The Neonatal Innate Immune Response to Sepsis: Checkpoint Proteins as Novel Mediators of This Response and as Possible Therapeutic/Diagnostic Levers.

Sepsis, a dysfunctional immune response to infection leading to life-threatening organ injury, represents a significant global health issue. Neonatal sepsis is disproportionately prevalent and has a cost burden of 2-3 times that of adult patients. Despite this, no widely accepted definition for neonatal sepsis or recommendations for management exist and those created for pediatric patients are significantly limited in their applicability to this unique population. This is in part due to neonates' reliance on an innate immune response (which is developmentally more prominent in the neonate than the immature adaptive immune response) carried out by dysfunctional immune cells, including neutrophils, antigen-presenting cells such as macrophages/monocytes, dendritic cells, etc., natural killer cells, and innate lymphoid regulatory cell sub-sets like iNKT cells, γδ T-cells, etc. Immune checkpoint inhibitors are a family of proteins with primarily suppressive/inhibitory effects on immune and tumor cells and allow for the maintenance of self-tolerance. During sepsis, these proteins are often upregulated and are thought to contribute to the long-term immunosuppression seen in adult patients. Several drugs targeting checkpoint inhibitors, including PD-1 and PD-L1, have been developed and approved for the treatment of various cancers, but no such therapeutics have been approved for the management of sepsis. In this review, we will comparatively discuss the role of several checkpoint inhibitor proteins, including PD-1, PD-L1, VISTA, and HVEM, in the immune response to sepsis in both adults and neonates, as well as posit how they may uniquely propagate their actions through the neonatal innate immune response. We will also consider the possibility of leveraging these proteins in the clinical setting as potential therapeutics/diagnostics that might aid in mitigating neonatal septic morbidity/mortality.

PD-1 cooperates with AIRE-mediated tolerance to prevent lethal autoimmune disease.

Immunological tolerance is established and maintained by a diverse array of safeguards that work together to protect against autoimmunity. Despite the identification of numerous tolerogenic processes, the basis for cooperation among them remains poorly understood. We sought to identify synergy among several well-defined tolerance mediators that alone provide protection only from mild autoimmune symptoms in C57BL/6 mice: BIM, AIRE, CBL-B, and PD-1. Survey of a range of compound mutant mice revealed that the combined loss of the autoimmune regulator, AIRE, with PD-1 unleashed a spontaneous, lethal autoimmune disease. Pdcd1−/−Aire−/− mice succumbed to cachexia before adulthood, with near-complete destruction of the exocrine pancreas. Such fatal autoimmunity was not observed in Pdcd1−/−Bim−/−, Bim−/−Aire−/−, or Cblb−/−Bim−/− mice, suggesting that the cooperation between AIRE-mediated and PD-1­mediated tolerance was particularly potent. Immune profiling revealed largely normal development of FOXP3+ regulatory T (Treg) cells in Pdcd1−/−Aire−/− mice, yet excessive, early activation of effector T cells. Adoptive transfer experiments demonstrated that autoimmune exocrine pancreatitis was driven by conventional CD4+ T cells and could not be prevented by the cotransfer of Treg cells from wild-type mice. The development of autoimmunity in mixed bone marrow chimeras supported these observations, indicating that failure of recessive tolerance was responsible for disease. These findings reveal a potent tolerogenic axis between AIRE and PD-1 that has implications for our understanding of how immune checkpoint blockade might synergize with subclinical defects in central tolerance to elicit autoimmune disease.

Comparative investigation of cell cycle and immunomodulatory genes in mucosal and cutaneous melanomas: Preliminary data suggest a potential promising clinical role for p16 and the PD-1/PD-L1 axis.

Mucosal melanomas arise from the mucosal lining of various organs. Their etiology is currently unknown and there are no tissue-based methods to differentiate it from cutaneous melanomas. Furthermore, prognostic and predictive markers (e.g. for immune checkpoint inhibition) are lacking. In this study, we aimed to assess the protein expression levels of cell cycle-associated proteins and immune checkpoint markers in a cohort of mucosal melanomas in comparison to cutaneous melanomas and evaluated the effect of potential regulatory mechanisms. We performed immunohistochemistry, DNA methylation analysis and copy number profiling of 47 mucosal and 28 cutaneous melanoma samples. Protein expression of CD117, Ki67 and p16 was higher in mucosal melanomas, while BCL2, Cyclin D1, PD-1 and PD-L1 were overexpressed in cutaneous melanomas. CDKN2A deletions were the most prevalent numeric chromosomal alterations in both mucosal and cutaneous melanoma and were associated with decreased p16 expression. KIT was frequently amplified in mucosal melanomas, but not associated with CD117 expression. On the other hand, amplification of CCND1 lead to Cyclin D1 overexpression. In mucosal melanoma patients high PD-1 expression and high PD-L1 promoter methylation levels were associated with improved survival. PD-L1 expression correlated with response to immune checkpoint inhibitor therapy in the combined group of melanoma patients. Mucosal and cutaneous melanomas show different expression levels of cell cycle-associated and immunomodulatory proteins that are partially regulated by DNA methylation and copy number alterations. PD-1 expression and PD-L1 promoter methylation levels might be a prognostic marker for mucosal melanomas.

