Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 ± 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 ± 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 ± 16 vs. 146 ± 19 pg/ml; TNFα 3.8 ± 1.0 vs. 6.6 ± 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFα and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 ± 4 vs. 7 ± 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.

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Atherosclerosis remains the leading cause of death in Western countries. Dipeptidyl peptidase (DPP) 4 has emerged as a novel target for the prevention and treatment of atherosclerosis. Family members DPP8 and 9 are abundantly present in macrophage-rich regions of atherosclerotic plaques, and DPP9 inhibition attenuates activation of human M1 macrophages in vitro. Studying this family in a mouse model for atherosclerosis would greatly advance our knowledge regarding their potential as therapeutic targets. We found that DPP4 is downregulated during mouse monocyte-to-macrophage differentiation. DPP8 and 9 expression seems relatively low in mouse monocytes and macrophages. Viability of primary mouse macrophages is unaffected by DPP4 or DPP8/9 inhibition. Importantly, DPP8/9 inhibition attenuates macrophage activation as IL-6 secretion is significantly decreased. Mouse macrophages respond similarly to DPP inhibition, compared to human macrophages. This shows that the mouse could become a valid model species for the study of DPPs as therapeutic targets in atherosclerosis.

Metastases rather than primary cancers determine nowadays the survival of patients. One of the most common primary malignancies is colorectal cancer and this type of tumor is characterized by a high tendency to spread metastases to the lung and liver. CD26/DPP4 is a transmembrane molecule with enzymatic functions which cleaves biologically active peptides. Recently, CD26/DPP4 has become the focus of cancer research and it was shown that CD26/DPP4-positive cancer cells display increased metastatic activity. Here, we tested if the CD26/DPP4-inhibitor Vildagliptin suppresses the development and growth of mouse colorectal lung metastases. This inhibitor of CD26/DPP4 was employed on mouse (C57BL/6) colorectal lung metastases, established by intravenous injection of the syngeneic cell line MC38. For mechanistic analysis, a subcutaneous tumor model was used. The treatment with Vildagliptin significantly suppressed both, the incidence and growth of lung metastases. Autophagy markers (LC3, p62, and ATF4) decreased, apoptosis increased (TUNEL, pH3/Ki-76), and the cell cycle regulator pCDC2 was inhibited. In conclusion, we here showed an anti-tumor effect of Vildagliptin via downregulation of autophagy resulting in increased apoptosis and modulation of the cell cycle. We therefore propose Vildagliptin for the evaluation as a new therapeutic approach for the treatment of colorectal cancer lung metastases.

Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.

Prostate cancer is the second leading cause of cancer-associated death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens which represented the different stages of disease progression and androgen sensitivity, including hormone sensitive and castration resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures representing activation of T cells and M1 macrophages.High fusion burden inversely correlated with immune suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer as it more closely associates with immunogenicity and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.