Merkel cell polyomavirus-specific and CD39+CLA+ CD8 T cells as blood-based predictive biomarkers for PD-1 blockade in Merkel cell carcinoma.

Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39+CLA+ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39+CD103+ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.

Prevention of Collagen-Induced Arthritis by an Anti-Glycan Monoclonal Antibody Reactive with 6-Sulfo Sialyl Lewis x in DBA/1 Mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial tissue inflammation, substantially impacting the quality of life of patients. The interaction between L-selectin and its glycoprotein ligands modified with 6-sulfo sialyl Lewis x (6-sulfo sLex) is known to mediate lymphocyte homing to initiate immune responses. Thus, this process could be a potential therapeutic target for RA. Herein, we explored the preventive effects of an anti-6-sulfo sLex monoclonal antibody (mAb), SF1, on collagen-induced arthritis (CIA) in DBA/1 mice. Mice were administered SF1 from day 21 postfirst immunization with type II collagen (CII), and the effects of SF1 on both clinical and histopathological disease progression evoked by the second immunization were examined. SF1 significantly suppressed clinical features and histological levels associated with arthritis severity. Enzyme-linked immunosorbent assay consistently indicated that SF1 inhibited the production of CII-specific IgG2a. Based on the reverse transcription-quantitative PCR analysis, SF1 suppressed the expression of interferon-γ, a T helper 1 cytokine, as well as that of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, in draining lymph nodes. Collectively, these results indicate that SF1, an anti-sulfated glycan mAb, could be beneficial in preventing CIA in mice and may afford as a novel agent to treat RA.

Tissue-Resident Memory CD8+ T Cells From Skin Differentiate Psoriatic Arthritis From Psoriasis.

OBJECTIVE: To compare immune cell phenotype and function in psoriatic arthritis (PsA) versus psoriasis in order to better understand the pathogenesis of PsA. METHODS: In-depth immunophenotyping of different T cell and dendritic cell subsets was performed in patients with PsA, psoriasis, or axial spondyloarthritis and healthy controls. Subsequently, we analyzed cells from peripheral blood, synovial fluid (SF), and skin biopsy specimens using flow cytometry, along with high-throughput transcriptome analyses and functional assays on the specific cell populations that appeared to differentiate PsA from psoriasis. RESULTS: Compared to healthy controls, the peripheral blood of patients with PsA was characterized by an increase in regulatory CD4+ T cells and interleukin-17A (IL-17A) and IL-22 coproducing CD8+ T cells. One population specifically differentiated PsA from psoriasis: i.e., CD8+CCR10+ T cells were enriched in PsA. CD8+CCR10+ T cells expressed high levels of DNAX accessory molecule 1 and were effector memory cells that coexpressed skin-homing receptors CCR4 and cutaneous lymphocyte antigen. CD8+CCR10+ T cells were detected under inflammatory and homeostatic conditions in skin, but were not enriched in SF. Gene profiling further revealed that CD8+CCR10+ T cells expressed GATA3, FOXP3, and core transcriptional signature of tissue-resident memory T cells, including CD103. Specific genes, including RORC, IFNAR1, and ERAP1, were up-regulated in PsA compared to psoriasis. CD8+CCR10+ T cells were endowed with a Tc2/22-like cytokine profile, lacked cytotoxic potential, and displayed overall regulatory function. CONCLUSION: Tissue-resident memory CD8+ T cells derived from the skin are enhanced in the circulation of patients with PsA compared to patients with psoriasis alone. This may indicate that aberrances in cutaneous tissue homeostasis contribute to arthritis development.

Near infrared photoimmunotherapy targeting the cutaneous lymphocyte antigen for mycosis fungoides.

Background: Mycosis fungoides (MF) is a low-grade T-cell lymphoma with primary cutaneous involvement accounting for more than half of all primary cutaneous lymphomas. The treatment of MF is very challenging due to the limited therapies available. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, the hydrophilic phthalocyanine IRdye 700DX® (IR700), and near infrared light. In this study, we investigated the effect of NIR-PIT on MF targeting the cell-surface antigen cutaneous lymphocyte antigen (CLA)Matherial and methods: MF derived My-La CD4+ cells were incubated with the anti-CLA antibody conjugated to IR700 and then irradiated with a 690 nm near-infrared light. Cell death was evaluated by propidium iodide staining and flow cytometry 24 hours after irradiation.Results: Treatment with anti-CLA or light irradiation exhibited very modest pro-death effects, whereas treatment with the anti-CLA antibody conjugated to IR700 and then irradiation with a 690 nm near-infrared light induced a substantial increase in death in the MF cell line.Conclusions: NIR-PIT targeting CLA to treat MF showed marked antitumour effects. As such, CLA-targeted NIR-PIT could be a promising treatment for MF and, possibly, other cutaneous diseases characterized by CLA+ skin infiltrating T-cells.

Ageing dangerously; homing of senescent CD8 T cells in cutaneous Leishmaniasis.

Both CD8+ T cells and NK cells contribute to the immune response against the protozoan Leishmania parasite. Both are able to generate IFN-γ and both display cytotoxic features. These features may enable them to not only contribute to parasite clearance but also to cause immune-mediated pathology. This pathology is evident, for example, in the Leismania-induced skin lesions found in patients with cutaneous leismaniasis (CL). Here we highlight new data demonstrating that CD8+ T cells and NK cells in CL display a highly cytotoxic senescent phenotype, and that the senescent T cells play a major role in mediating skin pathology. This is the first demonstration that senescent CD8 T cells contribute to immunopathology in vivo.

