Jared H. Rowe

Pregnancy imprints regulatory memory that sustains anergy to fetal antigen

Pregnancy is an intricately orchestrated process where immune effector cells with fetal specificity are selectively silenced. This requires the sustained expansion of immune-suppressive maternal FOXP3+ regulatory T cells (Treg cells), because even transient partial ablation triggers fetal-specific effector T-cell activation and pregnancy loss. In turn, many idiopathic pregnancy complications proposed to originate from disrupted fetal tolerance are associated with blunted maternal Treg expansion. Importantly, however, the antigen specificity and cellular origin of maternal Treg cells that accumulate during gestation remain incompletely defined. Here we show that pregnancy selectively stimulates the accumulation of maternal FOXP3+ CD4 cells with fetal specificity using tetramer-based enrichment that allows the identification of rare endogenous T cells. Interestingly, after delivery, fetal-specific Treg cells persist at elevated levels, maintain tolerance to pre-existing fetal antigen, and rapidly re-accumulate during subsequent pregnancy. The accelerated expansion of Treg cells during secondary pregnancy was driven almost exclusively by proliferation of fetal-specific FOXP3+ cells retained from prior pregnancy, whereas induced FOXP3 expression and proliferation of pre-existing FOXP3+ cells each contribute to Treg expansion during primary pregnancy. Furthermore, fetal resorption in secondary compared with primary pregnancy becomes more resilient to partial maternal FOXP3+ cell ablation. Thus, pregnancy imprints FOXP3+ CD4 cells that sustain protective regulatory memory to fetal antigen. We anticipate that these findings will spark further investigation on maternal regulatory T-cell specificity that unlocks new strategies for improving pregnancy outcomes and novel approaches for therapeutically exploiting Treg cell memory.

Foxp3(+) regulatory T cell expansion required for sustaining pregnancy compromises host defense against prenatal bacterial pathogens.

Although pregnancy confers unique susceptibility to infection, the pregnancy-associated immune defects that erode host defense remain largely undefined. Herein, we demonstrate that expansion of immune-suppressive Foxp3(+) regulatory T cells (Tregs) which occurs physiologically during pregnancy or when experimentally induced in transgenic mice caused enhanced susceptibility to prenatal pathogens including Listeria and Salmonella species. Reciprocally, infection susceptibility was uniformly reduced with Treg ablation. Importantly however, the sustained expansion of maternal Tregs was essential for maintaining immune tolerance to the developing fetus because even partial transient ablation of Foxp3-expressing cells fractured maternal tolerance to fetal antigen and triggered fetal resorption. Interestingly, Foxp3 cell-intrinsic defects in the immune-suppressive cytokine IL-10 alone were sufficient to override Treg-mediated infection susceptibility, while IL-10 was nonessential for sustaining pregnancy. Thus, maternal Treg expansion required for sustaining pregnancy creates naturally occurring holes in host defense that confer prenatal infection susceptibility.

Regulatory T Cell Suppressive Potency Dictates the Balance between Bacterial Proliferation and Clearance during Persistent Salmonella Infection

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3–4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3 GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3+ Treg suppressive potency. In complementary experiments using Foxp3 DTR mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

Foxp3(+) regulatory T cells, immune stimulation and host defence against infection.

The immune system is intricately regulated allowing potent effectors to expand and become rapidly mobilized after infection, while simultaneously silencing potentially detrimental responses that averts immune‐mediated damage to host tissues. This relies in large part on the delicate interplay between immune suppressive regulatory CD4+ T (Treg) cells and immune effectors that without active suppression by Treg cells cause systemic and organ‐specific autoimmunity. Although these beneficial roles have been classically described as counterbalanced by impaired host defence against infection, newfound protective roles for Treg cells against specific viral pathogens (e.g. herpes simplex virus 2, lymphocytic choriomeningitis virus, West Nile virus) have been uncovered using transgenic mice that allow in vivo Treg‐cell ablation based on Foxp3 expression. In turn, Foxp3+ Treg cells also provide protection against some parasitic (Plasmodium sp., Toxoplasma gondii) and fungal (Candida albicans) pathogens. By contrast, for bacterial and mycobacterial infections (e.g. Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis), experimental manipulation of Foxp3+ cells continues to indicate detrimental roles for Treg cells in host defence. This variance is probably related to functional plasticity in Treg cell suppression that shifts discordantly following infection with different types of pathogens. Furthermore, the efficiency whereby Treg cells silence immune activation coupled with the plasticity in Foxp3+ cell activity suggest that overriding Treg‐mediated suppression represents a prerequisite ‘signal zero’ that together with other stimulation signals [T‐cell receptor (signal 1), co‐stimulation (signal 2), inflammatory cytokines (signal 3)] are essential for T‐cell activation in vivo. Herein, the importance of Foxp3+ Treg cells in host defence against infection, and the significance of infection‐induced shifts in Treg‐cell suppression are summarized.

