The Influence Of Immunosuppressive Therapy On Murine Cytomegalovirus Reactivation Post Allo-HSCT

Cytomegalovirus(CMV)is known as a ubiquitous member of the β-herpesviridae family,leading to a prevalence of more than 50%.Primary infection of CMV is always characterized by self-limiting,appearing to be asymptomatic in immunocompetent individuals.However,it is believed to establish a lifelong latent infection,which has the ability to reactivate in immunocompromised hosts,such as transplant recipients and AIDS patients.Current antiviral drugs(ganciclovir and foscarnet for example)have been proven efficacious against CMV recurrence.Nevertheless,these drugs are limited seriously during clinical practice because of increased concern for thrombocytopenia,neutropenia and bacterial infection.Besides,the emergence of drug-resistant strains among CMV-infected patients following the repeated and prolonged use of antiviral drugs has been documented in detail.In clinical practice,CMV infection still poses a remarkable concern for morbidity and mortality following allogeneic hematopoietic stem cell transplantation(allo-HSCT),especially when associated with interstitial pneumonia.To date,several factors that influence CMV recurrence have been documented.However,some evidence may appear confused and conflicting,which call for further investigation,such as the relationship between CMV recurrence and immunosuppressive therapy.On one hand,immunosuppressive therapy may delay the CMV-specific immune recovery following allo-HSCT,resulting in relapses of CMV infection.Conditioning regiments which accelerate antiviral immune recovery are believed to be the most effective strategy to prevent CMV recurrence.On the other hand,the progress of graft-versus-host disease(GVHD),which serves as the major therapeutic target of immunosuppressive therapy,was widely reported to severely damage engraftment,hematopoiesis,and antiviral immune recovery.Moreover,one of the pro-inflammatory cytokines increased significantly during GVHD,tumor necrosis factor alpha(TNF-α),was reported to be a pivotal factor of immediate-early(IE)gene expression.Therefore,the clear role of immunosuppressive therapy in allo-HSCT is still pending.Because of the rigorous species specificity of human cytomegalovirus(HCMV),most of researchers considered murine cytomegalovirus(MCMV)as a powerful tool for the investigation of CMV latency and recurrence.MCMV-infected mice have proven to be an available surrogate model,as they are quite similar to HCMV infection with respect to the primary infection,latency,and viral recurrence.In our study,we establish the animal model of MCMV latency and reactivation.And on this basis,we investigate the multiple influences of immunosuppressive agents on MCMV reactivation by two different allo-HSCT models,one with mild GVHD and the other with lethal GVHD.We found immunosuppressive therapy alleviated recurrent MCMV loads in an allo-HSCT model with severe GVHD as well as rare immune recovery.Besides,we show evidence that the impairment of MCMV recurrence arising from immunosuppressive agents is associated with decreased level of TNF-α.The specific study contents as well as results are shown as follows:1.The establishment of animal model with MCMV latencyAfter viral propagation and titration,we infected BALB/c with 1×105 PFU MCMV,and sacrificed 3-4 mice on day 14,60,180 respectively.The blood and lungs were collected to determine viral latency.The quantitative real-time PCR(qPCR)for blood,TCID50(medium tissue culture infective dose)and RT-PCR(reverse transcription PCR)for lungs all showed a remarkable level of viral load on day 14.However,the MCMV infection was undecidable on day 60 and 180.The anti-MCMV IgG was measured by using a indirect ELISA.The results show that the levels of antibodies were increased obviously on day 14,and remained stable on day 60 and 180.In further experiments,the mice(3-4 wk)infected with 1×105 PFU MCMV and maintained for 180 d are considered to be latently infected animal.2.Immunosuppressive therapy enhanced the recurrence of MCMV in the allo-HSCT model with mild GVHDTo avoid the problem that GVHD impairs immune recovery following allo-HSCT,we study the influence of immunosuppressive therapy on viral recurrence in the allo-HSCT model with mild GVHD firstly.Latently infected BALB/c mice received irradiation and engraftment of 1 × 107 bone marrow cells from femurs of C57BL/6 with latent infection.Cyclosporin A(CsA)and rapamycin(RAPA),two current immunosuppressive drugs applied to clinical treatment,or placebo,were daily administrated from the first day following allo-HSCT.All mice showed mild symptoms of GVHD,along with less mortality.On day 56 following allo-HSCT,mice were all sacrificed.The lungs were collected from each animal for viral titration.Moreover,the viral genome in blood was also extracted on days 14,28,42,56 following allo-HSCT by a kit,and determined by qPCR.Results show that recipients treated with immunosuppressive agents had a increased viral load than those treated with placebo.These results exhibit the fact that immunosuppressive therapy enhanced viral reactivation in this model.3.Immunosuppressive therapy badly