CD74 is a regulator of hematopoietic stem cell maintenance.

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

SLAMF9 regulates pDC homeostasis and function in health and disease.

SLAMF9 belongs to the conserved lymphocytic activation molecule family (SLAMF). Unlike other SLAMs, which have been extensively studied, the role of SLAMF9 in the immune system remained mostly unexplored. By generating CRISPR/Cas9 SLAMF9 knockout mice, we analyzed the role of this receptor in plasmacytoid dendritic cells (pDCs), which preferentially express the SLAMF9 transcript and protein. These cells display a unique capacity to produce type I IFN and bridge between innate and adaptive immune response. Analysis of pDCs in SLAMF9-/- mice revealed an increase of immature pDCs in the bone marrow and enhanced accumulation of pDCs in the lymph nodes. In the periphery, SLAMF9 deficiency resulted in lower levels of the transcription factor SpiB, elevation of pDC survival, and attenuated IFN-α and TNF-α production. To define the role of SLAMF9 during inflammation, pDCs lacking SLAMF9 were followed during induced experimental autoimmune encephalomyelitis. SLAMF9-/- mice demonstrated attenuated disease and delayed onset, accompanied by a prominent increase of immature pDCs in the lymph node, with a reduced costimulatory potential and enhanced infiltration of pDCs into the central nervous system. These results suggest the crucial role of SLAMF9 in pDC differentiation, homeostasis, and function in the steady state and during experimental autoimmune encephalomyelitis.

CD74 is a novel transcription regulator.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.

SLAMF receptors on normal and malignant B cells.

The Signaling Lymphocyte Activation Molecule family (SLAMF) is a collection of nine surface receptors expressed mainly on hematopoietic cells, and was found to modulate the behavior of immune cells. SLAMF receptors are expressed on B cells in health and disease. Each SLAM receptor has a unique differential expression pattern during the development and activation of B cells. Furthermore, recent findings have revealed a principal role for this family of receptors in B cell malignancies, emphasizing their importance in the control of malignant cell survival, cell to cell communication within the tumor microenvironment, retention in the supporting niches and regulation of T cell anti-tumor response. This review summarizes the latest studies regarding SLAMF expression and behavior in B cells and in B cell pathologies, and discusses the therapeutic potential of these receptors.

Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects.

Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.

The cloning and inducible expression of the rainbow trout ERp57 gene.

ERp57 is a member of a protein disulfide isomerase family and is a chaperone responsible for the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum and in the assembly of the major histocompatibility complex class I in the endogenous pathway of antigen presentation. This study reports the identification of a full length ERp57 cDNA in rainbow trout that encodes a putative 477aa mature protein with an additional signal sequence of 16aa. The trout protein shared 75% identity with the human homolog, but interestingly did not include either a C terminal endoplasmic reticulum retention signal, Q/KEDL in humans, or a nuclear localization signal which is highly conserved in mammals. Amino acid sequence alignment revealed conservation of four classical domains in trout ERp57 and two conserved active CXXC redox motifs. Trout ERp57 protein was identified as a single band around 57 kDa. Southern blotting analysis revealed that there two copies of the ERp57 gene in the trout genome and northern blotting showed a wide tissue distribution of gene expression in various tissues with the highest expression in liver and egg. This study showed for the first time in teleost that ERp57 transcript is upregulated in response to immune stimuli such as double stranded RNA or phytohemagglutinin. Furthermore, upon treatment with ER stress inducer A23187, trout ERp57 protein expression levels were increased both in peripheral blood leukocytes and the RTS11 macrophage like cell line after 6 and 8 h respectively. These findings suggest a possible conserved function for trout ERp57 in the ER and during the activation of the immune response.

Bone marrow dendritic cells support the survival of chronic lymphocytic leukemia cells in a CD84 dependent manner.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes. The microenvironment of the CLL cells is a vital element in the regulation of the survival of these malignant cells. CLL cell longevity is dependent on external signals, originating from cells in their microenvironment including secreted and surface-bound factors. Dendritic cells (DCs) play an important part in tumor microenvironment, but their role in the CLL bone marrow (BM) niche has not been studied. We show here that CLL cells induce accumulation of bone marrow dendritic cells (BMDCs). Depletion of this population attenuates disease expansion. Our results show that the support of the microenvironment is partly dependent on CD84, a cell surface molecule belonging to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors. Our results suggest a novel therapeutic strategy whereby eliminating BMDCs or blocking the CD84 expressed on these cells may reduce the tumor load.

Tapasin's protein interactions in the rainbow trout peptide-loading complex.