Selective Priming and Expansion of Antigen-Specific Foxp3−CD4+ T Cells during Listeria monocytogenes Infection

The Foxp3-expressing subset of regulatory CD4+ T cells have defined Ag specificity and play essential roles in maintaining peripheral tolerance by suppressing the activation of self-reactive T cells. Similarly, during chronic infection, pathogen-specific Foxp3-expressing CD4+ T cells expand and actively suppress pathogen-specific effector T cells. Herein, we used MHC class II tetramers and Foxp3gfp knockin mice to track the kinetics and magnitude whereby pathogen-specific Foxp3+CD4+ and Foxp3−CD4+ cells are primed and expand after acute infection with recombinant Listeria monocytogenes (Lm) expressing the non-“self”-Ag 2W1S52–68. We demonstrate that Lm infection selectively primes proliferation, expansion, and subsequent contraction of Lm-specific Foxp3− effector CD4+ cells, while the numbers of Lm-specific Foxp3+CD4+ regulatory cells remain essentially unchanged. In sharp contrast, purified 2W1S52–68 peptide primes coordinated expansion of both Foxp3+ regulatory and Foxp3− effector T cells with the same Ag specificity. Taken together, these results indicate selective priming and expansion of Foxp3− CD4 T cells is a distinguishing feature for acute bacterial infection.

PDL-1 Blockade Impedes T Cell Expansion and Protective Immunity Primed by Attenuated Listeria monocytogenes

Infection with attenuated Listeria monocytogenes (Lm) is a robust in vivo model for examining how Ag-specific T cells are primed, and subsequent challenge with virulent Lm allows for the protective effects of T cell priming to be quantified. Herein, we investigated the role of programmed death ligand 1 (PDL-1) in T cell priming and immunity conferred after primary infection with Lm ΔactA followed by virulent Lm challenge. In striking contrast to the inhibitory role of PDL-1 on T cell immunity in other infection models, marked reductions in the magnitude of T cell expansion and the kinetics of T cell proliferation were observed with PDL-1 blockade after primary Lm ΔactA infection. More importantly, PDL-1 blockade beginning before primary infection and maintained throughout the experiment resulted in delayed bacterial clearance and T cell expansion after secondary challenge with virulent Lm. These results indicate that for immunity to intracellular bacterial infection, PDL-1 plays an important stimulatory role for priming and expansion of protective T cells.

Listeria monocytogenes cytoplasmic entry induces fetal wastage by disrupting maternal Foxp3+ regulatory T cell-sustained fetal tolerance.

Although the intracellular bacterium Listeria monocytogenes has an established predilection for disseminated infection during pregnancy that often results in spontaneous abortion or stillbirth, the specific host-pathogen interaction that dictates these disastrous complications remain incompletely defined. Herein, we demonstrate systemic maternal Listeria infection during pregnancy fractures fetal tolerance and triggers fetal wastage in a dose-dependent fashion. Listeria was recovered from the majority of concepti after high-dose infection illustrating the potential for in utero invasion. Interestingly with reduced inocula, fetal wastage occurred without direct placental or fetal invasion, and instead paralleled reductions in maternal Foxp3+ regulatory T cell suppressive potency with reciprocal expansion and activation of maternal fetal-specific effector T cells. Using mutants lacking virulence determinants required for in utero invasion, we establish Listeria cytoplasmic entry is essential for disrupting fetal tolerance that triggers maternal T cell-mediated fetal resorption. Thus, infection-induced reductions in maternal Foxp3+ regulatory T cell suppression with ensuing disruptions in fetal tolerance play critical roles in pathogenesis of immune-mediated fetal wastage.

Foxp3+ Regulatory T Cells Impede the Priming of Protective CD8+ T Cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3+ regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3+ Tregs unleashes the robust expansion and activation of peptide-stimulated CD8+ T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8+ T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8+ T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8+ T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3+ Treg suppressive potency. Therefore, Foxp3+ Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8+ T cells.

Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection

Typhoid fever is a persistent infection caused by host‐adapted Salmonella strains adept at circumventing immune‐mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen‐specific CD4+ T cells after virulent Salmonella infection requires SPI‐2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella‐expressed antigens, but extends to CD4+ T cells primed to expand by co‐infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen‐specific CD4+ T cells identified with MHC class II tetramer. Analysis of T‐cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T‐cell subsets after Salmonella co‐infection that is associated with the purging of antigen‐specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T‐cell subsets, which re‐shapes the repertoire of antigen‐specific T cells that persist later after infection.

Early eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection

Typhoid fever is a systemic, persistent infection caused by host-specific strains of Salmonella. Although the use of antibiotics has reduced the complications associated with primary infection, recurrent infection remains an important cause of ongoing human morbidity and mortality. Herein, we investigated the impacts of antibiotic eradication of primary infection on protection against secondary recurrent infection. Using a murine model of persistent Salmonella infection, we demonstrate protection against recurrent infection is sustained despite early eradication of primary infection. In this model, protection is not mediated by CD4(+) or CD8(+) T cells because depletion of these cells either alone or in combination prior to rechallenge does not abrogate protection. Instead, infection followed by antibiotic-mediated clearance primes robust levels of Salmonella-specific antibody that can adoptively transfer protection to naïve mice. Thus, eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection.