Immunotherapy-induced cytotoxic T follicular helper cells reduce numbers of retrovirus-infected reservoir cells in B cell follicles.

Antiretroviral therapy (ART) transformed HIV from a life-threatening disease to a chronic condition. However, eliminating the virus remains an elusive therapy goal. For several decades, Friend virus (FV) infection serves as a murine model to study retrovirus immunity. Similar to HIV, FV persists at low levels in lymph nodes B cell follicles avoiding elimination by immune cells. Such immune-privileged reservoirs exclude cytotoxic T cells from entry. However, CXCR5+ T cells are permitted to traffic through germinal centers. This marker is predominantly expressed by CD4+ follicular helper T cells (Tfh). Therefore, we explored immunotherapy to induce cytotoxic Tfh, which are rarely found under physiological conditions. The TNF receptor family member CD137 was first identified as a promising target for cancer immunotherapy. We demonstrated that FV-infected mice treatment with αCD137 antibody resulted in an induction of the cytotoxic program in Tfh. The therapy significantly increased numbers of cytotoxic Tfh within B cell follicles and contributed to viral load reduction. Moreover, αCD137 antibody combined with ART delayed virus rebound upon treatment termination without disturbing the lymph node architecture or antibody responses. Thus, αCD137 antibody therapy might be a novel strategy to target the retroviral reservoir and an interesting approach for HIV cure research.

A detailed analysis of F-MuLV- and SFFV-infected cells in Friend virus-infected mice reveals the contribution of both F-MuLV- and SFFV-infected cells to the interleukin-10 host response.

BACKGROUND: Friend virus (FV) is a complex of the Friend murine leukemia virus (F-MuLV) and the replication-defective, pathogenic spleen focus forming virus (SFFV). In the past, we used a fluorescently labeled F-MuLV to analyze FV target cells. To build on these findings, we have now created a double-labeled FV that contains a Katushka-labeled F-MuLV and an mTagBFP-labeled SFFV, which we have used to study the infection by the two individual viruses in the FV infection of highly susceptible BALB/c mice. RESULTS: Our data show that the target cells of SFFV largely mirror those of F-MuLV, with the highest virus loads in erythroblasts, B cells and myeloid cells. The early phase of infection was dominated by cells infected by either SFFV or F-MuLV, whereas double-infected cells became dominant later in the course of infection with increasing viral loads. In the late phase of infection, the frequency of double-infected cells was similarly high as the frequencies of SFFV or F-MuLV single-infected cells, and single- and double-infected cells outnumbered the uninfected cells in the most highly infected cell populations such as erythroblasts. FV and retroviruses in general have been shown to induce interleukin 10 (IL-10) as a means of suppressing immune responses. Interestingly, we found in infected IL-10-eGFP reporter mice that SFFV-infected cells contributed to the IL-10-producing cell pool much more significantly than F-MuLV-infected cells, suggesting that the truncated SFFV envelope protein gp55 might play a role in IL-10 induction. Even though BALB/c mice mount notoriously weak immune responses against FV, infection of mice with an ablation of IL-10 expression in T cells showed transiently lower viral loads and stronger T cell activation, suggesting that IL-10 induction by FV and by SFFV in particular may contribute to a suppressed immune response in BALB/c mice. CONCLUSION: Our data provide detailed information about both F-MuLV- and SFFV-infected cells during the course of FV infection in highly susceptible mice and imply that the pathogenic SFFV contributes to immune suppression.

Immune suppression of vaccine-induced CD8+ T-cell responses by gamma retrovirus envelope is mediated by interleukin-10-producing CD4+ T cells.

Retroviral envelope (Env) proteins have long been recognized to exhibit immunosuppressive properties, which affect the CD8+ T-cell response to an infection but also to immunization. Interestingly, we previously showed in the Friend murine leukemia virus (F-MuLV) model that the surface Env protein gp70 also plays a role in immunosuppression, in addition to the immunosuppressive function attributed to the transmembrane Env protein. We now demonstrate that immunization with F-MuLV Env leads to a significant increase in interleukin-10 (IL-10)-producing CD4+ T cells and that the induction of CD8+ T-cell responses in the presence of Env is rescued if the capacity of CD4+ T cells to produce IL-10 is abrogated, indicating a mechanistic role of IL-10-producing CD4+ T cells in mediating the Env-induced suppression of CD8+ T-cell responses in Env co-immunization. We found that CD8+ T-cell responses against different immunogens are not all equally affected. On the other hand, suppression of immunity was observed not only in co-immunization experiments but also for immune control of subcutaneous tumor growth after an Env immunization. Finally, we show that suppression of CD8+ T cells by the surface Env protein is observed not only for Friend MuLV Env but also for the Env proteins of other gamma retroviruses. Taken together, our results show that IL-10-producing CD4+ T cells mechanistically underlie the Env-mediated suppression of CD8+ T-cell responses and suggest the presence of an immunosuppressive motif in the surface Env protein of gamma retroviruses.

Cellular Immune Response Induced by DNA Immunization of Mice with Drug Resistant Integrases of HIV-1 Clade A Offers Partial Protection against Growth and Metastatic Activity of Integrase-Expressing Adenocarcinoma Cells.

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209-239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist.

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.