ORF 2), and with the classic ORF 1 AUG that initiates conventional gag polyprotein translation. These results have implications for the design of ARF-epitope-based vaccines, both to counter retroviral pathogenesis, as well as potentially more broadly, including in tumor systems.