Material Supplementary 6.DC1http://www.jimmunol.org/content/suppl/2010/07/16/jimmunol.100013

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.

Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.

Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

The maintenance of B-cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B-cell extrinsic mechanisms that regulate B-cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B-cell anergy. We now show that in Treg-cell-deficient mice, helper T cells are necessary and sufficient for loss of B-cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4 + cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC-T FH ) cells. These GC-T FH cells, but not those from Tregcell-sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T FH cells, especially GC-T FH cells, which support autoantibody production.Keywords: anergy r autoimmunity r B cells r T FH r Treg cells Supporting Information available online IntroductionThe production of autoantibodies due to loss of B-cell tolerance is central to the development of autoimmunity. Our understanding of the mechanisms that maintain B-cell tolerance, especially B-cell anergy, remains poorly defined. The absence of regulatory T (Treg) cells results in the development of IPEX, a fatal disease associated with the development of significant autoantibodies and autoimmunity [1,2]. The development of autoantibodies suggests that Treg cells play a key role in regulating B-cell tolerance, a statement supported by our previous study showing a role for Treg cells in the maintenance of B-cell anergy [3].Numerous signaling molecules have been identified whose absence prevents the establishment or maintenance of B-cell Correspondence: Dr. Stephen B. Gauld e-mail: sgauld@mcw.edu anergy at the intrinsic level (reviewed in [4]). However, our understanding of B-cell extrinsic mechanisms that regulate B-cell anergy remains poor. DCs have been shown to both positively and negatively regulate autoantibody production [5][6][7]. Helper T cells, or signals associated with T-cell help, appear to negatively regulate B-cell tolerance [8][9][10][11][12]. It remains unclear as to which helper T-cell populations can support autoantibody production.Follicular helper T (T FH ) cells are defined based on their expression of the chemokine receptor CXCR5, the transcription factor Bcl6, and play an essential role in providing help to B cells, promoting antibody production [13][14][15]. However, a role for T FH cells in the production of autoantibodies is less well defined. The expansion of T FH cells is known to be associated with a lupus-like disease in sanroque mice and CD4 + cells from the sanroque model support antichromatin autoantibody production in an adoptive transfer system [16,17]. A study by Simpson et al. [18] In our study, we use the Foxp3 DTR mouse model [20] to deplete the complete Treg-cell population in adult hosts and show that Treg cells are essential to prevent th...

SummaryViruses such as Epstein-Barr virus (EBV) have been linked to mechanisms that support autoantibody production in diseases such as systemic lupus erythematosus. However, the mechanisms by which viruses contribute to autoantibody production remain poorly defined. This stems in part, from the high level of seropositivity for EBV (> 95%) and the exquisite species specificity of EBV. In this study we overcame these problems by using murine gammaherpesvirus 68 (MHV68), a virus genetically and biologically related to EBV. We first showed that MHV68 drives autoantibody production by promoting a loss of B-cell anergy. We next showed that MHV68 infection resulted in the expansion of follicular helper T (Tfh) cells in vivo, and that these Tfh cells supported autoantibody production and a loss of B-cell anergy. Finally, we showed that the expansion of Tfh cells and autoantibody production was dependent on the establishment of viral latency and expression of a functional viral gene called Orf73. Collectively, our studies highlighted an unexpected role for viral latency in the development of autoantibodies following MHV68 infection and suggest that virus-induced expansion of Tfh cells probably plays a key role in the loss of B-cell anergy.

The absence of regulatory T cells (Tregs) results in significant immune dysregulation that includes autoimmunity. The mechanism(s) by which Tregs suppress autoimmunity remains unclear. We have shown that B cell anergy, a major mechanism of B cell tolerance, is broken in the absence of Tregs. In this study, we identify a unique subpopulation of CD4+ Th cells that are highly supportive of Ab production and promote loss of B cell anergy. Notably, this novel T cell subset was shown to express the germinal center Ag GL7 and message for the B cell survival factor BAFF, yet failed to express markers of the follicular Th cell lineage. We propose that the absence of Tregs results in the expansion of a unique nonfollicular Th subset of helper CD4+ T cells that plays a pathogenic role in autoantibody production.

Vaccination with heat-killed Saccharomyces cerevisiae (HKY) protects against experimental infection by pathogenic fungi of five genera. Here we tested whether purified Saccharomyces cell wall b-glucan could induce protection against systemic aspergillosis. CD-1 mice were given three weekly vaccine doses subcutaneously prior to intravenous infection with Aspergillus fumigatus. Mice received PBS, 2.5 mg HKY, whole glucan particles (WGP), WGP conjugated to BSA (0.06 to 12 mg per dose), a soluble medium molecular mass (MMW) b-glucan alone or MMW-BSA (¡24 mg per dose). Survival and c.f.u. were determined, and cytokine induction and anti-bglucan antibodies were assessed in vaccinated mice. Neither soluble MMW glucan, nor MMW-BSA was effective. HKY protected in two studies (survival and c.f.u. were reduced in brain and kidney organs, P,0.004). Six or 12 mg WGP or WGP-BSA prolonged survival (P¡0.004) and reduced c.f.u. in each organ (P¡0.015) in both experiments; 0.6 mg WGP or WGP-BSA prolonged survival (P¡0.015) and reduced c.f.u. (P¡0.015) in one experiment. Cytokine profiles in serum and bronchoalveolar lavage from uninfected vaccinated mice showed an innate and adaptive immune profile (i.e. upregulation of colony stimulating factors, interferons, TNF-a, chemokines such as MCP-1, MIP-1a, RANTES and KC, and Th17-activating cytokines such as IL-6, IL-1b, IL-17). No anti-b-glucan antibodies were in the sera, suggesting an adaptive T cellmediated, not a B cell-mediated, protective response. Vaccination with WGP or WGP-BSA proved protective against systemic aspergillosis, equivalent to that of HKY, supporting the potential of particulate b-glucans, alone or conjugated, as vaccines against aspergillosis.