Opposing roles by KRAS and BRAF mutation on immune cell infiltration in colorectal cancer - possible implications for immunotherapy.

BACKGROUND: The immune response has important clinical value in colorectal cancer (CRC) in both prognosis and response to immunotherapy. This study aims to explore tumour immune cell infiltration in relation to clinically well-established molecular markers of CRC. METHODS: Multiplex immunohistochemistry and multispectral imaging was used to evaluate tumour infiltration of cytotoxic T cells (CD8+), Th1 cells (T-bet+), T regulatory cells (FoxP3+), B cells (CD20+), and macrophages (CD68+) in a cohort of 257 CRC patients. RESULTS: We found the expected association between higher immune-cell infiltration and microsatellite instability. Also, whereas BRAF-mutated tumours displayed increased immune-cell infiltration compared to BRAF wild-type tumours, the opposite was seen for KRAS-mutated tumours, differences that were most prominent for cytotoxic T cells and Th1 cells. The opposing relationships of BRAF and KRAS mutations with tumour infiltration of cytotoxic T cells was validated in an independent cohort of 608 CRC patients. A positive prognostic importance of cytotoxic T cells was found in wild-type as well as KRAS and BRAF-mutated CRCs in both cohorts. CONCLUSION: A combined evaluation of MSI status, KRAS and BRAF mutational status, and immune infiltration (cytotoxic T cells) may provide important insights to prognosis and response to immunotherapy in CRC.

Signal regulatory protein alpha is present in several neutrophil granule populations and is rapidly mobilized to the cell surface to negatively fine-tune neutrophil accumulation in inflammation.

Signal regulatory protein alpha (SIRPα) is a cell surface glycoprotein with inhibitory functions, which may regulate neutrophil transmigration. SIRPα is mobilized to the neutrophil surface from specific granules, gelatinase granules, and secretory vesicles following inflammatory activation in vitro and in vivo. The lack of SIRPα signaling and the ability to upregulate SIRPα to the cell surface promote neutrophil accumulation during inflammation in vivo.

Lack of CD47 impairs bone cell differentiation and results in an osteopenic phenotype in vivo due to impaired signal regulatory protein α (SIRPα) signaling.

Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1α,25(OH)2-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47(-/-) mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)(+) osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor κß ligand) was reduced in CD47(-/-) BMC, as compared with CD47(+/+) BMC. The stromal cell phenotype in CD47(-/-) BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and α-1-collagen, and reduced mineral deposition, as compared with that in CD47(+/+) BMC. CD47 is a ligand for SIRPα (signal regulatory protein α), which showed strongly reduced tyrosine phosphorylation in CD47(-/-) bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRPα cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47(-/-) and non-signaling SIRPα mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47(-/-) mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts.

Neutrophil apoptosis is associated with loss of signal regulatory protein alpha (SIRPα) from the cell surface.

Cells of the innate immune system, including monocytes, macrophages, and neutrophils, play a major role in the development of inflammatory diseases. During inflammation, large numbers of neutrophils are recruited from the blood and subsequently undergo apoptosis, which involves changes in the cell surface expression of a number of receptors. Neutrophils express the Ig superfamily member, SIRPα, which is a receptor involved in regulating cell adhesion and migration. As apoptotic neutrophils down-regulate their capacity for adhesion and migration, we here investigated whether neutrophil expression of SIRPα was affected during apoptosis. We found that apoptotic neutrophils lost SIRPα from their cell surface with kinetics similar to the loss of CD16. The majority of neutrophils with reduced SIRPα also expressed PS on their surface, and the loss of the receptor was reduced proportional to the reduction of apoptosis by caspase inhibitors during Fas-induced apoptosis but less so during spontaneous apoptosis. Neutrophil loss of SIRPα or CD16 was inhibited by the protease inhibitor TAPI-2, as well as specific inhibitors of MMP3 or -8, suggesting that proteolytic mechanisms were involved. Finally, SIRPα was also found on smaller membrane vesicles released from the cells during apoptosis. Our data suggest that neutrophils reduce their SIRPα expression during apoptosis, which may be part of the functional down-regulation seen in apoptotic neutrophils.

An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support.

A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition ('click' reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into 'clickable' azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction.

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.

Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration.

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.