B1 lymphocytes and myeloid dendritic cells in lymphoid organs are preferential extratumoral sites of parvovirus minute virus of mice prototype strain expression

Abstract

Due to their oncolytic properties and apathogenicity, autonomous parvoviruses have attracted significant interest as possible anticancer agents. Recent preclinical studies provided evidence of the therapeutic potential of minute virus of mice prototype strain (MVMp) and its recombinant derivatives. In a murine model of hemangiosarcoma, positive therapeutic outcome correlated with high intratumoral expression of MVMp-encoded genes in tumors and lymphoid organs, especially in tumor-draining lymph nodes. The source and relevance of this extratumoral expression, which came as a surprise because of the known fibrotropism of MVMp, remained unclear. In the present study, we investigated (i) whether the observed expression pattern occurs in different tumor models, (ii) which cell population is targeted by the virus, and (iii) the immunological consequences of this infection. Significant MVMp gene expression was detected in lymphoid tissues from infected tumor-free as well as melanoma-, lymphoma-, and hemangiosarcoma-bearing mice. This expression was especially marked in lymph nodes draining virus-injected tumors. Fluorescent in situ hybridization analysis, multicolor fluorescence-activated cell sorting, and quantitative reverse transcription-PCR revealed that MVMp was expressed in rare subpopulations of CD11b (Mac1)-positive cells displaying CD11c+ (myeloid dendritic cells [MDC]) or CD45B (B220+ [B1 lymphocytes]) markers. Apart from the late deletion of cytotoxic memory cells (CD8+ CD44+ CD62L-), this infection did not lead to significant alteration of the immunological profile of cells populating lymphoid organs. However, subtle changes were detected in the production of specific proinflammatory cytokines in lymph nodes from virus-treated animals. Considering the role of B1 lymphocytes and MDC in cancer and immunological surveillance, the specific ability of these cell types to sustain parvovirus-driven gene expression may be exploited in gene therapy protocols.

FIG. 1.

MVMp distribution in vivo. (A) RT-PCR analysis of viral expression in vivo. MVMp-specific transcripts were evaluated by RT-PCR in spleens (Spl), livers (Liv), tumor-draining lymph nodes (DLN), and tumors (Tu) after extratumoral (i.p., upper panel) or intratumoral (lower panel) injections. The PCR products obtained were visualized by agarose gel electrophoresis with ethidium bromide, scanned, and subjected to densitometric analysis. Signal intensities were quantified with NIH Image 1.62 software and normalized to the sample's β-actin expression. The intensities are presented as arbitrary units above the gel images. (B) Tracing of transformed H5V cells in organs of hemangiosarcoma-bearing mice. Detection of PymT, a specific marker of H5V cells, was performed by RT-PCR followed by Southern blotting and ECL detection to ensure the specificity and sensitivity of the signal. The results show a signal detected in tissues derived from three different animals, infected or not, with MVMp.

FIG. 2.

MVMp expression by lymphocytes in vitro. Transformed A9 fibroblasts (positive control) and stimulated immunocytes (5 × 10⁶ per well in 24-well plates) were infected with MVMp at an MOI of 10 PFU/cell. (A) Southern blot analysis of ConA-stimulated immunocytes revealing the presence of viral replicative forms 24, 48, and 72 hpi (ss, single-stranded DNA; mRF, monomer replicative form; dRF, dimer replicative form). (B). RT-PCR analysis showing viral RNA expression in LPS-stimulated immunocytes derived from LN and spleen 24 hpi. (C). Western blot analysis presenting parvoviral nonstructural (NS1) and capsid (VP1 and VP2) proteins in cultures of A9 cells and PMA-ionomycin-stimulated splenocytes and cells after 2 and 48 hpi.

FIG. 3.

Identification of cells expressing MVMp upon infection in vivo. Single-cell suspensions prepared from the draining lymph nodes of naïve and MVMp-infected mice (labeled as MVMp− and MVMp+, respectively) were processed for in situ hybridization with NS1-specific probe (2.5 × 10⁵ cells/slide) and DAPI staining (A) or dual- or triple-color labeled for consequent FACS experiments (B), using different combinations of following antibodies: anti-TCRαβ, B220, Mac1, CD11c, and NK 1.1 (clone Dx5). The presence of MVMp-specific transcripts in the isolated populations was assessed with LightCycler QRT-PCR technology. Positive and negative populations are indicated in the figure by + and −, respectively. Quadrants in panel C indicate two rare populations harboring viral transcripts upon enrichment of cells derived from draining lymph nodes of tumor-free (left panel) and H5V hemangiosarcoma-bearing (right panel) mice.

FIG. 4.

Phenotypic changes linked to MVMp infection. (A) Level of memory CD8 (CD8⁺ CD62L⁻) cells in lymph nodes of noninfected (upper panel) and infected (6 weeks postinfection, lower panel) mice. Shown is the proportion (percent) of memory CTL in the total CD8⁺ lymphocyte population. (B) Expression levels of IP-10, IFN-γ, IL-10, and IL-12 p40 in lymph nodes of infected mice. mRNA was isolated from lymph nodes and analyzed by QRT-PCR. Data points represent the average number of normalized copies per microliter of cDNA. (C) IFN-γ protein production by stimulated splenocytes infected, or not, with MVMp. The experiments were repeated two to eight times.