Major histocompatibility complex (MHC) class I receptors play a key role in the immune system by presenting non-self peptides to T cell lymphocytes. In humans, the assembly of the MHC class I with a peptide is mediated by machinery in the endoplasmic reticulum referred as the peptide loading complex (PLC). Although, the identity of the PLC has been widely explored in humans, this complex has not been characterized in fish. Co-immunoprecipitation and mass spectrometry analysis revealed that the protein-protein interactions which exist in the human PLC are conserved in the monocyte/macrophage rainbow trout cell line (RTS11), in particular the interaction of tapasin with the transporter associated with antigen processing (TAP), MHC class I and ERp57. Importantly, a 20 kDa tapasin version that contains an intact C and N terminal domains was found to associate with ERp57 and form a 75 kDa heterodimer. These results suggest a possible novel alternative spliced version of tapasin may regulate the formation of the peptide-loading complex in teleosts.

CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.

Expression of tapasin in rainbow trout tissues and cell lines and up regulation in a monocyte/macrophage cell line (RTS11) by a viral mimic and viral infection.

Tapasin is a transmembrane glycoprotein that acts as a bridge between the transporter associated with antigen processing and the MHC class I receptor in mammals. Through the development of antibody against trout tapasin, this report demonstrates the detection of trout tapasin as a N-glycosylated 48 kDa protein. Tissue and cell line distribution revealed that tapasin protein is expressed mainly in immune system organs and in rainbow trout epithelial cell lines from gill (RTgill-W1), liver (RTL-W1), and intestine (RTgutGC). An additional 20 kDa band was observed in tissues and cell lines, and appeared to be most prominent in RTgutGC but was absent in peripheral blood leukocytes. Tapasin 48 kDa protein was most strongly expressed in RTS11 (monocyte/macrophage cell line) and its regulation following dsRNA stimulation was explored. Upon poly I:C treatment and Chum Salmon Reovirus (CSV) infection, tapasin protein expression was upregulated up to 3.5 fold and 3 fold respectively, in parallel with increased expression of the glycosylated MH class I heavy chain, whereas the expression of the 20 kDa form remained unchanged. Overall this work demonstrates the induction of tapasin protein by dsRNA stimulation, which implies its possible conserved regulation during viral infection in teleost cells.

Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins.

Calnexin (IP90/P88) is an integral membrane protein of the endoplasmic reticulum that binds newly synthesized N-linked glycoproteins during their folding in the ER including MHC class I molecule. This manuscript reports the identification of two unique cDNA clones of calnexin in rainbow trout. Both encode putative mature proteins of 579 and 592 aa respectively in addition to a 24 aa signal peptide. Sequence analysis revealed that only one of the two cDNA clones encodes a putative ER retention signal, K/QEDDL, followed by a serine phosphorylation site conserved with mammalian homologs. Amino acid sequence alignment illustrated conservation of the calnexin luminal domain, which consists of a globular and a P domain, in both copies. Southern blotting revealed that there are at least two copies of the calnexin gene in the trout genome and northern blotting showed a wide tissue distribution of an estimated 3 kbp calnexin transcript with an additional minor transcript of 2.3 kbp expressed only in head kidney, spleen PBLs and strongly in RTS11. Importantly, the smaller transcript was predominantly upregulated in RTS11 after a 24h treatment with the calcium ionophore A23187. In western blots, calnexin was detected primarily as a 120 kDa protein and upon A23187 treatment; a 100 kDa band was most prominently expressed. These results suggest that in salmonids there are two differentiated versions of the calnexin gene which encode proteins that may have diverged to perform unique biological functions.

Induction of rainbow trout MH class I and accessory proteins by viral haemorrhagic septicaemia virus.

Major histocompatibility (MH) class I receptors are glycoproteins which play a critical role during responses to intracellular pathogens by presenting endogenous peptides to cytotoxic T cell lymphocytes (CD8+). To date, little is known about MH class I regulation at the protein level during viral infections in fish. In this study, we characterised the MH class I pathway response to polyinosinic-polycytidylic acid (poly I:C) and upon infection with viral haemorrhagic septicemia virus (VHSV) genotype IVa using the rainbow trout monocyte/macrophage cell line RTS11. A 14-day challenge with VHSV IVa at 14°C demonstrated enhanced expression of the class I heavy chain, ß2 microglobulin (ß2M) and tapasin, while the expression of other accessory molecules ERp57 and calreticulin remained unchanged. However, when infection occurred at 2°C no change in expression levels of any of these molecules was observed. ß2M accumulated in the media of RTS11 over time, however the ß2M concentrations were 2 fold higher in cultures infected with VHSV 14 days post infection. Strikingly, when cells were maintained at 2°C the secretion of ß2M was significantly reduced in both infected and non-infected cultures. These results indicate that VHSV infection alters the kinetics of ß2M release as well as the expression of MH class I and suggests that cellular immunity against VHSV can be compromised at low temperatures which may increase host susceptibility to this virus during the winter.