Exploring the radiosynthesis and in vitro characteristics of [68Ga]Ga‐DOTA‐Siglec‐9

Vascular adhesion protein 1 is a leukocyte homing‐associated glycoprotein, which upon inflammation rapidly translocates from intracellular sources to the endothelial cell surface. It has been discovered that the cyclic peptide residues 283–297 of sialic acid‐binding IgG‐like lectin 9 (Siglec‐9) “CARLSLSWRGLTLCPSK” bind to vascular adhesion protein 1 and hence makes the radioactive analogues of this compound ([⁶⁸Ga]Ga‐DOTA‐Siglec‐9) interesting as a noninvasive visualizing marker of inflammation. Three different approaches to the radiosynthesis of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 are presented and compared with previously published methods. A simple, robust radiosynthesis of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 with a yield of 62% (non decay‐corrected) was identified, and it had a radiochemical purity >98% and a specific radioactivity of 35 MBq/nmol. Furthermore, the protein binding and stability of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 were analyzed in vitro in mouse, rat, rabbit, pig, and human plasma and compared with in vivo pig results. The plasma in vitro protein binding of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 was the lowest in the pig followed by rabbit, human, rat, and mouse. It was considerably higher in the in vivo pig experiments. The in vivo stability in pigs was lower than the in vitro stability. Despite considerable species differences, the observed characteristics of [⁶⁸Ga]Ga‐DOTA‐Siglec‐9 are suitable as a positron emission tomography tracer.     

Sialoadhesin – a macrophage‐restricted marker of immunoregulation and inflammation

Sialoadhesin (Sn, also known as Siglec‐1 and CD169) is a macrophage‐restricted cell surface receptor that is conserved across mammals. Sn is a member of the sialic acid‐binding IgG‐like lectin (Siglec) family of proteins characterized by affinity to specifically sialylated ligands, and under normal conditions is expressed on subsets of macrophages in secondary lymphoid tissues, such as lymph node and spleen. However, Sn‐positive macrophages can also be found in a variety of pathological conditions, including (autoimmune) inflammatory infiltrates and tumours. Sn has been shown to contribute to sialylated pathogen uptake, antigen presentation and lymphocyte proliferation, and to influence both immunity and tolerance. This review presents Sn as a macrophage‐specific marker of inflammation and immunoregulation with the potential to becoming an important biomarker for immunologically active macrophages and a target for therapy.     

CD22 and Autoimmune Disease

CD22 is a 140-kDa member of the Siglec family of cell surface proteins that is expressed by most mature B-cell lineages. As a co-receptor of the B-cell receptor (BCR), it is known to contribute to the sensitive control of the B-cell response to antigen. Cross-linking of CD22 and the BCR by antigen triggers the phosphorylation of CD22, which leads to activation of signaling molecules such as phosphatases. Signal transduction pathways involving CD22 have been explored in a number of mouse models, some of which have provided evidence that in the absence of functional CD22, B cells have a “hyperactivated” phenotype, and suggest that loss of CD22 function could contribute to the pathogenesis of autoimmune diseases. Modulating CD22 activity has therefore been suggested as a possible therapeutic approach to such diseases. For example, the novel CD22-targeting monoclonal antibody epratuzumab is currently under investigation as a treatment for the connective tissue disorder systemic lupus erythematosus (SLE).     

SIGLEC16 encodes a DAP12-associated receptor expressed in macrophages that evolved from its inhibitory counterpart SIGLEC11 and has functional and non-functional alleles in humans

Sialic acid binding immunoglobulin-like lectins (Siglec) are important components of immune recognition. The organization of Siglec genes in different species is consistent with rapid selection imposed by pathogens. We studied SIGLEC11 genes in human, rodent, dog, cow and non-human primates. The lineages of SIGLEC11 genes in these species have undergone dynamic gene duplication and conversion, forming a potential inhibitory (SIGLEC11)/activating (SIGLEC16) receptor pair in chimpanzee and humans. A cDNA encoding human Siglec-16, currently classed as a pseudogene in the databases (SIGLECP16), is expressed in various cell lines and tissues. A polymorphism screen for the two alleles (wild type and four-base pair deletion, 4bpDelta) of SIGLEC16 found their frequencies to be 50% amongst the UK population. A search for donor sequences for SIGLEC16 revealed a subfamily of activating Siglec with charged transmembrane domains predicted to associate with ITAM-encoding adaptor proteins. In support of this, Siglec-16 was expressed at the cell surface in the presence of DAP12, but not the FcRgamma chain. Using antisera specific to the cytoplasmic tail of Siglec-16, we identified Siglec-16 expression in CD14(+) tissue macrophages and in normal human brain, cancerous oesophagus and lung. This is the first activating human Siglec receptor found to have functional and non-functional alleles within the population.     