The diagnostic role of PD-1+ CXCR5+ follicular helper CD8+ T cell in renal allograft dysfunction.

BACKGROUND: The roles of PD-1+ CXCR5+ follicular helper CD8+ T cell were reported in different disease conditions, but their roles in transplantation are unclear. In this study, the association between PD-1+ CXCR5+ follicular helper CD8+ T cell and renal allograft dysfunction in kidney transplant recipients (KTRs) was investigated. METHODS: 82 KTRs were enrolled in this study. 45 KTRs were included in the chronic allograft dysfunction (CAD) group, and 37 KTRs were included in the stable recipients group. Among the CAD group, 12 cases of antibody-mediated rejection (ABMR) and 4 cases of T cell-mediated rejection (TCMR) were diagnosed by biopsy. The percentage of CXCR5+ CD8+ T cells and the co-expression of signal transducers and activators of transcription 4 (STAT4), STAT5, and PD-1 in peripheral blood were determined by flow cytometry. RESULTS: The expression of CXCR5 on CD3+ CD8+ T cells and the percentage of STAT5+ CXCR5+ cells in the CD3+ CD8+ T-cell population were significantly lower in the CAD group (p < 0.05), while the expression of PD-1+ CXCR5+ CD8+ T cells was significantly higher (p < 0.05). Through logistic regression analysis, we concluded that the percentage of PD-1+ CXCR5+ CD8+ T cells was an independent risk factor for renal dysfunction. Grouping by pathological type, PD-1+ CXCR5+ CD8+ T cells showed relatively good diagnostic efficacy for ABMR by ROC analysis. CONCLUSIONS: Our results suggested that PD-1+ CXCR5+ CD8+ T cells were a promising biomarker for distinguishing renal allograft dysfunction and different allograft pathological types. Also, our findings may provide new ways of identifying and treating allograft rejection.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

PD-1 preferentially inhibits the activation of low-affinity T cells.

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

The PD-L1/PD-1 Axis Blocks Neutrophil Cytotoxicity in Cancer.

The PD-L1/PD-1 axis mediates immune tolerance and promotes tumor growth and progression via the inhibition of anti-tumor immunity. Blocking the interaction between PD-L1 and PD-1 was clinically shown to be beneficial in maintaining the anti-tumor functions of the adaptive immune system. Still, the consequences of blocking the PD-L1/PD-1 axis on innate immune responses remain largely unexplored. In this context, neutrophils were shown to consist of distinct subpopulations, which possess either pro- or anti-tumor properties. PD-L1-expressing neutrophils are considered pro-tumor as they are able to suppress cytotoxic T cells and are propagated with disease progression. That said, we found that PD-L1 expression is not limited to tumor promoting neutrophils, but is also evident in anti-tumor neutrophils. We show that neutrophil cytotoxicity is effectively and efficiently blocked by tumor cell-expressed PD-1. Furthermore, the blocking of either neutrophil PD-L1 or tumor cell PD-1 maintains neutrophil cytotoxicity. Importantly, we show that tumor cell PD-1 blocks neutrophil cytotoxicity and promotes tumor growth via a mechanism independent of adaptive immunity. Taken together, these findings highlight the therapeutic potential of enhancing anti-tumor innate immune responses via blocking of the PD-L1/PD-1 axis.

Cellular and molecular regulation of the programmed death-1/programmed death ligand system and its role in multiple sclerosis and other autoimmune diseases.

