Christian T. Mayer

Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma.

Tumor-bearing individuals have been reported to harbor increased numbers of Foxp3(+) regulatory T cells (Treg), which prevent the development of efficient antitumor immune responses. Thus, Treg depletion has already been tested as a promising therapeutic approach in various animal models and entered clinical trials. However, the use of nonspecific Treg targeting agents such as CD25 depleting antibodies, which in addition to CD25(+) Tregs also deplete recently activated CD25(+) effector T cells, potentially masked the tremendous potential of this therapeutic strategy. To avoid such nonspecific effects, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3(+) Tregs even during ongoing immune responses. We showed that Foxp3(+) Treg depletion induced partial regression of established ovalbumin (OVA)-expressing B16 melanoma, which was associated with an increased intratumoral accumulation of activated CD8(+) cytotoxic T cells. The antitumor effect could be significantly enhanced when Treg depletion was combined with vaccination against OVA. To further assess whether this therapeutic approach would break self-tolerance, we crossed DEREG mice with RipOVA(low) mice, expressing OVA as neo-self-antigen under control of the rat insulin promoter. In these mice, combined Treg depletion and vaccination also induced tumor regression without the onset of diabetes. Together, our data suggest that selective Treg targeting strategies combined with vaccinations against tumor-associated (self) antigens have the potential to evoke efficient antitumor responses without inducing overt autoimmunity. These findings might have implications for future therapeutic interventions in cancer patients.

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (Treg cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff cell) function and gain of suppressive activity by Treg cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of Treg cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3′ untranslated region. Release of SATB1 from the control of Foxp3 in Treg cells caused loss of suppressive function, establishment of transcriptional Teff cell programs and induction of Teff cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining Treg cell functionality.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Summary Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

T cell help controls the speed of the cell cycle in germinal center B cells

B cells have a need for speed High-affinity antibodies provide long-lasting protective immunity against many infections. Generating such antibodies requires help, in the form of T cells, which interact with antibody-producing B cells. As B cells proliferate and mutate their antibody genes, T cells select the cells producing high-affinity antibodies. Gitlin et al. show in mice that B cells that receive T cell help transit through the cell cycle more quickly by increasing the speed at which replication forks progress. Such a rapid cell cycle transition gives high-affinity B cells a selective advantage. Science, this issue p. 643 T cells help select high-affinity antibodies by increasing the speed at which B cells progress through the cell cycle. The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.

CD8+Foxp3+ T cells share developmental and phenotypic features with classical CD4+Foxp3+ regulatory T cells but lack potent suppressive activity

“Suppressor T cells” were historically defined within the CD8+ T‐cell compartment and recent studies have highlighted several naturally occurring CD8+Foxp3− Treg populations. However, the relevance of CD8+Foxp3+ T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8+Foxp3− T cells is counter‐regulated by DC‐mediated co‐stimulation via CD80/CD86. CD8+Foxp3+ T cells fail to develop in TCR‐transgenic mice with Rag1−/− background, similar to classical CD4+Foxp3+ Tregs. Notably, both naturally occurring and induced CD8+Foxp3+ T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN‐γ production upon restimulation when compared with their CD8+Foxp3− counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3+ cells by eGFP reporter expression, we demonstrate that induced CD8+Foxp3+ T cells similar to activated CD8+Foxp3− T cells only mildly suppress T‐cell proliferation and IFN‐γ production. We therefore categorize CD8+Foxp3+ T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4+Foxp3+ Tregs, but lacking potent suppressive activity.

Nonfunctional regulatory T cells and defective control of Th2 cytokine production in natural scurfy mutant mice.

