T cell immunity directed against tumor-encoded amino acid substitutions (AAS) occurs in some melanoma patients. This implicates missense mutations (MM) as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as validated targets of anti-tumor immunity is lacking. Moreover, whether vaccination can augment such responses is unknown. Here we show that a dendritic cell vaccine increased naturally occurring and revealed new HLA class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor repertoire in terms of both TCRVβ usage and clonal composition. Our results demonstrate that vaccination directed at tumor AAS broadens the antigenic breadth and clonal diversity of anti-tumor immunity.
Objective. To assess the presence of fibroblast collagenase (MMP-l), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion.Methods. Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis.Results. MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor a stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13.Conclusion. These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.The matrix metalloproteinase (MMP) family of enzymes consists of at least 15 distinct entities, including
Background. Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but doselimiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ-matured, IL-12p70-producing DCs.Methods. 7 HLA-A*0201 + newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ-matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST.Results. 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen-derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine-derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients.Conclusion. These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen-specific CD8 + T cell immunity in humans with cancer.
BACKGROUND/OBJECTIVES: Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. METHODS: Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. RESULTS: Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. CONCLUSIONS: Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche.
SummaryTo better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two-to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes . These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites . Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection . In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack 02 microglobulin association, and display a slower rate of oligosaccharide maturation . In spite of their deficiencies, the non-ligandassociated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lyphocytes .Class I MHC molecules are highly polymorphic 45-kD membrane glycoproteins that associate noncovalently with a-2 microglobulin (02m), 1 a non-MHC-encoded, non-membrane-bound 11-kD polypeptide . Although each MHC haplotype of the mouse contains approximately 40 class I genes, only a few have known functions . For example, the H-2d haplotype, represented in the BALB/c inbred strain, expresses three class I molecules designated Kd, Dd, and Ld that function as classical transplantation antigens. The K, D, and L molecules on virus-infected or allogeneic cells function as recognition structures for CTL . Crystallographic studies revealed that the highly polymorphic ul and cr2 domains of the class I molecule combine in an intricate folding pattern to form a single potential binding site (1-3) . In fact the putative ligand binding site of the crystallized class I molecules was found to contain heterogeneous material estimated to be til-2 kD. In other studies using functional assays, virusspecific CTL were found to recognize a processed virus-derived peptide ligand in the context of a self class I molecule (4) . There are now several examples where the specific virus-derived peptide has been identified for a given CTL clone (e .g., refer-'Abbreviations used in this paper. Ag, antigen ; 02m, /3-2 microglobulin ; BEA, brefeldin A . ences 5, 6). Furthermore, these peptide ligands have been found to be between 5 and 20 amino acids in length . Thus, there is complete concordance between the crystallographic and functional studies. In spite of this knowledge, direct evidence of the binding of peptide to class I molecules has been very difficult to demonstrate using in vitro binding assays analogous to ones previously used to show class II...
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