Programmed Cell Death 1 (PD-1) receptor and its ligands (PD-Ls) are essential to maintain peripheral immune tolerance and to avoid tissue damage. Consequently, altered gene or protein expression of this system of co-inhibitory molecules has been involved in the development of cancer and autoimmunity. Substantial progress has been achieved in the study of the PD-1/PD-Ls system in terms of regulatory mechanisms and therapy. However, the role of the PD-1/PD-Ls pathway in neuroinflammation has been less explored despite being a potential target of treatment for neurodegenerative diseases. Multiple Sclerosis (MS) is the most prevalent, chronic, inflammatory, and autoimmune disease of the central nervous system that leads to demyelination and axonal damage in young adults. Recent studies have highlighted the key role of the PD-1/PD-Ls pathway in inducing a neuroprotective response and restraining T cell activation and neurodegeneration in MS. In this review, we outline the molecular and cellular mechanisms regulating gene expression, protein synthesis and traffic of PD-1/PD-Ls as well as relevant processes that control PD-1/PD-Ls engagement in the immunological synapse between antigen-presenting cells and T cells. Also, we highlight the most recent findings regarding the role of the PD-1/PD-Ls pathway in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), including the contribution of PD-1 expressing follicular helper T (TFH) cells in the pathogenesis of these diseases. In addition, we compare and contrast results found in MS and EAE with evidence reported in other autoimmune diseases and their experimental models, and review PD-1/PD-Ls-targeting therapeutic approaches.

PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment.

The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1-PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale.

PD-L1 signaling on human memory CD4+ T cells induces a regulatory phenotype.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

Low-Dose Decitabine Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PD-1 Methylation in Immune Thrombocytopenia.

Cytotoxic T lymphocytes (CTLs)-mediated platelet destruction plays an important role in the pathogenesis of primary immune thrombocytopenia (ITP). The programmed cell death protein 1 (PD-1) signaling can turn off autoreactive T cells and induce peripheral tolerance. Herein, we found that the expression of PD-1 and its ligand PD-L1 on CD8+ T cells from ITP patients was decreased. Activating PD-1 pathway by PD-L1-Fc fusion protein inhibited CTLs-mediated platelet destruction in ITP in vitro. PD-1 promoter hypermethylation in CD8+ T cells was found in ITP patients, resulting in decreased PD-1 expression. The demethylating agent decitabine at a low dose was proved to restore the methylation level and expression of PD-1 on CD8+ T cells and reduce the cytotoxicity of CTLs of ITP patients. The phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and AKT in CD8+ T cells were significantly downregulated by low-dose decitabine. Furthermore, blocking PD-1 could counteract the effect of low-dose decitabine on CTLs from ITP patients. Therefore, our data suggest that the aberrant PD-1/PD-L1 pathway is involved in the pathophysiology of ITP and enhancing PD-1/PD-L1 signaling is a promising therapeutic approach for ITP management. Our results reveal the immunomodulatory mechanism of low-dose decitabine in ITP by inhibiting CTLs cytotoxicity to autologous platelets through PD-1 pathway.

Quantification of biomarker functionality predicts patient outcomes.

Implementation of a quantitative molecular imaging method (iFRET), which determines receptor-ligand interactions, has led to the finding that patients with a low extent of PD-1/PD-L1 interaction in metastatic NSCLC, and malignant melanoma, display significantly worsened overall survival compared to those with a high level of interaction.

T-cell-based Immunotherapies for Haematological Cancers, Part A: A SWOT Analysis of Immune Checkpoint Inhibitors (ICIs) and Bispecific T-Cell Engagers (BiTEs).

Haematology has been at the vanguard of cancer immunotherapy. Immune checkpoint inhibitors (ICIs), bispecific T-cell engagers (BiTEs), allogeneic haematopoietic stem cell transplantation (allo-HSCT) and donor lymphocyte infusion (DLI), as well as adoptive T-cell therapies outside the setting of allo-HSCT, have been approved for distinct haematologic malignancies producing durable responses in otherwise untreatable patients. Despite recent advances, immunotherapies do not benefit most patients, due to resistance or lack of response, and are only approved in specific settings. Moreover, immunotherapies are expensive and may produce severe immune related adverse reactions. Combination therapy complicates the picture and requires further evaluation. This review considers the current status and future perspectives of ICIs and BiTEs approved for haematological malignancies by analysing their strengths, weaknesses, opportunities and threats (SWOT). The biological rationale for anti-cancer mechanisms, clinical data for specific haematological cancers, efficacy, toxicity, response and resistance profiles, novel strategies to improve these characteristics as well as the potential targets to enhance or expand the application of ICIs and BiTEs are also discussed.

High Levels of Thyroid Hormone Impair Regulatory T Cell Function Via Reduced PD-1 Expression.

