Programmed Cell Death-1 Engagement Research Articles

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.

Specific PD-1 Deletion on Regulatory T Cells Leads to Enhanced Anti-Tumor Responses

Programmed cell death 1 (PD-1) is a T cell inhibitory receptor and the most extensively exploited therapeutic target of checkpoint immunotherapy in cancer. Ligation of PD-1 by its ligands PD-L1 or PD-L2 leads to inhibition of CD4 + and CD8 + T cell responses. However, very little is known about the specific role of PD-1 in regulatory T (Treg) cells. Previous studies have shown that PD-1 engagement during induction of Treg polarization by TCR/CD28 signaling in the presence of IL-2 and TGF-β promoted the generation and expansion of Treg cells with potent suppressive function. It has also been shown that patients with high numbers of PD-1 + Treg cells have unfavorable responses to PD-1 checkpoint immunotherapy. Moreover, patients who developed hyperprogressive disease after PD-1 blocking immunotherapy exhibited enhanced Treg cell proliferative ability compared to patients with non-hyperprogressive disease. In the present study, we sought to understand the role of PD-1 on Treg cells in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with FoxP3Cre-YFP ( Pdcd1 f/fFoxP3Cre) resulting in specific deletion of PD-1 in Treg cells. Under steady-state conditions, the percentage of Treg cells in the spleen and lymph nodes remained comparable between Pdcd1 f/fFoxP3Cre and Pdcd1wt/wtFoxP3Cre control mice. However, PD-1-deficient Treg cells had a more activated phenotype compared to their counterparts in control mice, with higher expression of CD69, ICOS, and PD-L1. Furthermore, Treg cells in Pdcd1 f/fFoxp3Cre mice expressed increased levels of GITR, CTLA-4, ICOS, and PD-L1 compared to endogenous CD4 + T conventional cells (CD4 +Foxp3 -, Tconv) and CD8 + T cells. To investigate the implications of these immunological changes in anti-tumor responses, we used the MC38 colon adenocarcinoma syngeneic mouse model. We found a smaller tumor size in Pdcd1 f/fFoxP3Cre mice compared to their control counterparts. Treg cells from spleens and tumor-draining lymph nodes (tdLNs) showed minimal differences in their activation state, expression of checkpoint markers, and proliferation levels, as determined by direct ex vivo assessment. Similarly, CD4 + Tconv and CD8 + T cells from spleens and tdLNs showed a very low level of activation that was comparable between the two experimental groups. In the tumor site, however, Pdcd1 f/fFoxp3Cre tumor-bearing mice had a significant reduction in the fractions of CD4 + Tconv and Treg cells and a significant increase in the fraction of CD8 + T cells compared to control tumor-bearing mice. Detailed analysis revealed that in tumor-bearing Pdcd1 f/fFoxp3Cre mice intratumoral naive and central memory CD4 + T cells were reduced whereas CD4 + T effector cells were increased. Similarly, in Pdcd1 f/fFoxp3Cre tumor-bearing mice we noticed a reduction in naïve and central memory CD8 + T cells but a significant expansion in the effector CD8 + T cells . No significant differences were detected in the expression of activation markers in tumor infiltrating Treg cells between the two groups. In contrast, in Pdcd1 f/fFoxp3Cre tumor-bearing mice, tumor infiltrating CD4 + Tconv cells displayed an activated phenotype with upregulation of PD-L1, ICOS, TIM3, CD69, and CTLA-4, downregulated PD-1, and increased IFN-γ production. Tumor infiltrating CD8 + T cells also displayed an activated phenotype with upregulation of activation markers such as CD69, ICOS, PD-L1, TIM3, and LAG3 but reduced PD-1 expression. Notably, both CD4 + Tconv and CD8 + T cells proliferated more robustly in the Pdcd1 f/fFoxp3Cre tumor-bearing mice as determined by the high expression of the proliferation marker Ki67. These results indicate that selective ablation of PD-1 in Treg cells confers a less suppressive immunological environment and allows for expansion and activation of CD4 + and CD8 + T cells, leading to anti-tumor responses from CD4 + and CD8 + T effector cells.