Foxp3+ regulatory T cells (Tregs) are crucial for preventing autoimmunity. We have demonstrated that depletion of Foxp3+ Tregs results in the development of a scurfy-like disease, indicating that Foxp3− effector T cells are sufficient to induce autoimmunity. It has been postulated that nonfunctional Tregs carrying potentially self-reactive T cell receptors may contribute to scurfy (sf) pathogenesis due to enhanced recognition of self. Those cells, however, could not be identified in sf mutants due to the lack of Foxp3 protein expression. To address this issue, we crossed the natural sf mouse mutant with bacterial artificial chromosome transgenic DEREG (depletion of regulatory T cells) mice. Since DEREG mice express GFP under the control of an additional Foxp3 promoter, those crossings allowed proving the existence of “would-be” Tregs, which are characterized by GFP expression in the absence of functional Foxp3. Sf Tregs lost their in vitro suppressive capacity. This correlated with a substantial reduction of intracellular cAMP levels, whereas surface expression of Treg markers was unaffected. Both GFP+ and GFP− sf cells produced high amounts of Th2-type cytokines, reflected also by enhanced Gata-3 expression, when tested in vitro. Nevertheless, sf Tregs could be induced in vitro, although with lower efficiency than DEREG Tregs. Transfer of GFP+ sf Tregs, in contrast to GFP− sf T cells, into RAG1-deficient animals did not cause the sf phenotype. Taken together, natural and induced Tregs develop in the absence of Foxp3 in sf mice, which lack both suppressive activity and autoreactive potential, but rather display a Th2-biased phenotype.

Foxp3-Mediated Suppression of CD95L Expression Confers Resistance to Activation-Induced Cell Death in Regulatory T Cells

CD4+CD25++Foxp3+ regulatory T cells (Tregs) control self-reactive cells to maintain peripheral tolerance. Treg homeostasis has to be controlled tightly to ensure balanced Treg-mediated suppression. One mechanism that regulates the CD4+ T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the death ligand CD95L (FasL/APO-1L/CD178) in expanded T cells. These cells express the death receptor CD95 (Fas/APO-1), and binding of CD95L to CD95 results in AICD. In contrast, Tregs do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway. In this study, we show that human and murine Tregs express low levels of CD95L upon stimulation. Knockdown of the transcriptional repressor Foxp3 partially rescues CD95L expression and AICD in human Tregs. Moreover, upon stimulation Foxp3-mutant Tregs from Scurfy mice express CD95L similar to conventional T cells. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased GFP+ Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Tregs and demonstrates that Tregs are susceptible to homeostatic control by CD95 stimulation.

Selective and efficient generation of functional Batf3-dependent CD103+ dendritic cells from mouse bone marrow

Multiple subsets of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent dendritic cells (DCs) control T-cell tolerance and immunity. In mice, Batf3-dependent CD103(+) DCs efficiently enter lymph nodes and cross-present antigens, rendering this conserved DC subset a promising target for tolerance induction or vaccination. However, only limited numbers of CD103(+) DCs can be isolated with current methods. Established bone marrow culture protocols efficiently generate monocyte-derived DCs or produce a mixture of FLT3L-dependent DC subsets. We show that CD103(+) DC development requires prolonged culture time and continuous action of both FLT3L and granulocyte macrophage colony-stimulating factor (GM-CSF), explained by a dual effect of GM-CSF on DC precursors and differentiating CD103(+) DCs. Accordingly, we established a novel method to generate large numbers of CD103(+) DCs (iCD103-DCs) with limited presence of other DC subsets. iCD103-DCs develop in a Batf3- and Irf8-dependent fashion, express a CD8α/CD103 DC gene signature, cross-present cell-associated antigens, and respond to TLR3 stimulation. Thus, iCD103-DCs reflect key features of tissue CD103(+) DCs. Importantly, iCD103-DCs express high levels of CCR7 upon maturation and migrate to lymph nodes more efficiently than classical monocyte-derived DCs. Finally, iCD103-DCs induce T cell-mediated protective immunity in vivo. Our study provides insights into CD103(+) DC development and function.

Epigenetic targeting of activation-induced cytidine deaminase.

Significance Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme crucial for the generation of efficacious antibodies. AID also promiscuously introduces DNA lesions at cancer genes, leading to their chromosome translocation and lymphoma. However, how AID is recruited to these off targets is not well understood. Here, we compare AID-induced translocations in two different cell types, B cells and mouse embryonic fibroblasts. By analyzing the sites where AID is active in a cell type-specific manner, we find that, in addition to transcriptional activity, AID recruitment is mediated by specific epigenetic features associated with active enhancers and transcription elongation. Understanding AID’s targeting mechanism is a fundamental question of immunology with implications for the biology of cancer. Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.