impair the antiviral immune recovery in the mild GVHD modelWe subsequently investigate whether immunosuppressive agents enhanced viral reactivation by inhibition of anti-MCMV immunity.The PCR for chimerism shows the cells arising from both donors and recipients,indicating long-term survival of grafts following allo-HSCT.Tail vein blood was collected for absolute WBC as well as lymphocyte counts on days 14,28,42,56 following allo-HSCT.Immediately following allo-HSCT at 1 month,an obvious delay of the blood routine examination in groups receiving immunosuppressive agents was documented,which disappeared immediately at second month.To further observe antiviral immune reconstitution post-transplantation,both tetramer-positive MCMV-specific CD8+ T cells from blood and intracellular TNF-α-producing CD8+ T cells from spleens were determined by FACS analysis.Significantly decreased levels of CD8+ T cells both from blood and spleens were observed in the mice receiving CsA or RAPA.The impairment of antiviral recovery in mice treated with immunosuppressive agent accounts for the difference in MCMV reactivation among groups.4.Immunosuppressive therapy suppress the development of GVHD in the allo-HSCT model with lethal GVHDTo establish a model with severe GVHD,a revised regimen of 7 Gy irradiation,1 × 107 bone marrow cells plus 1 × 107 splenocytes from latent C57BL/6 were set as transplantation parameters.All mice were divided into three groups,and treated with CsA,RAPA,or placebo.Animal survival was documented,and GVHD scores were performed weekly.As we expected,the groups received CsA or RAPA exhibited reduced mortality along with statistically less GVHD score.For examination of histopathologic damage in the small intestine,aggravation of GVHD-damage was observed in the mice of control group,such as villous blunting and mucosal erosion in the lamina propria and so on.A semi-quantitative scoring system was applied with a blinded and objective fashion,suggesting a significant difference.The levels of TNF-α,IL-2(interleukin-2),and IFN-γ(interferon-γ)were also measured as they were usually considered as marker of severe GVHD.The results indicated that the levels of TNF-α and IL-2 were obviously lower in the recipients treated with CsA or RAPA.These results imply reduced severity of GVHD in mice receiving immunosuppressive therapy.5.Rare immune reconstitution in the model with badly GVHD despite of immunosuppressive therapyThe PCR for chimerism shows the cells arising from both donors and recipients,indicating long-term survival of grafts following allo-HSCT.The blood routine examination was performed on day 28.The results indicated a slight rose of leukocytes and poor recovery of lymphocytes.Furthermore,MCMV-specific immune recovery by FACS analysis showed rare antiviral immune response.The results exhibited poor immune reconstitution because of GVHD damage and despite of immunosuppressive therapy.Unlike the observation of first model,GVHD damage resulted in immune dysfunction instead of immunosuppressive agents.6.Immunosuppressive therapy exhibited an inhibitory effect on MCMV reactivation in the face of lethal GVHDWe subsequently determined viral recurrence.The results showed that recipients in control group had increased level of viral load in both lungs and blood.These indicate that immunosuppressive agents reduced recurrent viral loads in the allo-HSCT model with rare immune recovery and severe GVHD.7.Immunosuppressive therapy alleviated viral reactivation by inhibition of TNF-αThe results have shown pro-inflammatory cytokines may be involved in viral recurrence.To verify the hypothesis,the correlations between viral copies and TNF-α/IL-2 were examined.An obvious correlation was found between viral DNA copies and TNF-α(R2 = 0.81),while IL-2 didn’t appear a correlation.To verify the positive role of TNF-α as a pivotal mediator of the MCMV recurrence,lenalidomide(TNF-α inhibitor)was used to perform another set of experiments.Latent mice received irradiation and engraftment as described in the lethal GVHD model.The mice were randomly divided into placebo group,placebo and lenalidomide group,CsA and lenalidomide group,RAPA and lenalidomide group.On day 28 following allo-HSCT,immunohistochemical staining targeted MCMV-gB(murine cytomegalovirus glycoprotein B)were performed to evaluate MCMV recurrence in lungs,as well as collected data for IHC were documented by mean optical density.These results indicated the recipients in placebo group had higher reactivated virus in the lungs,and no statistical difference was observed among groups receiving lenalidomide,which is consistent with the examination of viral loads in blood.Moreover,examination of TNF-α of plasma indicated mice receiving lenalidomide had decreased levels of TNF-α.In conclusion,our investigation indicates the dual influences of immunosuppressive therapy on MCMV recurrence.Importantly,immunosuppressive therapy reduced MCMV reactivation in a model with lethal GVHD by decreasing TNF-α.Therefore,our studies benefit a improved understanding of the correlation between immunosuppressive therapy and MCMV recurrence,and provide novel antiviral strategies for clinical practice.