Resistance to friend virus-induced erythroleukemia in W/W(v) mice is caused by a spleen-specific defect which results in a severe reduction in target cells and a lack of Sf-Stk expression

Abstract

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (Fv2), dominant white spotting gene (W), and Steel gene (Sl), regulate the initial polyclonal expansion of infected erythroid progenitor cells. W and Sl encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively. W mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow, W/W(v) mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from W/W(v) mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit+ pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in W/W(v) mice and these cells fail to express Sf-Stk. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.

FIG. 1.

W/W^v bone marrow cells are susceptible to Friend virus infection in vitro. Total bone marrow cells from W/W^v and control mice were either mock infected and plated in methylcellulose medium containing IL-3 plus Epo (gray bars) or infected with Friend virus and plated in methylcellulose medium containing IL-3 (black bars). The numbers of BFU-E were counted. Bars represent the total number of colonies ± standard deviation (error bars) from one representative experiment of three independent experiments.

FIG. 2.

In vivo expansion of Friend virus-infected bone marrow cells occurs normally in W/W^v mice. Control (A) and W/W^v (B) mice were infected with Friend virus. At the indicated days after infection with Friend virus (FV), bone marrow cells were harvested and plated in methylcellulose medium containing IL-3 with or without Epo. Bars represent the total number of colonies ± standard deviation (error bars) from one representative experiment of three independent experiments. Values that are significantly different from day 0 values are indicated by asterisks (*, P < 0.01; **, P < 0.05).

FIG. 3.

W/W^v spleen cells infected with Friend virus in vitro exhibit impaired Epo^ind BFU-E formation. Spleen cells from W/W^v and control mice were either mock infected and plated in methylcellulose medium containing Epo and the indicated cytokines (gray bars) or infected with Friend virus and plated in methylcellulose medium containing only the indicated cytokines (black bars). Bars represent the total number of colonies ± standard deviation (error bars) from one representative experiment of three independent experiments. Values that are significantly different from the values for cultures containing IL-3 alone are indicated by an asterisk (*, P < 0.01).

FIG. 4.

In vivo expansion of Epo^ind BFU-E in the spleen is impaired in W/W^v mice. Control (A) and W/W^v (B) mice were infected with Friend virus, and spleen cells were harvested on the indicated days after infection with Friend virus (FV). Spleen cells were then plated in methylcellulose medium containing the indicated cytokines. Bars represent the total number of colonies ± standard deviation (error bars) from one representative experiment of three independent experiments. Values that are significantly different from day 0 values are indicated by asterisks (*, P < 0.01; **, P < 0.05).

FIG. 5.

W/W^v mice exhibit few Friend virus-infected cells in the spleen. Flow cytometry analysis of Friend virus-infected cells in the bone marrow and spleen. (A and B) MAb34 staining of the bone marrow (A) and spleens (B) of control and W/W^v mice infected with Friend virus (FV) for the indicated days. (C and D) Percentage of MAb34⁺ cells that are TER119⁺ in the bone marrow (C) and spleens (D) of control and W/W^v mice infected with Friend virus for the indicated days. (E) Spleen weights of control and W/W^v mice infected with Friend virus for the indicated days. The results from one representative experiment of two independent experiments are shown.

FIG. 6.

Transplantation of in vitro-infected W/W^v bone marrow cells into control recipients induces splenomegaly. (A) Transplantation experiments were done using bone marrow from W/W^v or control mice, infected in vitro with Friend virus, and transplanted into either control or W/W^v recipients. Spleens were harvested 14 days after transplant and weighed. The values for W/W^v cells transplanted into control mice are significantly different from the values for W/W^v cells transplanted into W/W^v mice (*, P < 0.05). The values for control cells transplanted into control mice are not significantly different from the values for control cells transplanted into W/W^v mice (**, P > 0.10). (B) Control bone marrow cells were infected in vitro and then irradiated with 1,200 rads prior to transplant into control and W/W^v recipients. Spleens were harvested 14 days postinfection and weighed. Values for irradiated control cells transplanted into control mice are significantly different from the values for irradiated control cells transplanted into W/W^v mice (***, P < 0.02).

FIG. 7.

Analysis of Sf-Stk expression in W/W^v mice. RT-PCR analysis of Stk (A) and Sf-Stk (B) expression in total bone marrow and spleen cells of W/W^v and control mice. The positions of Sf-Stk, Stk, and hypoxanthine phosphoribosyltransferase (HPRT) are shown; HPRT is included as a loading control.

FIG. 8.

MEPs from the spleen but not the bone marrow are targets for Friend virus in vitro. (A) In vitro infection of MEPs (Kit⁺ Sca1⁻ IL-7Rα⁻ CD34⁻ Lin⁻ FcγR^low) sorted from spleens and bone marrow isolated from control mice. MEPs were either mock infected and plated in methylcellulose medium containing the indicated cytokines (gray bars) or infected with Friend virus and plated in methylcellulose medium containing the indicated cytokines (white bars). Values that are significantly different from the values for colony assays containing Epo are indicated (*, P < 0.01). (B) Flow cytometry diagram depicting the sorting of CD34⁻ Sca1⁻ IL-7Rα⁻ Lin⁻ cells for Kit⁺ FcγR^low (MEPs) from control (left) and W/W^v (right) mice. The percentage of CD34⁻ Sca1⁻ IL-7Rα⁻ Lin⁻ Kit⁺ FcγR^low cells is indicated in the boxed area. The results of one representative experiment of four independent experiments are shown. PE, phycoerythrin; FITC, fluorescein isothiocyanate. (C) MEPs sorted from spleen express Sf-Stk, but spleen MEPs from W/W^v mice lack Sf-Stk expression. Sf-Stk expression was analyzed by RT-PCR using RNA isolated from the indicated cell populations. Hypoxanthine phosphoribosyltransferase (HPRT) was included as a loading control.

FIG. 8.

MEPs from the spleen but not the bone marrow are targets for Friend virus in vitro. (A) In vitro infection of MEPs (Kit⁺ Sca1⁻ IL-7Rα⁻ CD34⁻ Lin⁻ FcγR^low) sorted from spleens and bone marrow isolated from control mice. MEPs were either mock infected and plated in methylcellulose medium containing the indicated cytokines (gray bars) or infected with Friend virus and plated in methylcellulose medium containing the indicated cytokines (white bars). Values that are significantly different from the values for colony assays containing Epo are indicated (*, P < 0.01). (B) Flow cytometry diagram depicting the sorting of CD34⁻ Sca1⁻ IL-7Rα⁻ Lin⁻ cells for Kit⁺ FcγR^low (MEPs) from control (left) and W/W^v (right) mice. The percentage of CD34⁻ Sca1⁻ IL-7Rα⁻ Lin⁻ Kit⁺ FcγR^low cells is indicated in the boxed area. The results of one representative experiment of four independent experiments are shown. PE, phycoerythrin; FITC, fluorescein isothiocyanate. (C) MEPs sorted from spleen express Sf-Stk, but spleen MEPs from W/W^v mice lack Sf-Stk expression. Sf-Stk expression was analyzed by RT-PCR using RNA isolated from the indicated cell populations. Hypoxanthine phosphoribosyltransferase (HPRT) was included as a loading control.