Upregulation of programmed cell death 1 by interferon gamma and its biological functions in human monocytes

Programmed cell death 1 (PD-1) is a co-inhibitory checkpoint receptor expressed in various immune cells, especially in activated T cells. Engagement of PD-1 with its ligand leads to the exhausted T cells and impaired antitumor immunity. To date, PD-1 expression and its roles have been widely reported in T cells but not well defined in innate immune cells including monocytes. In this study, expression of PD-1 was investigated in human monocytes. Here we observed that among cytokines tested, IFN-γ significantly upregulated the PD-1 expression in both THP-1 cell line and human primary monocytes in a dose- and time-dependent manner. This effect was reduced by PI3K inhibitor, suggesting that the involvement of PI3K/AKT pathway. Furthermore, enrichment of active histone mark H3K4me3 in the Pdcd1 promotor was also observed in IFN-γ-induced THP-1, indicating that epigenetic regulation also plays a role in IFN-γ-induced PD-1 expression. To investigate the biological functions of PD-1, Pdcd1 was deleted in THP-1 cell line by CRISPR/Cas9 system and the phagocytic ability was investigated. The results showed that the PD-1 deficiency in THP-1 cell line resulted in significantly poor phagocytic potency against carboxylated-modified latex beads. Moreover, the PD-1 deficiency or blocking PD-1/PD-L1 interaction by immune checkpoint inhibitor resulted in an impaired induction of IL-4-induced CD163 expression in THP-1 cell line. Taken together, these results highlighted the importance of PD-1 expression in some of key monocyte functions.

In Vitro Assays to Study PD-1 Biology in Human T Cells.

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

PD-1 Dynamically Regulates Inflammation and Development of Brain-Resident Memory CD8 T Cells During Persistent Viral Encephalitis.

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1hi, tissue-resident memory population in the brains (bTRM) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expressed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limited their effector activity. NanoString gene expression analysis showed that neuroinflammation was higher in PD-L1−/− than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bTRM and the fraction of these cells expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1−/− mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection.

Evaluation of PD1/PDL1 Expression and Their Clinicopathologic Association in EBV-associated Lymphoproliferative Disorders in Nonimmunosuppressed Patients.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD)/lymphomas in nonimmunosuppressed patients represent a unique entity and have been proposed to be related to immune senescence. Engagement of programmed cell death 1 (PD1) by its ligand programmed death ligand 1 (PDL1) inhibits T-cell activation, and leads to T-cell exhaustion. In clinical trials, therapeutic antibodies that block the PD1-PDL1 axis have shown promising therapeutic activity in certain types of lymphomas. Although PD1/PDL1 has been extensively studied in variety of lymphomas, there are few reports characterizing their expression in EBV-positive LPD. As these group of patients are presumed to be associated with immunosenescence/immune dysregulation, we hypothesize that the immune checkpoint pathway might be relevant in this entity. We explored the expression of PD1, PDL1 and its clinicopathologic association in 6 patients with a total of 8 independent specimens of EBV-positive LPD/lymphomas. We also applied proximity assay, a novel technique, which can identify intermolecular interaction, to evaluate physical interaction or in situ engagement of PD1 and PDL1. We found that the malignant cells in the EBV-positive LPDs express PDL1. PD1-positive tumor-infiltrating lymphocytes can be seen in these tumors. Proximity assay suggests there is active engagement between PD1 and PDL1. To our knowledge, this is the first report on the utility of proximity assay to test the active engagement between PD1 and PDL1 in lymphomas. As some EBV-positive LPDs were positive for PDL1, this subgroup of EBV-positive LPDs might be suitable for PD1/PDL1 antibody therapies.

A new tool in the cancer immunotherapy toolbox?

