Peter T. Sage

The PD‐1 pathway in tolerance and autoimmunity

Summary:  Regulatory T cells (Tregs) and the PD‐1: PD‐ligand (PD‐L) pathway are both critical to terminating immune responses. Elimination of either can result in the breakdown of tolerance and the development of autoimmunity. The PD‐1: PD‐L pathway can thwart self‐reactive T cells and protect against autoimmunity in many ways. In this review, we highlight how PD‐1 and its ligands defend against potentially pathogenic self‐reactive effector T cells by simultaneously harnessing two mechanisms of peripheral tolerance: (i) the promotion of Treg development and function and (ii) the direct inhibition of potentially pathogenic self‐reactive T cells that have escaped into the periphery. Treg cells induced by the PD‐1 pathway may also assist in maintaining immune homeostasis, keeping the threshold for T‐cell activation high enough to safeguard against autoimmunity. PD‐L1 expression on non‐hematopoietic cells as well as hematopoietic cells endows PD‐L1 with the capacity to promote Treg development and enhance Treg function in lymphoid organs and tissues that are targets of autoimmune attack. At sites where transforming growth factor‐β is present (e.g. sites of immune privilege or inflammation), PD‐L1 may promote the de novo generation of Tregs. When considering the consequences of uncontrolled immunity, it would be therapeutically advantageous to manipulate Treg development and sustain Treg function. Thus, this review also discusses how the PD‐1 pathway regulates a number of autoimmune diseases and the therapeutic potential of PD‐1: PD‐L modulation.

The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood

CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR cells) inhibit humoral immunity mediated by CD4+CXCR5+Foxp3− follicular helper T cells (TFH cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of TFR cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of TFR cells in the lymph nodes and that those TFR cells had enhanced suppressive ability. We also found substantial populations of TFR cells in mouse blood and demonstrated that TFR cells in the blood homed to lymph nodes and potently inhibited TFH cells in vivo. TFR cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability

Foxp3+ regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3+CD25− cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

The coinhibitory receptor CTLA-4 Controls B cell Responses by Modulating T Follicular Helper, T Follicular Regulatory and T Regulatory Cells

The receptor CTLA-4 has been implicated in controlling B cell responses, but the mechanisms by which CTLA-4 regulates antibody production are not known. Here we showed deletion of CTLA-4 in adult mice increased Tfh and Tfr cell numbers and augmented B cell responses. In the effector phase, loss of CTLA-4 on Tfh cells resulted in heightened B cell responses, whereas loss of CTLA-4 on Tfr cells resulted in defective suppression of antigen-specific antibody responses. We also found that non-Tfr Treg cells could suppress B cell responses through CTLA-4 and that Treg and/or Tfr cells might downregulate B7-2 on B cells outside germinal centers as a means of suppression. Within the germinal center, however, Tfr cells potently suppress B cells through CTLA-4, but with a mechanism independent of altering B7-1 or B7-2. Thus, we identify multifaceted regulatory roles for CTLA-4 in Tfh, Tfr, and Treg cells, which together control humoral immunity.

Response to BRAF Inhibition in Melanoma Is Enhanced When Combined with Immune Checkpoint Blockade

Cooper, Juneja, Sage, and colleagues show that combining BRAF and PD-1/PD-L1 blockade slowed tumor growth and prolonged survival in a melanoma mouse model, with increased number and activity of tumor-infiltrating lymphocytes similar to that in a human melanoma patient treated with this regimen. BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8+ T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten−/− syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8+ T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8+ T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy. Cancer Immunol Res; 2(7); 643–54. ©2014 AACR.

T follicular regulatory cells in the regulation of B cell responses

High affinity antibodies result from interactions between B cells and T follicular helper (Tfh) cells in germinal centers (GCs). Recent studies have identified an effector subset of T regulatory cells termed T follicular regulatory (Tfr) cells that specifically controls GC responses by suppressing Tfh and B cells. The discovery of Tfr cells has shed new light on pathways regulating humoral immunity that enable potent and specific responses to pathogens while restricting autoimmunity. Here, we review the current understanding of the cellular and molecular mechanisms underlying the differentiation and function of Tfr cells. In this context we discuss recent insights into the role of Tfh cells in disease, how this knowledge may be translated therapeutically, and important areas of further research.

Circulating T follicular regulatory and helper cells have memory-like properties

Follicular Tregs (Tfr cells) inhibit antibody production, whereas follicular Th cells (Tfh cells) stimulate it. Tfr cells are found in blood; however, relatively little is known about the developmental signals for these cells or their functions. Here we demonstrated that circulating Tfr and Tfh cells share properties of memory cells and are distinct from effector Tfr and Tfh cells found within lymph nodes (LNs). Circulating memory-like Tfh cells were potently reactivated by DCs, homed to germinal centers, and produced more cytokines than did effector LN Tfh cells. Circulating memory-like Tfr cells persisted for long periods of time in vivo and homed to germinal centers after reactivation. Effector LN Tfr cells suppressed Tfh cell activation and production of cytokines, including IL-21, and inhibited class switch recombination and B cell activation. The suppressive function of this population was not dependent on specific antigen. Similar to LN effector Tfr cells, circulating Tfr cells also suppressed B and Tfh cells, but with a much lower capacity. Our data indicate that circulating memory-like Tfr cells are less suppressive than LN Tfr cells and circulating memory-like Tfh cells are more potent than LN effector Tfh cells; therefore, these circulating populations can provide rapid and robust systemic B cell help during secondary antigen exposure.

Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots

The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in vivo.

T follicular regulatory cells

Pathogen exposure elicits production of high‐affinity antibodies stimulated by T follicular helper (Tfh) cells in the germinal center reaction. Tfh cells provide both costimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. Under normal circumstances, the germinal center reaction results in antibodies that precisely target foreign pathogens while limiting autoimmunity and excessive inflammation. In order to have this degree of control, the immune system ensures Tfh‐mediated B‐cell help is regulated locally in the germinal center. The recently identified T follicular regulatory (Tfr) cell subset can migrate to the germinal center and inhibit Tfh‐mediated B‐cell activation and antibody production. Although many aspects of Tfr cell biology are still unclear, recent data have begun to delineate the specialized roles of Tfr cells in controlling the germinal center reaction. Here we discuss the current understanding of Tfr‐cell differentiation and function and how this knowledge is providing new insights into the dynamic regulation of germinal centers, and suggesting more efficacious vaccine strategies and ways to treat antibody‐mediated diseases.

Antigen Recognition Is Facilitated by Invadosome-like Protrusions Formed by Memory/Effector T Cells

Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich “invadosome/podosome-like protrusions” (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.