CD22 serves as a receptor for soluble IgM

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB).     

Transitional and marginal zone B cells have a high proportion of unmasked CD22: implications for BCR signaling

CD22, a B cell-specific member of the Siglec family, is an important inhibitor of B cell signaling. The first Ig-like domain of CD22 specifically binds to alpha2,6-linked sialic acids. Through these interactions CD22 can mediate adhesion to other cells in trans, but can also bind endogenous ligands on the B cell surface in cis. Cis binding of CD22 to sialylated ligands enhances the efficiency of inhibition and thereby reduces the BCR signaling strength. In this study we used a newly developed oligomeric streptavidin-based sialylated probe as an artificial CD22 ligand. We found that CD22 is bound to ligands in cis on most B cells. However, there is a proportion of B cells with unbound (unmasked) CD22. The subpopulation with unmasked CD22 is 2-fold increased in transitional and marginal zone B cells in the spleen and on B1 cells in the peritoneum, when compared to mature B cells. Also, B cells with unmasked CD22 have an activated phenotype. Unmasking of CD22 could be functionally involved in lowering the signaling threshold on developmental checkpoints such as transitional B cells and during B cell activation or could be a consequence of such activation processes.     

固有免疫细胞上Siglec在肿瘤中作用的研究进展与展望

免疫治疗给恶性肿瘤患者带来了新的希望。然而，常见的免疫治疗靶点，例如细胞毒性T淋巴细胞相关抗原4（cytotoxic T lymphocyte-associated antigen-4，CTLA-4）、程序性死亡[蛋白]配体-1（programmed death ligand-1，PD-L1）在患者中阳性表达率低，只有少部分患者能从免疫治疗中获益，因此寻找新的免疫治疗靶点对于提高免疫治疗的响应率和效果极其重要。近期的研究提示唾液酸结合免疫球蛋白型凝集素（sialic acid-binding immunoglobulin-type lectin，Siglec）家族在巨噬细胞、自然杀伤（natural killer，NK）细胞和树突状细胞（dendritic cell，DC）等固有免疫细胞上表达丰富，并与肿瘤的发生、发展存在许多联系，有可能成为免疫治疗的下一个新靶点。在巨噬细胞上不同的Siglecs发挥不同的作用，Siglec-1增强巨噬细胞的抗原呈递作用，从而增强CD8⁺T细胞的杀伤作用；Siglec-7和Siglec-9诱导肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）向免疫抑制性TAM分化从而影响免疫微环境；Siglec-10与CD24相互作用保护肿瘤细胞免受巨噬细胞的攻击，Siglec-15也表现出类似PD-L1的相关作用，这些研究提示其有可能是全新的免疫检查点。NK细胞上高表达Siglec-7和Siglec-9，通过消除NK细胞表面的顺式相互作用，这些Siglec展现出免疫抑制作用。肿瘤表面高表达的唾液酸糖蛋白与NK细胞表面的Siglec相结合从而抑制NK细胞的杀伤作用，通过消除肿瘤表面的唾液酸可以增强NK细胞的杀伤力。有方案设想通过构建高亲合顺式配体来增强NK细胞杀伤力，但发现顺式配体的浓度会对NK细胞的杀伤力造成截然不同的影响，提示唾液酸存在某种动态平衡从而影响免疫反应。DC表面的Siglec-3和Siglec-9会与肿瘤黏蛋白相结合，诱导DC凋亡和减少免疫相关分子的产生进而造成免疫逃逸。在小鼠实验中证实相关Siglec影响DC的抗原呈递和免疫相关因子的表达。本综述总结了近期在固有免疫系统中，Siglec与肿瘤之间的相互作用以及Siglec影响肿瘤发生、发展的相关研究进展，讨论该家族成员作为免疫治疗新靶点的可能。     

