Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis

Abstract

Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.

Keywords: Cytokine; Immunotherapy; Mycobacterium tuberculosis; T Helper Cells; Vaccine.

FIGURE 1.

Immunomodulators enhance the capacity of a BCG vaccine to reduce bacterial burden upon M. tuberculosis challenge. A, schematic diagram of the immunization scheme, treatment with immunomodulators, and infection of BALB/c mice. I.P., immunoprecipitate; S.C., subcutaneous. B, after immunization BALB/c mice were treated daily with D4476 (TGFβRI inhibitor) and/or suplatast tosylate (Th2 inhibitor) at 16 nmol/g of body weight for a total of 10 days, and the mice were then rested for 20 days before aerosol challenge with a low dose of M. tuberculosis (H37Rv) (∼100 cfu). Bacterial burdens (cfu) were measured in lungs and spleens at 60 days post infection. Data represent the mean ± S.D. values of four mice per group per time point, and the experiment was repeated twice. C, proliferation of splenocytes in response to complete soluble antigen was measured by a [³H]thymidine incorporation assay. CSA, complete soluble antigen. D, photomicrographs (×10) of histological lung sections (6 μm) at 60 days after infection of the indicated mice, stained with hematoxylin and eosin. Results shown here are representative of three independent experiments.

FIGURE 2.

Induction of multifunctional CD4⁺ T cells after BCG immunization and immunomodulator treatment of mice. A and B, CD4⁺ and CD8⁺ T cell counts at 60 days post infection of immunized and immunomodulator-treated mice. C and D, expression of different surface phenotypes (CD44^hiCD62L^lo, CD44^hiCD62L^hi, CD44^loCD62L^hi) by multifunctional CD4⁺ T cells. E, prevalence of IFN-γ-producing cells among CD44^hiCD62L^hi cells at 60 days post infection of immunized and immunomodulator-treated mice. Results shown here are representative of three independent experiments.

FIGURE 3.

BCG immunization plus immunomodulator treatment induces Th1 responses. A and B, intracellular staining for IFN-γ, IL-4, IL-17, and FoxP3 (CD4⁺ CD25⁺) of CD4⁺ T lymphocytes isolated from the spleens of different groups (infected, BCG immunized, BCG immunized, and treated) of mice. C and D, intracellular cytokine staining for the production of IL-6, IL-10, IL-12, and TNF-α from different groups (infected, BCG-immunized, BCG-immunized, and treated) of mice. Results shown here are representative of three independent experiments.

FIGURE 4.

T lymphocytes from mice immunized with BCG and treated with immunomodulators can adoptively transfer protective immunity against M. tuberculosis infection. A, diagrammatic sketch of adaptive transfer of CD4⁺ T cells from Thy1.2⁺ donor mice to γ-irradiated Thy1.1⁺ mice. Vaccinated mice were used for CD4⁺ T cell isolation before adoptive transfer. B, after immunization Thy1.1⁺ mice were treated daily with D4476 (TGFβRI inhibitor) and/or suplatast tosylate (Th2 inhibitor) at 16 nmol/g of body weight for a total of 10 days and after a 20-day rest period T lymphocytes were isolated from lymph node and injected intraperitoneally (5 × 10⁶ cells) to γ-irradiated Thy1.2⁺ mice before aerosol challenge with a low dose of M. tuberculosis (H37Rv) (∼100 cfu). Bacterial burdens (cfu) were measured in lungs and spleens at 15, 30, and 60 days post infection. Data represent the mean ± S.D. values of four mice per group per time point, and the experiment was repeated twice. AT, adoptive transfer.

FIGURE 5.

Frequency of T cells and cytokine-producing cells in mice adoptively transferred with T cells from animals immunized with BCG and treated with immunomodulators. A, profile of CD4⁺ and CD8⁺ T cell counts at 60 days post infection of mice that received T cells from BCG-immunized and immunomodulator-treated mice. B and C, intracellular staining for IFN-γ and IL-4 of CD4⁺ T lymphocytes isolated from the spleens of different groups of adoptively transferred mice. D and E, frequency of IL-6, IL-10, IL-12, and TNF-α intracellular cytokine-producing cells of different groups of adoptively transferred mice. Results shown here are representative of three independent experiments. AT, adoptive transfer.