T cells differentiate upon Ag recognition by integrating multiple signals from the TCR, costimulatory molecules, and cytokines and transducing signals via intracellular molecules, including extracellular regulated kinases (Erks). Chang et al. (p. 721) used a reverse genetics approach to assess the role of Erk2 during T cell differentiation. Ex vivo stimulation of mouse Erk2−/− CD4+ T cells revealed a defect in proliferation relative to wild-type (WT) cells, which was overcome by increased costimulation via CD28 or mitogen stimulation. Th1, but not Th2 or Th17, differentiation was impaired in Erk2−/− CD4+ T cells stimulated in vitro. Mice with Erk2−/− CD4+ T cells showed diminished Th1 responses following LCMV infection, compared with WT mice. Gene expression analysis revealed that the Th1 transcription factor T-bet was Erk2 dependent whereas Erk2 suppressed expression of the Th2 transcription factor Gata3. A greater proportion of Erk2−/− CD4+ T cells differentiated into Foxp3+ induced regulatory T cells (iTreg) in vitro in the presence of Treg-skewing cytokines relative to WT T cells, and Erk2 expression was associated with reduced Gata3 and Foxp3 expression and increased Smad signaling in WT iTregs. Together, these results identify the contrasting ways Erk2 influences differentiation of T cell subsets.

Anti-fetus CD8+ T cells have been identified in maternal circulation after pregnancy, and Lissauer et al. (p. 1072) characterized HY-specific CD8+ T cell responses throughout pregnancy in women pregnant with male fetuses to better understand the kinetics and phenotype of these cells. Staining of PBMCs with an MHC-peptide dextramer specific to an HLA*0201-restricted HY minor histocompatibility Ag expressed by males identified HY-specific CD8+ T cells in 50% of women carrying male fetuses. HY-specific CD8+ T cells could be detected as early as the first trimester, increased throughout pregnancy, and were detectable after delivery. A predominant fraction of HY-specific CD8+ T cells had an effector memory phenotype, and HY-specific T cell clones retained HY-specific cytotoxicity and were able to proliferate and produce IFN-γ. Together, these data suggest that anti-fetus CD8+ T cell responses are found during and after pregnancy. These findings may also contribute to a better understanding of immunological responses associated with pregnancy loss and miscarriage.

Th1 differentiation is controlled at the genetic level by multiple transcription factors, including Runx3, STAT4, and T-bet. Pham et al. (p. 832) identified a role for the transcriptional repressor Twist1 as a negative regulator of Th1 differentiation through its effects on Th1-promoting transcription factors. Twist1 expression was highest in Th1 cells relative to other T cell subsets and increased upon TCR stimulation. Ectopic expression of Twist1 in Th1 cells correlated with decreased expression of Th1-oriented transcription factors including Hlx2, Runx3, and T-bet, as well as decreased IFN-γ and TNF-α production. In contrast, short hairpin RNA-mediated knockdown of Twist1 correlated with increased expression of these transcription factors and cytokines. Il12rb2 gene expression also negatively correlated with Twist1 expression. Ectopic coexpression of Runx3 and Twist1 reversed the negative regulatory effects of Twist1 on IFN-γ and TNF-α expression independently of T-bet and STAT4. Twist1 expression was linked to diminished binding of Runx3 and T-bet to the Ifng locus as well as alterations in the chromatin conformation within this locus. Moreover, binding of Twist1 to Ifng required complex formation with Runx3. These results suggest that Twist1 influences Th1 differentiation through its negative regulatory effects on Th1 transcription factors, particularly Runx3.

One strategy developed to induce tolerance against autoimmune epitopes involves treatment with an autoimmune Ag or peptide conjugated to donor splenocytes (SPs) via crosslinking with 1-ethyl-3-(3′-dimethylaminopropyl)-carboiimide (ECDI). Kheradmand et al. (p. 804) applied this method to induce islet transplantation tolerance in mice. Donor ECDI-SPs were detected within APCs of recipient mice soon after injection, and CD11c+ DCs with upregulated levels of negative costimulatory molecules were identified as the APC subset essential for inducing islet allograft tolerance. Uptake of donor ECDI-SPs by APCs induced a significant expansion and an ensuing deletion of recipient CD4+ T cells with indirect allospecificity in the spleen, mostly likely through an IFN-γ–dependent mechanism. Residual T cells with indirect allospecificity were retained in the spleen and did not migrate to the islet graft or graft-draining lymph node. Free ECDI-SPs not taken up by APCs were able to induce anergy of CD4+ T cells with direct allospecificity. Donor ECDI-SP infusion also induced an increase in regulatory T cells in the graft and secondary lymphoid organs. These findings suggest that ECDI-SP infusion may be a viable approach to inducing transplant tolerance by limiting allospecific CD4+ T cell-mediated graft destruction by multiple mechanisms and promoting regulatory T cell expansion.

Autophagy is an immune defense pathway used by phagocytes to eliminate harmful pathogens. Autophagy can be triggered by cytokines like IFN-γ or binding of pattern recognition receptors by pathogen-derived ligands. Matsuzawa et al. (p. 813) used an autophagosome reporter with a tandem fluorescently tagged LC3b (tfLC3) to distinguish autophagosomes together with short hairpin RNA-mediated knockdown of different signaling molecules to discern the molecular components required for IFN-γ–mediated activation of autophagy in macrophages. IFN-γ induced a dose-dependent increase in the number, size, and maturation status of autophagosomes expressing tfLC3, which was dependent on the kinases JAK1/2, PI3K, and p38 MAPK. Surprisingly, autophagosome formation was not dependent on either STAT1 or the guanosine triphosphatase Irgm1, a molecule that has been linked to autophagy activation in other studies. These observations present a new perspective of the mechanisms by which IFN-γ triggers autophagy and suggest that Irgm1 is not an essential component of all autophagy pathways.

TLR signaling is critical to innate defense against pathogens in part through activation of NF-κB–driven proinflammatory responses. Leishmania donovani, an intracellular parasite that infects macrophages, has been shown to downregulate innate responses despite the presence of a TLR2 ligand on the parasite’s surface. Srivastav et al. (p. 924) show that L. donovani thwarts TLR2-mediated responses by inhibiting NF-κB signaling and proinflammatory cytokine production through upregulation of the deubiquitinating enzyme A20. L. donovani-infected macrophages had a defect in TRAF6 complex formation with TAK1–TAB2, which is necessary for TLR2 signaling-driven NF-κB activation. Lys 63-linked ubiquitination of TRAF6 is required for complex formation with the TAK1–TAB2 complex and was significantly reduced in L. donovani-infected macrophages, concurrent with a significant increase in the level of the deubiquitinating enzyme A20. Small-interfering RNA-mediated knockdown of A20 restored TRAF6 ubiquitination and NF-κB–driven proinflammatory cytokine production. A20 knockdown was also associated with reduced parasite survival in macrophages, and A20 silencing in BALB/c mice enhanced proinflammatory cytokine responses and parasite clearance. Thus, L. donovani subversion of TLR2 signaling appears to be linked to a disruption in TRAF6 complex formation through its effect on A20 expression.

Different subsets of regulatory T cells (Tregs) have been defined based on phenotypic markers and functional differences, and previous studies have suggested that the integrin CD103 is expressed on the surface of effector/memory Tregs. Chang et al. (p. 567) investigated the suppressive capabilities of CD103+ Tregs in vitro and in tumor-bearing mice. They confirmed that CD103+ Tregs have an effector/memory phenotype. In contrast to previous findings, CD103+ Tregs and CD103 Tregs were similarly effective at suppressing proliferation and IFN-γ production by CD4+CD25 T cells in vitro. CD103+ Tregs isolated from the spleens of mice with colon tumors also had an effector/memory phenotype and transfer of these cells, but not CD103 Tregs, into mice shortly after tumor inoculation significantly inhibited antitumor CD8+ T cell activity. Within tumors, CD103+ Tregs coexpressed the chemokine receptor CCR5, which facilitated migration to CCL5-expressing tumor sites and subsequent inhibition of antitumor CD8+ T cell responses. Together, these results reveal how migration is fundamental to the ability of CD103+ Tregs to suppress antitumor responses in vivo.

The Sle1c sublocus within the Sle1 lupus susceptibility locus of the NZM2410 mouse strain has been associated previously with lupus pathogenesis and increased CD4+ T cell activation and proliferation. Perry et al. (p. 793) mapped a region within Sle1c to a gene encoding an orphan nuclear receptor that contributes to lupus pathogenesis through its effects on CD4+ T cell metabolism. A 675 kB interval within the centromeric region, designated Sle1c2, was linked to the CD4+ T cell activation phenotype through screening of multiple Sle1c congenic mouse strains. Congenic mice carrying this genetic region, termed B6.Sle1c2 mice, also showed a tendency toward Th1 skewing. B6.Sle1c2 CD4+ T cells promoted chronic graft-versus-host disease, and NZB hybrid mice generated from a NZB x B6.Sle1c2 cross showed increased B cell activation, autoantibody production, and immune complex deposition, which together strongly linked Sle1c2 to lupus pathogenesis. The estrogen-related receptor gamma (Esrrg) gene, which encodes an orphan nuclear receptor that regulates mitochondrial function and metabolism, was the only gene within the Sle1c2 interval that was expressed in T cells. Relative to wild-type B6 mice, Esrrg expression was reduced in B6.Sle1c2 CD4+ T cells, which was associated with increased mitochondrial dysfunction, reduced mitochondrial mass, and altered oxidative metabolism. Together, these data indicate that Esrrg deficiency may contribute to lupus susceptibility due to CD4+ T cell metabolic dysfunction.

Summaries written by Christiana N. Fogg, Ph.D.