Loading of oncolytic vesicular stomatitis virus onto antigen-specific T cells enhances the efficacy of adoptive T-cell therapy of tumors.

Although adoptive T-cell therapy has shown clinical success, efficacy is limited by low levels of T-cell trafficking to, and survival in, the immunosuppressive environment of an established tumor. Oncolytic virotherapy has recently emerged as a promising approach to induce both direct tumor cell killing and local proinflammatory environments within tumors. However, inefficient systemic delivery of oncolytic viruses remains a barrier to use of these agents against metastatic disease that is not directly accessible to the end of a needle. Here we show that the ability of antigen-specific T cells to circulate freely, and to localize to tumors, can be exploited to achieve the systemic delivery of replication-competent, oncolytic vesicular stomatitis virus (VSV). Thus, VSV loaded onto OT-I T cells, specific for the SIINFEKL epitope of the ovalbumin antigen, was efficiently delivered to established B16ova tumors in the lungs of fully immune-competent C57Bl/6 mice leading to significant increases in therapy compared to the use of virus, or T cells, alone. Although OT-I T-cell-mediated delivery of VSV led to viral replication within tumors and direct viral oncolysis, therapy was also dependent upon an intact host immune system. Moreover, VSV loading onto the T cells increased both T-cell activation in vitro and T-cell trafficking in vivo. The combination of adoptive T-cell transfer of antigen-specific T cells, along with oncolytic virotherapy, can, therefore, increase the therapeutic utility of both approaches through multiple mechanisms and should be of direct translational value.

Progressive human immunodeficiency virus-specific immune recovery with prolonged viral suppression.

The degree of immune recovery achievable with anti-human immunodeficiency virus (HIV) therapy remains to be established. The effects of potent antiretroviral therapy, including ritonavir and saquinavir, on immune function were studied for a prolonged period in 41 patients. After 96 weeks, 88% of patients had plasma HIV RNA levels below the limit of quantitation. There were continuous increases in CD4 lymphocyte counts and in CD4:CD8 ratios over time. About half the patients developed lymphoproliferative responses to HIV p24 antigen, and nearly all developed responses to phytohemagglutinin. This occurred in parallel with increases in interleukin-12 production and expression of CD28 on CD8 lymphocytes, despite potential antiproliferative effects of protease inhibitors. Transient increases in virus load were temporally associated with loss of proliferative responses. The improved immune function, including HIV-specific immunity in many subjects, demonstrates the potential reversibility of HIV-induced immunodeficiency and does not identify a limit to immune recovery.

Decreased HIV-associated T cell apoptosis by HIV protease inhibitors.

Antiretroviral treatment of patients infected with HIV results in improvements in CD4+ T cell number. Emerging evidence suggests that some of the improvements in CD4+ T cell number that occur in response to protease inhibitor (PI) therapy may not be accounted for solely by enhanced viral suppression, implying that PI may directly affect T cell survival. Since HIV T cell depletion is associated with enhanced apoptosis, we analyzed the effect of PIs on T cell apoptosis. In vitro treatment of peripheral blood lymphocytes (PBLs) from HIV-infected but untreated patients with reverse transcriptase inhibitors (RTIs) does not alter apoptosis, whereas PI treatment rapidly reduces CD4+ and CD8+ T cell apoptosis. In contrast, PI treatment does not alter apoptosis in PBL blasts from HIV-negative patients, or in Jurkat T cells. Consistent with this observation, 8 days of PI therapy in HIV-infected patients does not significantly alter plasma viremia, yet results in significant inhibition of CD4+ and CD8+ T cell apoptosis. The inhibitory effects of PI on apoptosis have implications concerning the treatment of HIV and its pathogenesis.

Sustained suppression of plasma HIV RNA is associated with an increase in the production of mitogen-induced MIP-1alpha and MIP-1beta.

The chemokine receptor CCR5 has been shown to be a major coreceptor for HIV-1. The chemokines that bind to this receptor (MIP-1alpha, MIP-1beta, and RANTES) are potent inhibitors of HIV replication and may play an important role in the pathophysiology of HIV disease. We investigated the effect of potent antiretroviral therapy (ritonavir and saquinavir) on the production of MIP-1alpha, MIP-1beta, and RANTES in 19 HIV-infected patients who had sustained decreases in plasma HIV RNA levels (<200 copies/ml). Chemokine concentrations were measured in serum, plasma, and PHA-stimulated PBMCs at baseline and 24 and 48 weeks after initiating therapy. MIP-1alpha, MIP-1beta, and RANTES levels in serum and plasma did not significantly change in the 48-week period. In contrast, MIP-1alpha and MIP-1beta secreted by PHA-stimulated PBMCs increased at 24 weeks, with this increase sustained at 48 weeks, whereas no significant change was observed in PHA-induced RANTES production. A significant positive correlation was found between the changes in PHA-induced chemokine production and baseline CD4+ T cell counts. These data demonstrate that sustained suppression of viral replication by potent antiretroviral therapy has a potentially beneficial effect on chemokine production and early initiation of this therapy appears to confer a more favorable chemokine profile.

Dynamic correlation of apoptosis and immune activation during treatment of HIV infection.

T cells from HIV infected patients undergo spontaneous apoptosis at a faster rate than those from uninfected patients, are abnormally susceptible to activation induced cell death (AICD), and undergo increased apoptosis in response to Fas receptor ligation. These observations have led to the hypothesis CD4 T cell apoptosis may be a mechanism of CD4 T cell depletion and the pathogenesis of AIDS. Successful treatment of HIV infected patients is accompanied by quantitative and qualitative improvements in immune function reflecting at least partial reversibility of the underlying pathogenesis of HIV. In this report we correlate improvements in markers of immune function with a decrease in apoptosis, and changes in its regulation. Therapy with nelfinavir plus saquinavir in combination with two nucleoside analogue inhibitors of reverse transcriptase dramatically reduces plasma viremia and increases CD4 T cell counts. Coincident with these improvements, CD38 and HLA-DR coexpression on both CD4 and CD8 T cells decrease, and CD45RA and CD62L coexpression increase. Furthermore, spontaneous apoptosis decreases in both CD4 and CD8 T cells (CD4 apoptosis 17.4 vs 2.6%, P=0.005; CD8 apoptosis 15.0 vs 1.0%, P<0.001), as does both Fas mediated apoptosis (CD4 apoptosis 19.0 vs 3.5%, P=0.03; CD8 apoptosis 13.7 vs 1.5%, P=0.002) and CD3 induced AICD (CD4 apoptosis 13.7 vs 3.2%, P=0.001; CD8 apoptosis 29 vs 2.2%, P=0.08). Changes in apoptosis are not associated with changes in Fas receptor expression, but are significantly correlated with changes in activation marker profiles. Although this suggests a possible regulatory role for the apoptosis inhibitory protein FLIP, direct assessment did not reveal quantitative differences in FLIP expression between apoptosis resistant PBL's from HIV negative patients, and apoptosis sensitive PBL's from HIV positive patients. These findings support the hypothesis that apoptosis mediates HIV induced CD4 T cell depletion, but indicate the need for further studies into the molecular regulation of HIV induced apoptosis.

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness.

Immune unresponsiveness in HIV-1 infection can result from impaired signals delivered by the costimulatory CD28-B7 pathway and the altered production of immunoregulatory cytokines, in particular IL-10, whose production is altered in HIV-1 infection. In this study we investigate IL-10 regulation in T cells and monocytes from HIV+ individuals, and its association with CD28-mediated T cell proliferation. IL-10 production as analysed in T cell- and monocyte-depleted peripheral blood mononuclear cells (PBMC), and by intracellular staining at the single-cell level, reveals a defect in IL-10 production by CD4+ and CD8+ T cells, whereas monocytes constitute the major IL-10-producing cell type. To investigate the impact of IL-10 on immune responsiveness, CD28-mediated proliferative responses in HIV+ individuals were correlated with PHA-induced IL-10 production. CD4+ T cells expressed CD28, yet exhibited markedly reduced CD28-mediated cell proliferation. This CD28-mediated CD4+ T cell proliferation was found to be inversely associated with the levels of PHA-induced IL-10 production and could be restored, at least in part, by anti-IL-10 antibodies. These results suggest that IL-10 production is differentially regulated in T cells and monocytes of HIV+ individuals, and that IL-10 may have a role in inducing immune unresponsiveness by modulating the CD28-B7 pathway.

Improvement in cell-mediated immune function during potent anti-human immunodeficiency virus therapy with ritonavir plus saquinavir.

Inhibiting human immunodeficiency virus (HIV) replication with potent antiretroviral therapy may result in improved immune function, and this may lead to favorable outcomes, independent of changes in CD4+ lymphocyte count. The effect of combination protease inhibitor therapy (ritonavir plus saquinavir) on functional measures of cell-mediated immunity in 41 HIV-infected patients from one center of a multicenter trial was investigated. After 24 weeks, median plasma virus load decreased from 4.74 log10 copies/mL to below the detection limit of the assay (2.30 log10), and mean CD4+ lymphocyte count increased from 284 cells/microL to 413 cells/microL. Proliferative responses to phytohemagglutinin developed in 21 of 34 patients in whom responses were absent at baseline. Increases were observed in interleukin-2, -12, and -10 production and in the expression of CD28 on CD8+ lymphocytes. Initiation of potent anti-HIV therapy results in a degree of immune restoration, suggesting that HIV-induced immune suppression is a dynamic and potentially reversible process.