From the Cover: Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline

The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer’s dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer’s disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin.Natural selection operates on differential survival and reproductive success. Accordingly, most vertebrates lose fecundity as they age and die soon after their reproductive periods end. Humans, orcas, and pilot whales are the only vertebrate species known to have prolonged postreproductive lifespans (1, 2). In orcas, older members of the group influence the reproductive success and survival of subsequent generations, implying that activities of postreproductive individuals increase their inclusive fitness (3). Similarly, older humans communicate cultural and ecological information and often make influential decisions within groups and wider social networks. These contributions require maintenance of cognitive capacity (4–6). Dementia (defined as a decline in memory or other thinking skills severe enough to reduce a person''s ability to perform everyday activities) negates the informational value of postreproductive individuals, clouds critical decision-making by elders, sometimes results in disruptive behavior, and eventually diverts group resources toward the care of affected individuals. Behaviors and social structures that enhance the effectiveness of postreproductive individuals might therefore be expected to select specifically for the retention of cognitive capacity. In contrast, ancestral alleles that directly enhance survival and reproductive success during fertile years are favored, even if they limit retention of cognitive capacity in postreproductive age and predispose individuals to dementia. Extended postreproductive lifespans may thus evolve as a balance between these two opposing selective forces.The brains of humans and other related primates differ not only in size and capability but also in their susceptibility to particular diseases. For instance, late-onset Alzheimer’s disease (LOAD) is considered to be unique to humans (7). In fact, whereas humans accumulate amyloid beta deposits and neurofibrillary tangles composed of hyperphosphorylated tau protein after age 40 y, postmortem brain samples from age-matched chimpanzees and other great apes do not show the complete pathology of LOAD (8–10). Human-unique neurodegenerative diseases could be byproducts of major differences in brain development that evolved along the human lineage. If so, derived protective alleles may be compensatory and restore functions that were altered as a consequence of human-specific brain evolution.Genome-wide studies have associated the rs3865444 SNP in the promoter region of the CD33 gene with LOAD susceptibility (11–13). CD33 is the canonical member of the family of CD33-related SIGLEC genes, which have undergone multiple unique genetic and expression changes in the human lineage (14). CD33-related Siglecs are predominantly expressed on cells of the innate immune system and modulate cellular reactivity upon recognition of cell surface sialic acids that serve as “self-associated molecular patterns” (15–17). CD33 encodes a type 1 transmembrane protein with two Ig-like extracellular domains, a single transmembrane span, and two intracellular inhibitory motifs (18). Engagement of sialic acid-containing ligands by the outermost V-set Ig-like domain of CD33 results in the phosphorylation of cytosolic domains, which leads to inhibition of proinflammatory cascades in monocytes and macrophages (19, 20). Human CD33 produces two alternative splice forms: a full-length CD33M and a minor form CD33m that lacks the exon 2 encoding the V-set domain (Fig. 1A) (21, 22). Thus, CD33M and CD33m differ both in molecular weight and in their ability to bind sialic acids.Open in a separate windowFig. 1.The LOAD-protective allele rs3865444A is unique to humans. (A) Schematic of the human CD33 genomic region, messenger RNAs generated by alternative splicing, and CD33M and CD33m proteins. Exon 2 encoding the V-set domain is indicated in red. (B) Alignments of the sequences surrounding the rs3865444 and rs12459419 SNPs reveal that the rs3865444C and rs12459419C alleles are ancestral. Data for humans are from the 1000 Genomes database. Neanderthal and Denisovan alleles are from the respective genome assemblies, accessed using the University of California, Santa Cruz, genome browser. Primate data are from ref. 28. The number of alleles available for each species is indicated.A series of recent studies has provided a molecular link between the rs3865444 SNP and LOAD. First, CD33 was detected in brain microglia, phagocytic immune cells that respond to cellular damage in the central nervous system (23–25). Then, the rs3865444 SNP associated with LOAD was found to be in complete linkage disequilibrium with rs12459419, a SNP located at the third base pair of CD33 exon 2 (25, 26). Notably, the rs12459419 SNP was shown to influence the efficiency of exon 2 splicing. Humans homozygous for the LOAD-risk allele marked by rs3865444C have greater cell surface expression of CD33M compared with homozygotes with the LOAD-protective allele rs3865444A (25). Thus, both rs3865444 and rs12459419 polymorphisms predict the ratio of the two CD33 isoforms. Existing studies suggest that CD33M suppresses microglial uptake of amyloid beta peptide, which otherwise accumulates in the central nervous system and contributes to LOAD (27).Here, we compare the regulation of CD33 in humans and chimpanzees and assess the splicing and CD33 expression of alleles that predispose and protect against LOAD. We ask whether the protective allele of CD33 is derived and human-unique and survey alleles of other human genes that are also reported to protect against cognitive decline.     

The identification and developmental requirements of colonic CD169+ macrophages

CD169‐positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169‐positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115⁺ F4/80^lo CD11c^lo macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll‐like receptor signalling in mucosal homeostasis, the presence of CD169⁺ macrophages was not affected in mice that were deficient in MyD88‐mediated Toll‐like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169⁺ macrophages was dependent on lymphotoxin α, colonic CD169⁺ macrophages were present in normal numbers in lymphotoxin α‐deficient mice. In contrast, reduced numbers of CD169⁺ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169⁺ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169⁺ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.