CD4+ T cell-intrinsic T-bet sustains protective CNS immunity and parasite control during Toxoplasma gondii infection

Technical Summary: CD4⁺ T cell-intrinsic T-bet sustains protective CNS immunity and parasite control during Toxoplasma gondii infection

Problem Statement
The transcription factor T-bet (Tbx21) is established as the master regulator of type 1 immunity and is essential for host resistance to intracellular pathogens like Toxoplasma gondii. Global T-bet deficiency leads to profound susceptibility and failure to control parasite replication. However, the specific cell-intrinsic requirement for T-bet within CD4⁺ T cells remains unclear. Previous studies in global knockout models are confounded by the loss of T-bet in other critical populations, such as innate lymphoid cells (ILC1s) and NK cells, which are necessary for maintaining inflammatory dendritic cells (cDC1s) and IL-12 production. Furthermore, while T-bet is known to regulate IFN-γ, CD4⁺ T cells in global T-bet-deficient mice can still produce IFN-γ, suggesting that T-bet-dependent programs extend beyond canonical cytokine production. The specific role of CD4⁺ T cell-intrinsic T-bet in sustaining long-term immunity, particularly within the central nervous system (CNS) where the parasite establishes chronic infection, has not been defined.

Methodology
The authors utilized a conditional knockout approach to isolate the role of T-bet specifically within CD4⁺ T cells. They generated mice with CD4⁺ T cell-specific deletion of Tbx21 (CD4-Cre × Tbx21^fl/fl; CD4-Tbx21^−/−). These mice were compared against wild-type (WT) controls and global Tbx21^−/− mice.

Key experimental strategies included:

• Infection Models: Mice were infected intraperitoneally (i.p.) with ME49 cysts to model acute and chronic infection, and orally to model physiologically relevant mucosal infection.
• Temporal Analysis: Parasite burden, cytokine levels, and immune cell phenotypes were assessed at acute (8 days post-infection, dpi) and chronic/transition (14 dpi) time points.
• Adoptive Transfer: To distinguish between cell-intrinsic defects and environmental factors, splenocytes from infected WT or global Tbx21^−/− mice were transferred into Rag2^−/− recipients prior to infection. Additionally, WT CD4⁺ T cells were transferred into infected CD4-Tbx21^−/− recipients to test for rescue of CNS immunity.
• Cellular and Molecular Assays: The study employed flow cytometry to quantify T cell populations, IFN-γ production, and differentiation markers (CD44, Ly6C, CD11a). Quantitative real-time PCR (RT-qPCR) was used to measure parasite burden (B1 gene), tachyzoite markers (TgSag1), and IFN-γ-inducible immunity-related GTPases (IRGs: Irga6, Irgb6). Brain cysts were visualized and quantified via immunofluorescence using Dolichos biflorus agglutinin (DBA).

Key Results

1. Dispensability in Acute Systemic Control: CD4⁺ T cell-intrinsic T-bet was largely dispensable for controlling acute systemic infection. CD4-Tbx21^−/− mice maintained comparable systemic IFN-γ and TNF-α levels to WT mice at 8 dpi and exhibited similar parasite burdens in the liver, spleen, peritoneal exudate, and heart. Unlike global Tbx21^−/− mice, which succumbed by day 10, CD4-Tbx21^−/− mice survived the acute phase, though they eventually succumbed by day 25.
2. IFN-γ Production and Differentiation: While CD4-Tbx21^−/− mice generated antigen-experienced IFN-γ-producing CD4⁺ T cells, the magnitude of this response was reduced. Crucially, T-bet was required for the acquisition of a fully differentiated inflammatory effector state. CD4-Tbx21^−/− mice showed a significant reduction in Ly6C expression on CD4⁺IFN-γ⁺ T cells, a hallmark of terminally differentiated Th1 cells, despite normal expression of CD11a.
3. Critical Role in CNS Immunity: As infection transitioned to the chronic stage (14 dpi), the absence of T-bet in CD4⁺ T cells led to a failure in sustaining cerebral immunity. CD4-Tbx21^−/− mice exhibited significantly reduced accumulation of CD4⁺ T cells in the brain and diminished cerebral IFN-γ production, while CD8⁺ T cell responses remained intact.
4. Parasite Control and Latency: The loss of CD4⁺ T cell-intrinsic T-bet resulted in impaired IFN-γ-dependent antimicrobial mechanisms in the brain, evidenced by reduced expression of IRGs (Irga6, Irgb6). Consequently, CD4-Tbx21^−/− mice failed to restrict tachyzoite activity, showing elevated TgSag1 transcripts and fewer, smaller brain cysts compared to WT, indicating a failure to properly transition parasites to the latent bradyzoite stage.
5. Rescue by WT CD4⁺ T Cells: Transfer of WT CD4⁺ T cells into infected CD4-Tbx21^−/− mice restored cerebral CD4⁺ T cell accumulation, IFN-γ production, IRG expression, and tachyzoite control, confirming that the defect is cell-intrinsic to CD4⁺ T cells.

Significance and Claims
The paper establishes that the requirement for T-bet in CD4⁺ T cells during T. gondii infection is temporally distinct. While T-bet is not strictly required to initiate the IFN-γ response or control acute systemic replication, it becomes indispensable for sustaining protective immunity as the infection progresses to the chronic stage. Specifically, CD4⁺ T cell-intrinsic T-bet is critical for:

• Promoting the full differentiation of Th1 effector cells (Ly6C⁺ phenotype).
• Sustaining CD4⁺ T cell accumulation and effector function within the CNS.
• Driving IFN-γ-dependent antimicrobial programs (IRGs) that restrict tachyzoite persistence and facilitate the transition to latency.

The authors conclude that long-term resistance to T. gondii depends on T-bet-dependent programs in CD4⁺ T cells that extend beyond simple IFN-γ production, highlighting a non-redundant role for these cells in maintaining CNS immunity against persistent intracellular pathogens.