Expression of and immune responses to leukemia antigens in patients with hematological malignancies

Leukemia cells are characterized by differentially expressed leukemia-associated antigens (LAAs). We wondered whether the expression of the LAAs WT1 and RHAMM as well as the T cellular immune response to these LAAs correlated with the clinical outcome of patients suffering from leukemia. We investigated the expression of WT1 and RHAMM at RNA level using qPCR before and after treatment. We concluded that WT1 is a suitable marker for MRD after allogeneic SCT and that a WT1-specific T cell response might contribute to the maintenance of CR

Block of death-receptor apoptosis protects mouse cytomegalovirus from macrophages and is a determinant of virulence in immunodeficient hosts.

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system

Apoptosis inhibition is required for viral dissemination to distant organs.

<p>RAG1^−/− mice were (A) i.p. or (B) s.c. infected with 10⁵ PFU of indicated virus and monitored for survival (n = 4–6/group). Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight. (C) Infectious virus was determined by plaque assay on MEF cells in spleen (top panel), lungs (middle panel), and salivary glands (SG, bottom panel) of i.p. infected mice on day 13 after infection with 10⁵ PFU of indicated virus. Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection.</p

IFNγ controls ΔM36 growth by acting on the IFNγ receptor on fibroblasts, not on macrophages.

<p>(A) Experimental setup: MEF cells depleted for CD11b positive cells (MEF ΔCD11b) and BM-derived macrophages (BMM) were obtained from IFNγRec^−/− (IFNγR^−/−) or wild-type (WT) mice and cocultured (10% of macrophages, 90% of fibroblasts in cell culture) in all possible combinations. Cells were infected with ΔM36 or M36rev in the presence of Zymosan (30 µg/ml) and IFNγ (100 ng/ml) and virus titer in the supernatants was established at day 4 post infection. (B) Infectious titer of ΔM36 (white bars) or M36rev MCMV (grey bars) is shown as mean+standard deviation from three independent experiments. The combination of cells used in the infectious experiment is indicated below the x-axis, * p<0.05.</p

ΔM36 grows poorly in the presence of macrophages.

<p>(A) CD11b positive cells were removed from MEF cell preparations by monoclonal antibodies coupled to magnetic beads, upon which the cells were infected with ΔM36 (white bars) or M36rev (grey bars), alone or in the presence of Zymosan (30 µg/ml) or IFNγ (100 ng/ml). Virus titer in the supernatant of cells depleted of macrophages was compared to macrophage-undepleted MEF preparations at day 4 post infection. (B) Upon macrophage depletion, primary fibroblasts were cultured with indicated amounts of ANA-I macrophages (MΦ), in the presence or absence of Zymosan (30 µg/ml) and IFNγ (100 ng/ml). Infectious virus titer in supernatants was established at day 4 post infection. Histograms indicate mean values from three separate experiments, error bars show standard deviation, * p<0.05.</p

Diagram of the proposed mechanism of action.

<p>Activated macrophages secrete TNFα (and possibly additional cytokines) which synergize with IFNγ in fibroblasts to block virus growth by a mechanism that is dependent on caspase signaling. M36 blocks the caspase-dependent signaling pathway and thus prevents apoptosis and rescues the virus growth.</p

Macrophage, but not NK cell, depletion rescues ΔM36 MCMV <i>in vivo</i>.

<p>In a combined experiment to elucidate the role of (A) NK cells and (B) macrophages in the control of ΔM36 MCMV growth, RAG1^−/− and RAGγC^−/− mice received injections of 200 µl liposome encapsulated (A) PBS or (B) clodronate 48 hours (i.v.) and 24 hours (i.p.) prior to viral infection. Following liposome injection mice were i.p. injected with 10⁵ PFU ΔM36 (○) or M36rev (•) MCMV (n = 4–5/group). At day 3 post infection infectious virus was determined by plaque assay on MEF cells in spleen (top panels), lungs (middle panels) and liver (bottom panels). Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection. *p<0.05; **p<0.01.</p

TNFα secreted from macrophages synergizes with IFNγ to impair ΔM36 growth by a caspase-dependent mechanism.

<p>(A) Experimental setup: ANA-I cells were treated for 5 days with Zymosan and IFNγ in the presence of ΔM36 MCMV, M36rev MCMV, or no virus, upon which the supernatants were filtered to prevent virus carryover and transferred to CD11b-depleted MEF cells infected with ΔM36 or M36rev. (B) Infectious titer of ΔM36 (white bars) or M36rev-MCMV (grey bars) at 5 days post infection. Legends below the x-axis indicate the medium used during infection – control medium (DMEM), supernatant from ANA-I cells infected with ΔM36, M36rev or no virus (MOCK). Where indicated, the ANA-I supernatant was supplemented with neutralizing anti-TNFα (1 µg/ml) antibodies or z-VAD-fmk (33 µM). Histograms indicate mean values from three separate experiments, error bars show SD, * p<0.05.</p

ΔM36 MCMV applied locally is avirulent in RAG1^−/− mice.

<p>RAG1^−/− mice were infected by (A) intravenous, (B) intraperitoneal, (C) subcutaneous, or (D) intranasal administration with 10⁵ PFU of ΔM36 (○) or M36rev (•) MCMV (n = 6–15/group) and monitored for weight loss and survival. Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight.</p

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMV(UL36) inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo