Objective Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular T helper (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently identified that interleukin 21 (IL-21) promoted Tfh differentiation in autoimmune BXD2 mice that develop spontaneous GCs. The objective of this study was to determine the modulatory effects of IL-21 on Tfr and the Tfr/Tfh balance in BXD2 mice. Methods The percentage and phenotype of Tfr were determined in BXD2 and BXD2-Il21−/− mice. The effects of IL-21 on Tfr and the ratio of Tfr/Tfh were evaluated. Sorted Tfr cells from BXD2-Il21−/− mice were co-cultured with Tfh and B cells, or transferred into BXD2 mice to determine their function. Results GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-Il21−/− mice than in WT-BXD2. Adenovirus-IL-21 administration to BXD2-Il21−/− mice decreased Tfr and the ratio of Tfr/Tfh but increased GC B cells in the spleen. rmIL-21 suppressed Foxp3 and significantly reduced Tgfb1, Il2 and Gitr but enhanced Il21, Il6, Pd1, Cxcr5 and Icos in Tfr cells. IL-21 also counteracted Tfr-mediated inhibition of antibody secretion in the Tfh-B cell co-culture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased autoantibody-producing B cells. Conclusion High levels of IL-21 selectively enhanced Tfh differentiation but inhibited Tfr commitment and their suppressive function on Tfh and B cells, suggesting that IL-21 skews the balance from Tfr to Tfh to promote autoreactive GC reactions in BXD2 mice.

A major obstacle to the success of gene therapy strategies with ME180 tumors. Treatment of tumors with Ad.TK RC that directly target cancer cells is the poor vector distriwithout GCV resulted in a similar antitumor effect, conbution within solid tumors. To address this problem, we firming that the replicating vector has an oncolytic effect. developed an E1b 55 kDa attenuated, replicationWhen GCV was initiated 3 days after Ad.TK RC injection, competent adenovirus (Ad.TK RC ) which expresses the hersurvival of mice with each tumor type was greatly propes simplex-1 thymidine kinase (HSVtk) gene to sensitize longed, with 60% of animals with ME180 tumors surviving tumors to ganciclovir (GCV). Efficacy of this combined for over 160 days. These results confirm that both the strategy was tested in nude mice with subcutaneous oncolysis caused by a replicating virus and suicide/prodrug human A375 melanoma and ME180 cervical carcinomas.gene therapy with HSVtk/GCV have potent antitumor Intratumoral injection of a replication-defective adenoviral effects. When combined, these two approaches are compvector expressing HSVtk (Ad.TK) followed by GCV treatlementary resulting in a significantly improved treatment ment resulted in doubling of the survival time of mice bearoutcome. ing A375 tumors and 20% long-term survival of mice

In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Background-Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. Methods and Results-Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5ϫ10 9 pfu, 50 L/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5Ϯ2.8 in Ad.HSP70-injected hearts compared with 41.9Ϯ2.8 and 42.7Ϯ2.5 in the vehicle-and Ad.LacZ-treated hearts (PϽ0.01). The infarct size was not different between the vehicle-and Ad.LacZ-treated hearts (PϾ0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1Ϯ5.2, 47.7Ϯ3.5, and 53.3Ϯ2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (PϾ0.05). Conclusions-Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart. (Circulation.2001;103:877-881.)