Programmed cell death-1 (PD-1), a member of the CD28 superfamily, is primarily expressed on the surface of T cells. The normal function of PD-1 is to dampen the immune activity and promote self-tolerance of activated CD8 + cytotoxic T cells by suppressing T cell inflammatory reactions. Binding of PD-1 with its ligand PD-L1 (or B7-H1) or PD-L2 (or B7-DC) excessively leads to the exhaustion of CD8 + cytotoxic T cells (1). In most of patients with advanced cancers, CD8 + cytotoxic T cells are exhausted owing to the engagement of PD-1 by the aberrantly expressed PD-L1 on tumor cells and/or nurse cells in the tumor microenvironment.

Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy.

Programmed cell death-1 (PD1) has become a significant target for cancer immunotherapy. PD1 and its receptor programmed cell death 1 ligand 1 (PDL1) are key regulatory physiological immune checkpoints that maintain self-tolerance in the organism by regulating the degree of activation of T and B cells amongst other immune cell types. However, cancer cells take advantage of these immunosuppressive regulatory mechanisms to escape T and B cell-mediated immunity. PD1 engagement on T cells by PDL1 on the surface of cancer cells dramatically interferes with T cell activation and the acquisition of effector capacities. Interestingly, PD1-targeted therapies have demonstrated to be highly effective in rescuing T cell anti-tumor effector functions. Amongst these the use of anti-PD1/PDL1 monoclonal antibodies are particularly efficacious in human therapies. Furthermore, clinical findings with PD1/PDL1 blockers over several cancer types demonstrate clinical benefit. Despite the successful results, the molecular mechanisms by which PD1-targeted therapies rescue T cell functions still remain elusive. Therefore, it is a key issue to uncover the molecular pathways by which these therapies exert its function in T cells. A profound knowledge of PDL1/PD1 mechanisms will surely uncover a new array of targets susceptible of therapeutic intervention. Here, we provide an overview of the molecular events underlying PD1-dependent T cell suppression in cancer.

In situ and in silico kinetic analyses of programmed cell death-1 (PD-1) receptor, programmed cell death ligands, and B7-1 protein interaction network

Programmed cell death-1 (PD-1) is an inhibitory receptor with an essential role in maintaining peripheral tolerance and is among the most promising immunotherapeutic targets for treating cancer, autoimmunity, and infectious diseases. A complete understanding of the consequences of PD-1 engagement by its ligands, PD-L1 and PD-L2, and of PD-L1 binding to B7-1 requires quantitative analysis of their interactions at the cell surface. We present here the first complete in situ kinetic analysis of the PD-1/PD-ligands/B7-1 system. Consistent with previous solution measurements, we observed higher in situ affinities for human (h) than murine (m) PD-1 interactions, stronger binding of hPD-1 to hPD-L2 than hPD-L1, and comparable binding of mPD-1 to both ligands. However, in contrast to the relatively weak solution affinities, the in situ affinities of PD-1 are as high as those of the T cell receptor for agonist pMHC and of LFA-1 (lymphocyte function-associated antigen 1) for ICAM-1 (intercellular adhesion molecule 1) but significantly lower than that of the B7-1/CTLA-4 interaction, suggesting a distinct basis for PD-1- versus CTLA-4-mediated inhibition. Notably, the in situ interactions of PD-1 are much stronger than that of B7-1 with PD-L1. Overall, the in situ affinity ranking greatly depends on the on-rate instead of the off-rate. In silico simulations predict that PD-1/PD-L1 interactions dominate at interfaces between activated T cells and mature dendritic cells and that these interactions will be highly sensitive to the dynamics of PD-L1 and PD-L2 expression. Our results provide a kinetic framework for better understanding inhibitory PD-1 activity in health and disease.

Understanding the role of PD-L1/PD1 pathway blockade and autophagy in cancer therapy.

