We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 E1-, E2b--E6/E7) combined with ...programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 E1-, E2b--E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 E1-, E2b--E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 E1-, E2b--E6/E7 treated mice revealed elevated CD8(+) tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1(+) TILs. When Ad5 E1-, E2b--E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8(+) TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1(+) TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.
Objectives
Three leucoreduction filters were evaluated – when used alone or combined with centrifuge leucoreduction (C‐LR) – to prevent alloimmune platelet refractoriness in a dog platelet ...transfusion model.
Materials and Methods
Donor platelet‐rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F‐LR) or F‐LR/C‐LR, 51Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non‐leucoreduced (non‐LR) platelets to determine whether donor‐specific tolerance had been induced.
Results
Acceptance of F‐LR PRP transfusions ranged from 29% to 66%. F‐LR/C‐LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F‐LR/C‐LR transfusions was 83%. Tolerance to subsequent non‐LR transfusions occurred in 45% of the F‐LR‐/C‐LR‐accepting recipients unrelated to DR‐B compatibility between donors and recipients (P = 0·18).
Conclusion
In a dog platelet transfusion model, acceptance of donor platelets required combining F‐LR with C‐LR as apparently each process removes different immunizing WBCs.
SUMMARY
One hundrcd and fifteen overlapping synthetic peptides spanning the entire sequence of the isoallergen clonelA of Lol p I from rye grass Lolium perenne were synthesized by the multi‐pin ...technique. The peptides were overlapping 12mers, offset by two residues and overlapping by 10 residues. Sets of six adjacent overlapping peptides (except pool‐l,15,20) were pooled and were used in vitro and in vivo to map the T cell epilopes on Lol p I. Six atopics who were skin test and RAST positive to rye grass showed T cell responses to L. perenne extract (LPE) and its major fraction (Lol p I). Five out of six showed T cell responses in vitro to peptide pool‐17, while five non‐atopics did not respond to any of the peptide pools. By testing the individual peptides of pool‐17, we have located the T cell epitope on Lol p I. Interestingly, when we tested pool‐17 and its single peptides in vivo by intradermal skin testing we found in one patient a typical DTH after 24–48 h to pool‐17 and its peptides (peptides 3 and 4) which exactly matched the in vitro responses. By defining the T cell epitopes in this way a greater understanding of the allergic response to pollen will be obtained, and a more effective and less dangerous vaccine may be possible for treating patients with hay fever.
Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ...ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.
Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on ...hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2(-/-) mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-gamma production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2(-/-) non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell-mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1-PD-L1 interactions in mediating tissue tolerance.
Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of ...tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient ( PD- L1-/-) mice. CD4+and CD8+T cell responses were markedly enhanced in PD- L1-/-mice compared with wild-type mice in vitro and in vivo. PD- L1-/-dendritic cells stimulated greater wild-type CD4+T cell responses than wild-type dendritic cells, and PD- L1-/-CD4+T cells produced more cytokines than wild-type CD4+T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8+T cells responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8+T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4+T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD- L1-/-recipients led to exacerbated disease. Disease was even more severe in PD- L1-/-recipients of PD- L1-/-T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.
Newer members of the B7‐CD28 superfamily include the receptor PD‐1 and its two ligands, PD‐L1 and PD‐L2. Here, we characterize the expression of PD‐1, PD‐L1, and PD‐L2 in tissues of naive miceand in ...target organs from two models of autoimmunity, the pancreas from non‐obese diabetic (NOD) mice and brain from mice with experimental autoimmune encephalomyelitis (EAE). In naive mice, proteiexpression of PD‐1, PD‐L1, and PD‐L2 was detected in the thymus, while PD‐1 and PD‐L1 were detected in the spleen. PD‐L1, but not PD‐L2, was also detected at low levels on cardiac endothelium, pancreatic islets, and syncyciotrophoblasts in the placenta. In pre‐diabetic NOD mice, PD‐1 and PD‐L1 were expressed on infiltrating cells in the pancreatic islets. Furthermore, PD‐L1 was markedly up‐regulated on islet cells. In brains from mice with EAE, PD‐1, PD‐L1, and PD‐L2 were expressed on infiltrating inflammatory cells, and PD‐L1 was up‐regulated on endothelium within EAE brain. The distinct expression patterns of PD‐L1 and PD‐L2 led us to compare their transcriptional regulation in STAT4–/–, STAT6–/–, or NF‐κB p50–/–p65+/– dendritic cells (DC).PD‐L2, but not PD‐L1, expression was dramatically reduced in p50–/–p65+/– DC. Thus, PD‐L1 and PD‐L2 exhibit distinct expression patterns and are differentially regulated on the transcriptional level.
Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, ...which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) BALB/c.129 and Slamf2(-/-) BALB/c.129 do not develop disease. Surprisingly, Slamf1(-/-) B6.129 mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) B6.129 mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.
Abstract
The CD48 molecule belongs to a subfamily of the Ig superfamily that also includes the CD2, CD58, 2B4, Signaling lymphocyte activation molecule (SLAM), and Ly-9 molecules. Receptor-ligand ...interactions are known to occur between several members of this family, and these interactions can strengthen cell to cell adhesion. In mice, the CD48 molecule can bind to CD2. To search for additional ligands of murine CD48, we have generated a chimeric fusion protein consisting of the extracellular domain of murine CD48 and the C region of human IgG1. The results of immunofluorescence and immunoprecipitation experiments in which this reagent was used identify the 2B4 molecule as a novel counter-receptor of CD48.
Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 ...(PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.
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