Donor CD8+ T cells prevent Toxoplasma gondii de-encystation but fail to rescue the exhausted endogenous CD8+ T cell population

Abstract

Functional exhaustion of CD8(+) T cells due to increased expression of inhibitory molecule PD-1 (Programmed Death-1) causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from a primarily encysted, quiescent bradyzoite to a fast-replicating, highly motile tachyzoite. Thus, reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting gamma interferon (IFN-γ), a cytokine known to control latent toxoplasmosis, in chronically infected prerecrudescent mice. Since CD8(+) T cells (an important source of IFN-γ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8(+) T cells from acutely infected donors to the chronically infected prerecrudescent hosts could impede parasite de-encystation and rescue exhausted CD8(+) T cells. While the transfer of immune CD8(+) T cells temporarily restricted the breakdown of cysts, the exhausted endogenous CD8(+) T cell population was not rescued. Over time, the donor population got deleted, resulting in parasite de-encystation and host mortality. Considering that donor CD8(+) T cells fail to become long-lived, one of the cardinal features of memory CD8(+) T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover, our data strongly suggest that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in the chronic phase by itself is insufficient for rescue of exhausted CD8(+) T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis.

Fig 1

Decreased cyst burden during late-chronic T. gondii infection correlates with parasite reactivation. (A) C57BL/6 mice were infected with 10 ME49 cysts orally, and the number of brain cysts was enumerated at weeks 3, 5, 7, and 10 postinfection. A similar trend in cyst kinetics was observed in 3 independent experiments. (B and C) SAG-1 and BAG-1 relative expression levels were measured in brain at week 7 pi. The transcript level at day 10 was taken as 1. The data represent at least two experiments with at least 4 mice per group. Error bars represent standard deviations throughout.

Fig 2

Reduced cyst burden and increased leukocyte parasitization in mice treated with αIFN-γ during early-chronic infection. (A) αIFN-γ antibody was injected daily, starting at day 31 pi (n = 7). Animals were sacrificed 4 days after initiation of treatment. (B) The number of brain cysts was enumerated in isotype- or αIFN-γ antibody-treated mice. (C and D) T. gondii-infected cells in brain leukocyte-gated samples (n = 5) were assayed by flow cytometry, and the results are depicted as histograms (C) or a bar graph (D). The data represent two experiments with at least 4 mice per group.

Fig 3

Control of parasite reactivation by adoptive transfer of immune CD8⁺ T cells. (A) Splenic CD8⁺ T cells (4 × 10⁶) purified from donor mice at week 2 pi were adoptively transferred via the i.v. route into chronically infected recipient mice at week 5 pi. (B) Cyst burden in brains of recipient mice (n = 5) was evaluated 2 weeks posttransfer. (C and D) Frequencies of KLRG1-expressing recipient CD8⁺ T cells (n = 4) in brain are shown as density plots (C) (2 weeks posttransfer) or as a bar graph (D). (E) Cyst burden in brains of recipient mice was assessed at 4 weeks posttransfer. (F and G) T. gondii-infected cells in brain and blood leukocyte-gated samples from recipient mice (n = 5) were assayed by flow cytometry. (F) A histogram depicts frequency of parasite-infected leukocytes at 2 weeks posttransfer. (G) A bar graph (left panel, brain; right panel, blood) shows percentages of infected leukocytes at weeks 2 and 4 posttransfer in recipient animals that received immune CD8⁺ T cell transfer or PBS. (H) Survival of immune CD8⁺ T cell transfer recipients (n = 8) and saline-treated controls (n = 7) was monitored on a daily basis. Data represent two experiments.

Fig 4

Donor CD8⁺ T cells do not ameliorate endogenous CD8⁺ T cell exhaustion during late-chronic toxoplasmosis. (A and B) PD-L1 expression was assayed on total splenic and brain leukocytes by flow cytometry at weeks 2 and 4 posttransfer. Data are presented as representative histograms (A) or bar graphs (B). (C) The absolute number of endogenous CD8⁺ T cells in spleen and brain in the recipient mice was computed. (D) A bar graph represents the absolute number of donor CD8⁺ T cells in spleen (left panel) and brain (right panel) in immune CD8⁺ T cell transfer recipients. N.D. denotes “not detectable.” (E and F) PD-1 expression on endogenous CD8⁺ T cells in spleen and brain was evaluated by flow cytometry. Data are presented as a histogram (E) or as a bar graph (F) (left panel, spleen; right panel, brain). The data represent 2 experiments with at least 4 mice per group.

Fig 5

Donor CD8⁺ T cells fail to rescue endogenous CD8⁺ T cell functionality during late-chronic toxoplasmosis. (A and B) In vivo endogenous CD8⁺ T cell proliferation was assessed in spleen and brain of recipient animals at 2 weeks posttransfer by measuring Ki-67 expression via intracellular staining. Data are presented as pseudocolor plots (A) or as bar graphs (B) (left panel, spleen; right panel, brain). (C and D) Splenocytes and brain mononuclear cells were stimulated with TLA as described in Materials and Methods and then evaluated for IFN-γ production by endogenous CD8⁺ T cells at 2 weeks posttransfer. Data are depicted as a pseudocolor plot (C) (left panel, unstimulated; right panel, TLA stimulated) or as bar graphs (D) (left panel, spleen; right panel, brain). The data are representative of 2 experiments with 3 to 4 mice per group.

Fig 6

Donor memory CD8⁺ T cells fail to rescue endogenous CD8⁺ T cells during late-chronic toxoplasmosis. (A) Donor CD45.1 mice were infected with T. gondii. Four weeks later, brain CD8⁺ T cells were purified from donor CD45.1 mice and subsequently sorted into effector and memory T cell subsets. Sorted cells (1.5 × 10⁴) were transferred into chronically infected CD45.2 recipient mice (n = 4) at week 5 postinfection via the i.v. route. (B) T. gondii-infected cells in brain leukocyte-gated samples from recipient mice were assayed by flow cytometry. A bar graph shows percentages of parasite-infected leukocytes at 5 weeks posttransfer (10 weeks postinfection) in recipient animals that received effector CD8⁺ T cells or memory CD8⁺ T cells. (C) PD-L1 expression was assayed on total brain leukocytes by flow cytometry at week 5 posttransfer in recipient animals. (D) A bar graph represents the absolute number of endogenous CD8⁺ T cells in brain of CD8⁺ T cell recipients. (E) PD-1 expression on endogenous CD8⁺ T cells in brain was evaluated by flow cytometry. (F) Brain mononuclear cells were stimulated with TLA and then evaluated for IFN-γ production by endogenous CD8⁺ T cells at 5 weeks posttransfer. Data were acquired using a Cytek-upgraded 8-color BD FACSCalibur flow cytometer which accounts for differences in fluorescence scale.