Immunoglobulin Fc fragment tagging allows strong activation of endogenous CD4 T cells to reshape the tumor milieu and enhance the antitumor effect of lentivector immunization

Abstract

A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits, mainly owing to multiple immune suppressions in established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to influence the suppressive tumor milieu and antitumor effect. We engineered a lentivector (lv) to express a nominal fusion Ag composed of hepatitis B surface protein and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response but, more importantly, to induce effective coactivation of CD4 T cells. We found that, remarkably, immunization with HBS-Fc-lv caused significant regression of established tumors. Immunologic analysis revealed that, compared with HBS-lv without Fc fragment, immunization with HBS-Fc-lv markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor while substantially decreasing the regulatory T cell ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of CD4 T cells from the HBS-Fc-lv-immunized mice could activate endogenous CD8 T cells in an IFN-γ-dependent manner. We conclude that endogenous CD4 T cells can be activated by lv expressing Fc-tagged Ag to provide another layer of help--that is, creating a Th1/Tc1-like proinflammatory milieu within the tumor lesion to boost the effector phase of immune responses in enhancing the antitumor effect.

Fig. 1. lv expressing Fc tagged Ag elicits more potent CD8 and CD4 T cell immune responses

C57BL/6 mice were immunized with either HBS-lv or HBS-Fc-lv. Non-immunized mice were used as control. Two weeks later, HBsAg specific CD8 and CD4 T cell responses in the peripheral blood were determined by intracellular staining of IFNγ after brief stimulation ex vivo with S_190-197 peptide (for CD8 response) or whole HBsAg (for CD4 responses). Only CD8 or CD4 T cells were gated and shown. Data from 5 mice in each group are summarized and presented on the right. The experiment was repeated three times with similar results.

Fig. 2. HBS-Fc-lv immunization results in regression of established B16-S tumors

(A): The experimental design of tumor treatment with lv immunization. (B): The growth curve of lv treated and control tumors. Partial and complete tumor regressions were observed.

Fig. 3. Tumor lesion is skewed toward a Th1/Tc1-like immune stimulatory microenvironment after HBS-Fc-lv immunization

Eighteen days after immunization, total RNA was extracted from the tumor tissues of control and immunized mice (three tumor lesions in each group were combined together), and qRT-PCR was performed using primers for indicated cytokines, transcription factors, and chemokines. The result is presented as how many folds of increase of cytokines or chemokines in the immunized tumor over the control tumors. Each sample was done in triplicate; the average and SD are shown. The experiment was repeated 3 times with similar results.

Fig. 4. HBS-Fc-lv immunization markedly increases tumor infiltration of CD4 and CD8 T cells and decreases the Treg ratio in the tumor lesions

Mice bearing 5 day tumors were immunized with either HBS-lv or HBS-Fc-lv, or left untreated. The tumor lesions were collected on day 17–20 after immunization and analyzed for tumor infiltrating CD8 and CD4 T cells and the Treg ratio. The absolute numbers of CD8 and CD4 T cells and the Treg ratios in the tumor lesions of control and treated mice from a cohort of two studies are summarized.

Fig. 5. CD4 and CD8 TIL in the tumors treated with HBS-Fc-lv possess better effector function

(A): The effector function of CD8 TIL was measured by intracellular staining of IFNγ after brief ex vivo peptide stimulation. Representative dot plots of CD8 TIL from HBS-lv or HBS-Fc-lv immunized tumors are shown. Dot plots from control tumors are not shown because only few CD8 TILs could be collected. Only the CD8 T cells were gated and shown. Data of 5 mice are summarized. (B): Effector cytokine production by CD4 TILs is shown after stimulation with PMA/Ionomycin. Only the CD4 T cells were gated and shown. These experiments examining the TIL and their effector functions were repeated 3 times with similar results. (C): Degranulation of CD8 TIL was measured by CD107a staining. A summary of data from 5 tumors in each group is presented. This experiment was repeated twice with similar observation.

Fig. 6. Immunologic changes in the tumors and the antitumor effect of HBS-Fc-lv immunization are dependent on CD4 activation and FcγR

Wt and FcRγ KO mice were inoculated with B16-S tumor cells. Five days later, tumor bearing mice were immunized with HBS-Fc-lv. (A): CD8 and CD4 responses in the peripheral blood were determined 2 weeks after immunization by intracellular staining of IFNγ. The summary data of 5 mice was shown on the right. (B): The Treg ratio in the tumor was analyzed 3 weeks after tumor inoculation and the data of 5 mice was summarized at the right column. (C): Cytokines in the tumor lesions of wt and KO mice were analyzed by qRT-PCR. (D): The tumor growth curve and the tumor weight were recorded. Two experiments were conducted with similar results.

Fig. 7. Adoptive transfer of CD4 T cells can activate endogenous CD8 TIL in tumors and generate antitumor effects

(A): The experimental scheme: Activated CD4 and CD8 T cell subsets were isolated from HBS-Fc-lv immunized Thy1.1 congenic mice and 10 million cells were adoptively transferred into mice bearing 5 day B16-S tumor after a low dose (5Gy) irradiation. Two weeks after adoptive transfer, the tumor lesions were collected and the granzyme B (GrzB) expression of TILs was analyzed. (B): The GrB expression of endogenous CD8 TIL from mice that were transferred with activated CD4 or CD8 T cells. Irradiated mice without adoptive transfer of T cells were used as control. A summary of data of total CD8 TIL and percentage of GrzB expression was also presented. (C): The GrzB expression of exogenous adopted CD4 and CD8 TILs. A summary of data was presented on the right. (D): Tumor growth curve and tumor weight. A summary of data from 5 mice is presented. Two experiments were conducted with similar results.

Fig. 8. IFNγ expression play a critical role in the antitumor effect of CD4 T cell adoptive transfer

(A): The experimental design is shown. WT and IFNγ KO mice (Thy1.2) were immunized with HBS-Fc-lv. Two weeks later, activated CD4 T cells were isolated and 5 million cells of cells were injected into irradiated B16-S tumor bearing mice (Thy1.1). Mice with irradiation only without cell transfer were used as control. (B): The activation of CD4 T cells by HBS-Fc-lv immunization in both wt and IFNγ KO mice was determined by examining TNFα expression. A similar percent of CD4 T cells of wt and IFNγ KO mice produced TNFα in responses to Ag stimulation. (C): Two weeks after transfer, tumor lesions were collected and analyzed for IFNγ production by the endogenous CD8 T cells. The absolute number of IFNγ producing CD8 TIL of 5 mice in two experiments is summarized. (D): The tumor weights were recorded when the mice were sacrificed at the end of experiment.