CONTEXT: Regulatory T cell (Treg) dysfunction plays an important role in the development and progression of Graves' disease (GD). Programmed cell death 1 (PD-1) prompts FoxP3 in Treg expression and enhances the suppressive activity of Tregs. Whether abnormal expression of PD-1 contributes to the breakdown of Tregs and the role of thyroid hormone in the PD-1 expression of Tregs in GD remain substantially undefined. OBJECTIVE: To evaluate the role of PD-1 in Treg function and triiodothyronine (T3) in PD-1 expression in patients with GD and mice treated with T3. METHODS: We recruited 30 patients with GD and 30 healthy donors. PD-1 expression in Tregs and Treg function were determined. To evaluate the effects of thyroid hormone on PD-1 expression in Tregs, we used T3 for the treatment of human peripheral blood mononuclear cells (PBMCs). We then treated mice with T3 to confirm the effect of thyroid hormone on PD-1 expression in Tregs and Tregs function in vivo. RESULTS: PD-1 expression in Tregs and the suppressive function of Tregs significantly decreased in patients with GD. T3 reduced PD-1 expression in human Tregs in a concentration- and time-dependent manner in vitro. High levels of circulating T3 reduced PD-1 expression in Tregs, impaired Treg function, and disrupted T-helper cell (Th1 and Th2) balance in mice treated with T3. CONCLUSION: Treg dysfunction in GD patients might be due to downregulation of PD-1 expression in Tregs induced by high levels of serum T3.

PD-1/PD-L1 affects Graves progression through lymphocytes on the proliferation, apoptosis and inflammatory cytokine secretion of thyroid follicular epithelial cells.

We aimed to investigate the role of programmed cell death protein 1 (PD-1) and T lymphocytes in the proliferation, apoptosis and secretion of cells from patients and mice with Graves' disease (GD). The levels of serum hormones, related antibodies and inflammatory cytokines in GD patients were determined by electrochemiluminescence immunoassay and ELISA. The percentages of CD4 and CD8 T-lymphocytes and PD-1 expression were examined by flow cytometry. A GD mouse model, a thyroid follicular epithelial cell, and a CD4+PD-1+, CD4+PD-1- and CD8+PD-1+, CD8+PD-1- T lymphocyte co-culture system were constructed. The viability, apoptosis-related markers, serum hormones, related antibodies and inflammatory cytokines in thyroid follicular epithelial cells were determined by CCK-8, Western blot, qTR-PCR, electrochemiluminescence immunoassay and ELISA. Elevated free thyroid hormones (FT3, FT4), thyroid hormone antibodies (TRAb, TPOAb and TGAb), inflammatory cytokines, and inhibited TSH were observed in GD patients. The percentage of CD4+ T cells was increased, while that of CD8+ T cells was reduced in GD patients. PD-1 expression level was lifted in both CD4+ and CD8+ cells from GD patients. In mouse thyroid follicular epithelial cells co-cultured with CD4+PD-1+ and CD8+PD-1+ T lymphocytes, the cell viability, TH and TRAb levels and inflammatory cytokines level were the highest, while the TSH level and apoptosis were the lowest. PD-1 positive T lymphocytes were able to promote viability and inhibit apoptosis of thyroid follicular epithelial cells, which further caused a more accelerated development of GD.

The possible regulatory effect of the PD-1/PD-L1 signaling pathway on Tregs in ovarian cancer.

Aim of this study was to investigate the possible regulatory effect of the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway on Tregs in ovarian cancer. Immunohistochemistry was used to detect the expression of PD-L1 and PD-1 and the presence of FOXP3+ Tregs in ovarian cancer. Then, ovarian cancer HO8910 cells were subjected to transfection with PD-L1 siRNA in vitro. CCK-8, Transwell and wound healing assays were performed to detect the biological behaviors of ovarian cancer cells. Human T-cells isolated from human peripheral blood were cocultured with HO8910 cells, which were divided into the Control, TGF-ß, and TGF-ß+ anti-PD-L1 groups. The proportion of differentiated Tregs was detected by flow cytometry. Mouse models of ovarian cancer were established, and PD-L1 antibody therapy was administered. Tumor growth and Treg recruitment were observed. PD-L1, PD-1 and FOXP3+ Tregs were found in ovarian cancer tissue. Patients with tumors with an advanced stage and low differentiation and lymph node metastasis had significantly higher levels of PD-1, PD-L1 and FOXP3+ Tregs. After transfection with PD-L1 siRNA, HO8910 cells showed a significant reduction in PD-L1 expression, proliferation, migration and invasion. After T-cells were cocultured with ovarian cancer cells, the TGF-ß+ anti-PD-L1 group showed a substantial decline in the differentiation of T-cells into Tregs compared with the TGF-ß group. Moreover, mice in the anti-PD-L1 group had significantly reduced tumor growth rates, Treg proportions in the tumor microenvironment, and FOXP3 expression.

