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. 2015 Dec 30;90(6):2818-29.
doi: 10.1128/JVI.02774-15.

Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus

Wadzanai P Mboko  1 , Horatiu Olteanu  2 , Avijit Ray  3 , Gang Xin  3 , Eric J Darrah  1 , Suresh N Kumar  4 , Joseph M Kulinski  1 , Weiguo Cui  3 , Bonnie N Dittel  3 , Stephen B Gauld  5 , Vera L Tarakanova  6
Affiliations

Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus

Wadzanai P Mboko et al. J Virol. .

Abstract

Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis.

Importance: Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.

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Figures

FIG 1
FIG 1
IRF-1 suppresses the establishment of gammaherpesvirus latency. BL6 or IRF-1−/− mice were intranasally infected with 500 PFU of MHV68. The frequencies (A) and absolute numbers (B) of viral genome-positive splenocytes, the frequency (C) and absolute numbers (D) of splenocytes in which virus was reactivated in culture, and the frequency of persistent virus in lungs (E) and spleens (F) were measured at 16 days postinfection. Three to five mice per experimental group were used in each experiment, and data from at least three independent experiments were pooled.
FIG 2
FIG 2
IRF-1 restricts gammaherpesvirus-driven germinal center reaction during the establishment of viral latency. Mice of the indicated genotypes were infected as described in the Fig. 1 legend. (A to I) At 16 days postinfection, splenocytes harvested from individual animals were subjected to flow cytometry studies. Splenocytes were stained for surface expression of B220, IgM, and IgD (panel A shows representative results) to identify class-switched B cells (IgM negative [IgM−] IgD negative [IgD−]), which were quantified, and the result was expressed as a percentage of the number of B220+ cells (B) and as the absolute number per mouse spleen (C). Germinal center B cells (B220+) were identified by positive surface staining for GL7 and CD95 (panel D shows representative results), and the number of cells was expressed as a percentage of B220+ cells (E) and as the absolute number (F). Plasma cells were identified by surface CD138 and intracellular IgG staining (panel G shows representative results), and the number of cells was expressed as a percentage of IgM− IgD− B220+ cells (H) and as the absolute number (I). Each symbol represents the result for an individual spleen, and data from 2 to 4 independent experiments were pooled. (J) Sera collected from infected BL6 and IRF-1−/− mice at several times postinfection were diluted 1:50 and analyzed for virus-specific antibodies by ELISA. (K and L) Sera from mock- and MHV68-infected (16 days postinfection) mice were diluted, as indicated, and analyzed for total IgM (K) and IgG (L) levels. Data from two to four independent experiments were pooled.
FIG 3
FIG 3
IRF-1 is not a general suppressor of the germinal center response and likely exerts both B cell-intrinsic and -extrinsic functions to suppress MHV68-driven germinal center reaction. (A) Naive mice of the indicated genotypes were immunized with SRBCs, germinal center B cells were assessed at 9 days postimmunization, and the results were compared to those for naive mice. Each symbol represents the result for an individual spleen. (B) Mice of the indicated genotypes were infected with LCMV (Armstrong) or mock treated. Germinal center B cells were analyzed at 9 days postinfection. (C) B cells from naive BL6 or IRF-1−/− mouse spleens were sorted using CD19 magnetic beads and cultured in vitro without additional treatment in the presence of anti-CD40 (2 μg/ml) or following MHV68 infection (MOI = 1). Cell numbers were assessed on the indicated day of culture, and each condition was evaluated in quadruplicate cultures within each experiment; the data shown were pooled from two independent experiments. (D to F) Mice of the indicated genotypes were infected as described in the Fig. 1 legend, and follicular helper CD4 T cells were analyzed at 16 days postinfection. Splenocytes were stained for surface expression of CD4, CXCR5, and GL-7 (panel D provides representative results) to identify follicular helper T cells (CXCR5+, GL-7 positive [GL-7+]), which were quantified and expressed as a percentage of CD4+ cells (E) and the absolute number of cells per mouse spleen (F). Each symbol represents the result for an individual animal.
FIG 4
FIG 4
IRF-1 deficiency does not alter MHV68 tropism for splenic B cells. BL6 or IRF-1−/− mice were infected for 16 days as described in the Fig. 1 legend. Splenocytes were stained for the surface expression of the indicated markers and sorted to isolate germinal center B cells (A), nongerminal center B cells (B), and non-B cells (C). The frequency of genome-positive cells in each splenic compartment was determined by limiting dilution nested PCR. Splenocytes from each experimental group were pooled, and combined data from three to four independent experiments are shown.
FIG 5
FIG 5
IRF-1 suppresses long-term gammaherpesvirus infection and germinal center reaction. BL6 or IRF-1−/− mice were infected as described in the Fig. 1 legend. (A to C) The frequencies (A) and absolute numbers (B) of viral genome-positive splenocytes and the frequency of splenocytes reactivating virus in culture (C) were measured at 42 days postinfection. Three to five mice per experimental group were used in each experiment, and data from three independent experiments were pooled. (D to L) Splenocytes were also subjected to flow cytometry studies. Class-switched B cells (D to F), germinal center B cells (G to I), and Tfh cells (J to L) were identified as described in the Fig. 2 and 3 legends. Each symbol represents the result for an individual spleen, and data from two to four independent experiments were pooled.
FIG 6
FIG 6
Comparative spleen histology in BL6 and IRF-1-deficient mice. BL6 or IRF-1−/− mice were mock infected or infected with MHV68 as described in the Fig. 1 legend. Spleens were harvested at 42 days postinfection. (A and B) Mock-infected (A) and MVH68-infected (B) BL6 mice; (C and D) mock-infected (C) and MHV68-infected (D) IRF-1−/− mice; arrows, frequent germinal centers; (E and F) GL-7 (a germinal B cell marker) (E) and B220 (all B cells) (F) immunohistochemistry of the sample shown in panel D highlighting follicular hyperplasia; (G) PALS atypical lymphoid hyperplasia (outlined); (H) high-power magnification (500×) of the same lesion shown in panel G, showing atypical plasmacytoid cells (arrowheads). H&E staining was used. Magnifications, ×40 (A to D), ×100 (G), and ×500 (H).
FIG 7
FIG 7
IRF-1 expression in human PTLD. IRF-1 protein levels were assessed in 44 PTLD cases. (A, B) The proportion of malignant cells with any IRF-1 staining above the background was evaluated in each PTLD case. (A) The findings for two representative cases of PTLD in which <25% and >75% of all malignant cells were positive for IRF-1 expression are demonstrated. (B) The cases were further grouped on the basis of the EBV infection status of malignant cells. (C) The intensity of IRF-1 staining in malignant B cells was evaluated. IRF-1 staining comparable to that of tumor-infiltrating lymphocytes was deemed normal, and a significant decrease or the absence of IRF-1 staining compared to that for normal lymphocytes was defined as low/weak. Cases were grouped on the basis of the EBV infection status of malignant cells. The P value was determined using chi-square analysis.
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