Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection.

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.

Dynamic adoption of anergy by antigen-exhausted CD4+ T cells.

Exhausted immune responses to chronic diseases represent a major challenge to global health. We study CD4+ T cells in a mouse model with regulatable antigen presentation. When the cells are driven through the effector phase and are then exposed to different levels of persistent antigen, they lose their T helper 1 (Th1) functions, upregulate exhaustion markers, resemble naturally anergic cells, and modulate their MAPK, mTORC1, and Ca2+/calcineurin signaling pathways with increasing dose and time. They also become unable to help B cells and, at the highest dose, undergo apoptosis. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of T cell receptor (TCR) signals over a period of weeks. Upon antigen removal, the cells recover their functionality while losing exhaustion and anergy markers. Our data suggest an adjustable response of CD4+ T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease.

A "hotspot" for autoimmune T cells in type 1 diabetes.

The ability of a single T cell antigen receptor (TCR) to cross-react with multiple antigens allows the finite number of T cells within an organism to respond to the compendium of pathogen challenges faced during a lifetime. Effective immune surveillance, however, comes at a price. TCR cross-reactivity can allow molecular mimics to spuriously activate autoimmune T cells; it also underlies T cell rejection of organ transplants and drives graft-versus-host disease. In this issue of the JCI, Cole and colleagues provide insight into how an insulin-reactive T cell cross-reacts with pathogen-derived antigens by focusing on a limited portion of the peptides to provide a hotspot for binding. These findings dovetail with recent studies of alloreactive and autoimmune TCRs and suggest that the biochemical principles that govern conventional protein-protein interactions may allow the specificity and cross-reactivity profiles of T cells to be predicted.

The Timing of T Cell Priming and Cycling.

The proliferation of specific lymphocytes is the central tenet of the clonal selection paradigm. Antigen recognition by T cells triggers a series of events that produces expanded clones of differentiated effector cells. TCR signaling events are detectable within seconds and minutes and are likely to continue for hours and days in vivo. Here, I review the work done on the importance of TCR signals in the later part of the expansion phase of the primary T cell response, primarily regarding the regulation of the cell cycle in CD4(+) and CD8(+) cells. The results suggest a degree of programing by early signals for effector differentiation, particularly in the CD8(+) T cell compartment, with optimal expansion supported by persistent antigen presentation later on. Differences to CD4(+) T cell expansion and new avenues toward a molecular understanding of cell cycle regulation in lymphocytes are discussed.

Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Differential kinetics of antigen dependency of CD4+ and CD8+ T cells.

Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4+ and CD8+ T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4+ T cell proliferation depended on prolonged Ag presence, whereas CD8+ T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4+ T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8+ T cell proliferation was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8+ T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4+ and CD8+ T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II-restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4+ T cells require continuous stimulation for clonal expansion, whereas CD8+ T cells can divide following a much shorter TCR signal.

Role of antigen persistence and dose for CD4+ T-cell exhaustion and recovery.

It is currently not understood how some chronic infections exhaust antigen-specific T cells over time and which pathogen components contribute to exhaustion. Here, we dissected the behavior of primed CD4(+) T cells exposed to persistent antigen using an inducible transgenic mouse system that allowed us to control antigen presentation as the only experimental variable, independent of the persistent inflammation and disease progression that complicate infectious models. Moreover, this system restricted antigen presentation to dendritic cells (DCs) and avoided confounding B, CD8(+) T, or innate cell responses. When antigen presentation was extended beyond the expansion phase, primed CD4(+) T cells survived, but exhibited reduced memory functionality in terms of their proliferative capacity and cytokine expression potential. The effect was antigen dose and time dependent, not associated with increased PD-1 expression or reduced calcium influx, but impaired Jun phosphorylation in response to TCR engagement. Upon antigen removal, the cells regained the ability to proliferate, but remained unable to produce high levels of IL-2 and TNF-α. These data show that persistent antigen by itself rapidly induces a dysfunctional state in CD4(+) T cells that is only partially reversible upon antigen removal. These findings have implications for vaccine optimization and for the possible reinvigoration of CD4(+) T cells during chronic infection.

Making antigen invisible: a coinhibitory molecule regulates the interaction between T cells and dendritic cells.