Autophagy is a vital, physiological catabolic process for cell survival by which cells clear damaged organelles and recycle nutrients when homeostasis is maintained. Cancer is a complex disease with uncontrolled growth of cancer cells. Recent studies have suggested the role of autophagy in cancer. A complex relationship exists between autophagy and cancer, since autophagy can contribute to the survival or the destruction of malignant cells depending on the stage of tumor development. In this review, we describe in detail the mechanism underlying autophagy in cancer cells and the intricate involvement of the programmed cell death-1 (PD1) receptor with its ligand (PD-L1). The overexpression of PD-L1 receptors on cancer cell membranes has been observed in several types of cancers. The interaction of PD-L1 on cancer cells with PD1 on the surface of T-cells causes cancer cells to escape from the immune system by preventing the activation of new cytotoxic T-cells in the lymph nodes and subsequent recruitment to the tumor. In addition to its immunopathogenicity, PD1 has been related to autophagy. Reduction of this receptor due to treatment increases autophagy, therefore promoting the recycling of nutrients and clearance of toxic species, consequently promoting cell survival. In addition, PD-L1/PD1 engagement can induce autophagy in nearby T-cells due to a decrease in the amino acids tryptophan and arginine and due to the deprivation of nutrients such as glucose followed by a reduction in glucose metabolism. Resistance to cancer therapies is attributed to various pathways in oncogenesis including, inhibition of tumor suppressors, alteration of the tumor metabolic environment, and upregulation of autophagy. Here we explore the interaction between the immunosuppressive PD-L1/PD1 engagement and autophagy mechanisms, and evaluate the impact of inhibition of these pathways in augmenting antitumor efficacy.

ニボルマブの作用機序，効果，副作用と日本大学医学部附属板橋病院皮膚科での使用経

Malignant melanomas easily metastasize and are often resistant to conventional classical therapies, i.e., surgery, chemotherapy and radiotherapy, in patients with advanced/metastatic malignant melanoma. In recent years, rapid advances have been made in the immunotherapy of malignant melanoma. New medicines, which have been approved by Federal Drug Administration (FDA), have dramatically improved the clinical outcomes for patients with advanced/metastatic melanoma. Nivolumab is an immune checkpoint inhibitor that targets programmed cell death-1 (PD-1) receptors. PD-1 is expressed on many immune cells, including T cells, B cells and natural killer cells. Engagement of PD-1 with its ligands (PD-L1 and PD-L2) induces functional exhaustion of the cytotoxic immune response. Nivolumab inhibits the PD-1 pathway, and thus activates the cytotoxic immune response. Although the immune checkpoint inhibitor tends to take a few months until it exhibits efficacy, once established, the efficacy often lasts for a long time. However, immune checkpoint inhibitors can have many adverse effects, including autoimmune-related inflammation. In particular, relevant severe adverse effects include interstitial pneumonia, colitis, liver dysfunction, thyroid disorders, and infusion reaction. Other affected organs include the skin, eyes, kidneys and nerves. Furthermore, several cases of fulminant type 1 diabetes mellitus have been reported in 2015 and 2016. Because we cannot predict what kinds of adverse effects will occur or when they will occur, we must observe patients carefully in order to detect any adverse events early on, and initiate appropriate treatments. The development of a number of new therapies will provide benefits for patients with malignant melanoma. Dermatologists must use these new drugs appropriately after determining the correct diagnostic information and providing supporting evidence.

A novel method for identifying downstream signals in tumor-reactive T cells following PD-1 engagement and monitoring endogenous tumor immunity and immunotherapy.