Targeting immune checkpoints in hematological malignancies.

Immune checkpoint blockade (ICB) therapies such as anti-programmed death 1 (PD-1) and anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) have dramatically transformed treatment in solid tumor oncology. While immunotherapeutic approaches such as stem cell transplantation and anti-cancer monoclonal antibodies have made critical contributions to improve outcomes in hematological malignancies, clinical benefits of ICB are observed in only limited tumor types that are particularly characterized by a high infiltration of immune cells. Importantly, even patients that initially respond to ICB are unable to achieve long-term disease control using these therapies. Indeed, primary and acquired resistance mechanisms are differentially orchestrated in hematological malignancies depending on tumor types and/or genotypes, and thus, an in-depth understanding of the disease-specific immune microenvironments will be essential in improving efficacy. In addition to PD-1 and CTLA-4, various T cell immune checkpoint molecules have been characterized that regulate T cell responses in a non-redundant manner. Several lines of evidence suggest that these T cell checkpoint molecules might play unique roles in hematological malignancies, highlighting their potential as therapeutic targets. Targeting innate checkpoint molecules on natural killer cells and/or macrophages has also emerged as a rational approach against tumors that are resistant to T cell-mediated immunity. Given that various monoclonal antibodies against tumor surface proteins have been clinically approved in hematological malignancies, innate checkpoint blockade might play a key role to augment antibody-mediated cellular cytotoxicity and phagocytosis. In this review, we discuss recent advances and emerging roles of immune checkpoint blockade in hematological malignancies.

CTLA-4 immunotherapy exposes differences in immune response along with different tumor progression in colorectal cancer.

Tumor growth is accompanied by a changing tumor microenvironment and mutations that increase the resistance to therapy. Here, we used syngeneic models to evaluate the drug response of tumors of the same type of different sizes. We used the in vivo efficacy and Ki-67 immunohistochemistry (IHC) assay to assess the difference in responses in response to treatment with the same concentration of anti-CTLA-4. Flow cytometry analysis revealed changes in the immune subpopulations changes the spleen, peripheral blood, lymph node, and tumor tissue across different tumor growth phases. For example, naive CD4+T, CD4+TCM, CD8+TEM, T, B, Treg, CD8+TCM exhibited different percentages depending on the specific immune organ. To further expose the changes in the immune microenvironment, the level of expression of PD-1 and CTLA-4 showed statistically significant difference in related subsets for each four immune tissues in different tumor sizes. In addition, the ratios of CD4 + Teff/ CD4 + Treg and CD8 + T/Treg in corresponding immune tissue were also associated with statistically significant differences alongside tumor growth in different animal models. These results reveal the ongoing changes in the immune microenvironment during tumor progression and anti-CTLA-4 antibody immunotherapy effect depends on the expression level of immune factors.

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.

SBRT combined with PD-1/PD-L1 inhibitors in NSCLC treatment: a focus on the mechanisms, advances, and future challenges.

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1), programmed cell death ligand-1 (PD-L1), and others have shown potent clinical efficacy and have revolutionized the treatment protocols of a broad spectrum of tumor types, especially non-small-cell lung cancer (NSCLC). Despite the substantial optimism of treatment with PD-1/PD-L1 inhibitors, there is still a large proportion of patients with advanced NSCLC who are resistant to the inhibitors. Preclinical and clinical trials have demonstrated that radiotherapy can induce a systemic antitumor immune response and have a great potential to sensitize refractory "cold" tumors to immunotherapy. Stereotactic body radiation therapy (SBRT), as a novel radiotherapy modality that delivers higher doses to smaller target lesions, has shown favorable antitumor effects with significantly improved local and distant control as well as better survival benefits in various solid tumors. Notably, research has revealed that SBRT is superior to conventional radiotherapy, possibly because of its more powerful immune activation effects. Thus, PD-1/PD-L1 inhibitors combined with SBRT instead of conventional radiotherapy might be more promising to fight against NSCLC, further achieving more favorable survival outcomes. In this review, we focus on the underlying mechanisms and recent advances of SBRT combined with PD-1/PD-L1 inhibitors with an emphasis on some future challenges and directions that warrant further investigation.