Antigen-specific downregulation of T-cell effector function is critical for maintaining self-tolerance but it can promote pathogen persistence in chronic infections; consequently, the restoration of T-cell effector functions is a major goal of therapeutic vaccines against chronic viral infections and malignancies. Recently, a number of T-cell inhibitory receptors, most prominently programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4, have been described that are associated with T-cell exhaustion and tolerance. Blocking these receptors can restore T-cell function and, depending on the model, lead to autoimmune disease or successful viral elimination. Antibodies to PD-1 and cytotoxic T-lymphocyte antigen-4 are currently being tested in clinical trials in several malignant diseases and chronic hepatitis C as they are promising candidates for combination with both prophylactic and therapeutic vaccines. Given the central role of T-cell inhibitory receptors in the regulation of immune responses, understanding their molecular mode of action is of major importance. In the report from Fife and colleagues, two-photon laser scanning microscopy of mouse lymphoid and peripheral tissue has been employed to study the interaction of tolerized PD-1-expressing T cells with antigen-bearing dendritic cells in vivo. While tolerized T cells moved freely and did not make prolonged contacts with dendritic cells, addition of an antibody that blocked the interaction between PD-1 and its ligand PD-L1 lowered T-cell motility, enhanced T-cell-dendritic cell contacts and caused autoimmune disease in the nonobese diabetic mouse model of autoimmune diabetes. The authors conclude that PD-1-PD-L1 interactions mediate peripheral tolerance by inhibiting T-cell receptor-induced stop signals.

Sustained antigen presentation can promote an immunogenic T cell response, like dendritic cell activation.

Activation of dendritic cells (DCs) enhances their ability to prime naïve T cells. How activation renders them immunogenic rather than tolerogenic is unclear. Here, we show, using temporally regulated expression of a transgene-encoded neoself antigen in DCs, that either prolonged antigen presentation or DC activation could elicit full expansion, effector cytokine production, and memory-cell differentiation. Microarray analysis of gene expression in T cells showed that all changes linked to DC activation through CD40 could be reproduced by persistent antigen delivery, suggesting that stabilization of antigen presentation is an important consequence of DC activation in vivo. In this system, DC activation by CD40 engagement indeed extended their ability to present antigen to CD4(+) T cells in vivo, although different results were obtained with antigen delivered to DCs by means of endocytosis from the cell surface. These results suggest that antigen persistence may be an important discriminator of immunogenic and tolerogenic antigen exposure.

Adaptation of TCR repertoires to self-peptides in regulatory and nonregulatory CD4+ T cells.

Currently, it is not understood how the specificity of the TCR guides CD4(+) T cells into the conventional lineage (Tconv) vs directing them to become regulatory (Treg) cells defined by the Foxp3 transcription factor. To address this question, we made use of the "Limited" (LTD) mouse, which has a restricted TCR repertoire with a fixed TCRbeta chain and a TCRalpha chain minilocus. The TCR repertoires of Tconv and Treg cells were equally broad, were distinct, yet overlapped significantly, representing a less strict partition than previously seen between CD4 and CD8 T cells. As a group, the CDR3alpha motifs showed a significant trend to higher positive charge in Treg than in Tconv cells. The Tconv and Treg repertoires were both reshaped between thymus and periphery. Reducing the array of peptides presented by MHC class II molecules by introducing the H2-DM(o/o) mutation into the LTD mouse led to parallel shifts in the repertoires of Tconv and Treg cells. In both cases, the CDR3alpha elements were entirely different and strikingly shortened, relative to normal LTD mice. These peculiar sequences conferred reactivity to wild-type MHC class II complexes and were excluded from the normal repertoire, even among Treg cells, indicating that some forms of self-reactivity are incompatible with selection into the Treg lineage. In conclusion, the Treg repertoire is broad, with distinct composition and characteristics, yet significantly overlapping and sharing structural constraints with the repertoire of conventional CD4(+) T cells.

Gene expression microarrays: glimpses of the immunological genome.

Successful microarray experimentation can generate enormous amounts of data, potentially very rich but also very unwieldy. Bold outlooks and new methods for data analysis and presentation should yield additional insight into the complexities of the immune system.

Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response.

For CD8(+) T cells, a relatively short antigen pulse seems sufficient for antigen-presenting cells to drive clonal expansion and differentiation. It is unknown whether the requirement for antigen is similarly ephemeral for CD4(+) T cells. To study the dependence of a CD4(+) T cell response on antigen persistence in a quantitatively and temporally controlled manner in vivo, we engineered a mouse line expressing a major histocompatibility complex class II-restricted epitope in dendritic cells under the control of a tetracycline-inducible promoter. Experiments tracking the proliferation of CD4(+) T cells exposed to their cognate antigen in various amounts for different time periods revealed that the division of such cells was contingent on the presence of antigen throughout their expansion phase, even in the presence of an inflammatory stimulus. This previously unrecognized feature of a CD4(+) T cell response contrasts with the proliferative behavior of CD8(+) T cells that has been documented, and it implies that the two T cell subsets might require different strategies for efficient vaccination.