3049 Background: Therapies targeting programmed cell death 1 (PD-1) or its ligand B7-H1 (aka PD-L1) have yielded promising therapeutic effects in various solid and hematologic malignancies. A reliable method to determine whether PD-1 on T cells has engaged its ligand B7-H1 on tumor cells is highly desirable as it may have significance as a predictive marker for PD-1 targeted therapy. Methods: Endogenous tumor-reactive CD8+ T cells were identified by their high expression of PD-1 and CD11a CD8+ T cells and their expression of Bim, a pro-apoptotic molecule, in the peripheral blood of 26 patients with metastatic melanoma and 11 patients with metastatic prostate cancer or healthy donors using flow cytometry. Soluble PD-L1 in patients’ sera was quantified using ELISA. Results: The target-cell engaged CD8+ T cell population was significantly higher in patients with melanoma and prostate cancer compared to healthy donors, as assessed by CD11a and PD-1 expression. Bim expression was higher in CD8+ T cells in both cancer patients than healthy controls (p<0.01). In patients with advanced melanoma, Bim was significantly higher in the PD-1(+) target cell-engaged CD8+ T cells than the PD-1(-) CD-8+ T cells (P<0.01). Bim levels above the base line levels of healthy donors in the CD8+ T cells correlated with decreased survival in patients with melanoma (8.9 months v. 13.8 months, p<0.05). Our data suggest that the Bim level is dependent on PD-1 expression, and that higher Bim expression may be indicative of PD-1(+) CD8+ T cells which have engaged B7-H1 (PD-L1), either soluble (peripheral blood) or membrane bound (tumor cells), and are susceptible to apoptotic death. Conclusions: PD-1+ CD11a high CD8+ T cells identify a unique population of effector cells responsive to target tumor cells in cancer patients. Elevated Bim expression in CD8+ T cells may indicate the active immune inhibiting effects of PD-1/B7-H1 signaling on endogenous antitumor T cell responses. Bim expression can serve as a signaling biomarker for PD-1 function and may be useful in selecting patients who may benefit from checkpoint blockade therapy.

Mechanisms of costimulation

The immune system has the remarkable ability to defend against a diversity of microbial pathogens, yet not respond to self. The discovery of the T-cell antigen receptor (TCR) provided an explanation for the specificity and diversity of T-cell responses. Yet, functional studies of Lafferty and colleagues (1) suggested that antigen alone was not sufficient to drive the activation of naive T cells and led to the two signal concept of T-cell activation. According to this model, productive T-cell activation requires a first signal provided by the interaction of antigenic peptide/major histocompatibility complex (MHC) with the TCR and a second, antigen-independent co-signal, the ‘costimulatory signal’. In addition, seminal studies of Jenkins and Schwartz (2, 3) demonstrated that TCR-mediated activation of T cells in the absence of costimulation resulted in antigen-specific unresponsiveness (termed T-cell anergy), rendering the T cells unable to respond to subsequent exposure to antigen. Thus, costimulation was postulated to have a pivotal role in determining whether the outcome of T-cell encounter with antigen would be activation or anergy. The critical role of costimulation in regulating the immune response has given impetus to the study of costimulation and resulted in incredible growth of this field, since an understanding of costimulation is of fundamental and therapeutic interest. Early studies defined the function of the CD28 receptor as a stimulatory receptor for the activation of naive T cells (2), and identified the B7 family members, B7-1 (CD80) and B7-2 (CD86) as its ligands (5–7). The interaction between CD28 and B7-1/B7-2 fulfilled many of the requirements for the costimulatory signal postulated by Lafferty, Schwarz, and colleagues (1). The CD28 homolog cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (3) was found to be a higher affinity binding partner for B7-1 and B7-2, as compared to CD28 (4). It was presumed that CTLA-4 would also be a stimulatory receptor; however, the dramatic fatal inflammatory phenotype of CTLA-4 deficient (−/−) mice revealed the critical inhibitory function of CTLA-4 (10, 11). Furthermore, the phenotype of CTLA-4−/− mice demonstrated that T-cell costimulatory receptors could deliver inhibitory, as well as stimulatory, second signals for T-cell responses, and provided the first indication that inhibitory second signals (co-inhibitory) could regulate T-cell tolerance. While the B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized T-cell costimulatory pathway, there are now many additional costimulatory pathways. These pathways fall into two major families: the immunoglobulin (Ig) superfamily, which includes the B7/CD28, CD2, and T-cell Ig and mucin domain (TIM) families, and the tumor necrosis family (TNF)/TNF receptor (TNFR) family. Reviews in this issue of Immunol Rev discuss the functions of pathways within all of these families. These articles not only review the current understanding of costimulation on the fundamental level, but also the therapeutic opportunities and challenges of targeting costimulatory pathways for treating autoimmunity, graft rejection, cancer and infectious diseases. In this introduction, I provide perspectives on key functions of costimulatory pathways, highlighting recent advances, and discuss how this progress is changing our view of the functions of T-cell costimulation.