The OX40 Axis is Associated with Both Systemic and Local Involvement in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic, or chronically relapsing, inflammatory skin disease associated with asthma and allergic rhinitis, and is dominated by Th2 cells. The co-stimulatory T-cell receptor OX40 and its ligand, OX40L, play a central role in the pathogenesis of AD, as their interactions are crucial for the generation of TH2 memory cells. Using enzyme-linked immunoassay (ELISA) and flow cytometry on blood samples from patients with AD and healthy volunteers, this study shows that the serum level of soluble (s) OX40 is decreased in patients with AD, and the expression of OX40 by activated skin-homing CD4+ T cells is increased. This study further shows, using immunofluorescence on skin biopsies, that OX40+ and OX40L+ cells are co-located within the dermis, indicating local activity of OX40/OX40L. Serum levels of sOX40 were associated with atopic diseases and, together, these results support that the OX40 system is important for chronic inflammation in AD skin.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

Targeting the cutaneous lymphocyte antigen (CLA) in inflammatory and neoplastic skin conditions.

Introduction: The cutaneous lymphocyte antigen interacts with E-selectin on endothelial cells and is expressed on 15% of circulating T-cells. Skin-homing T-cells express the cutaneous lymphocyte antigen and play a role in local cutaneous immunity in inflammatory reactions and neoplastic conditions.Areas covered: Lymphocyte extravasation is the essential para-physiological mechanism enabling immune surveillance of tissues for tumors as well as effector cell recruitment to inflammatory sites.The authors focused on skin inflammatory disorders, on cutaneous lymphoproliferative disease, and on other skin malignancies.Expert opinion: Interfering with leukocyte extravasation has been regarded as an attractive strategy in skin disorders, in the past for inflammatory conditions and more recently for cutaneous T-cell lymphomas. Therapeutic blocking of skin-homing interactions has been attempted in psoriasis and atopic dermatitis and has been achieved in the treatment of cutaneous T-cell lymphomas. Cutaneous lymphocyte antigen is a potential molecular target for both systemic and skin-directed therapy for cutaneous T-cell lymphomas.

T lymphocyte phenotype of contact-allergic patients: experience with nickel and p-phenylenediamine.

BACKGROUND: There is considerable interest in understanding the immunological variables that have the greatest influence on the effectiveness of sensitization by contact allergens, particularly in the context of developing new paradigms for risk assessment of novel compounds. OBJECTIVES: To examine the relationship between patch test score for three different contact allergens and the characteristics of T cell responses. METHODS: A total of 192 patients with confirmed nickel, p-phenylenediamine (PPD) or methylisothiazolinone (MI) allergy were recruited from the Contact Dermatitis Investigation Unit at Salford Royal Hospital. Severity of allergy was scored by the use of patch testing, peripheral blood lymphocytes were characterized for T cell phenotype by flow cytometry, and proliferative activity was characterized by radiolabelled thymidine incorporation. Comparisons were drawn with buffy coat samples from healthy volunteers. RESULTS: Patch test positivity for nickel, PPD and MI was associated with changes in the phenotype of peripheral blood T cells: increases in naïve cells, decreases in regulatory T cell frequency and the CD4+ /CD8hi ratio, and increased expression of the skin-homing marker cutaneous lymphocyte antigen (CLA), particularly for those patients with a +++ patch test score. CONCLUSIONS: This increased understanding of the characteristics of the T cell responses to contact allergens may provide parameters with which to better measure health risks associated with skin sensitization.

Circulating pathogen-specific plasmablasts in female patients with upper genital tract infection.

Mucosal antibodies constitute the first line of adaptive immune defence against invaders in the female genital tract (FGT), yet the sequence of events leading to their production is surprisingly poorly characterized. We explored the induction of pathogen-specific antibody-secreting cells (ASC) as a response to an acute infection in the upper FGT. We recruited 12 patients undergoing surgery due to an upper FGT infection (7/12 blood culture positive, 5/12 negative) and six healthy controls. Pathogens were sampled during surgery and PBMC collected in the acute phase of the disease (days 7-10). We searched by ELISPOT circulating pathogen-specific ASC and explored their frequency, immunoglobulin isotype distribution, and expressions of homing receptors (α4ß7, L-selectin, and CLA). All patients had circulating ASC specific to the infective bacteria; the geometric mean was 434 (95%CI 155-1234) ASC (IgA + IgG + IgM)/106 PBMC. IgA ASC predominated in 7/12, IgG ASC in 3/12, and IgM ASC in 2/12 cases. Of all the pathogen-specific ASC, 60% expressed α4ß7, 67% L-selectin, and 9% CLA. This study is the first to show induction of pathogen-specific ASC in the peripheral blood in bacterial infection in the human FGT. Our findings reveal that such FGT-originating pathogen-specific ASC are predominated by IgA ASC and exhibit a homing receptor profile resembling that of ASC in acute urinary tract infection. The data thus suggest a characteristic profile shared by the urogenital tract.

Role of sialyl 6-sulfo Lewis X in antitumor immunity against oral squamous cell carcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) reportedly play a pivotal role in antitumor immunity against oral squamous cell carcinoma (OSCC); however, mechanisms governing TIL recruitment to OSCC tissues remain to be clarified. This study was undertaken to assess a potential association between TILs and high endothelial venule (HEV)-like vessels that express sialyl 6-sulfo Lewis X (LeX). METHODS: OSCC tissue sections (n=41) were subjected to immunohistochemistry for sialyl 6-sulfo LeX and CD34 to allow quantitation of HEV-like vessels. Triple immunohistochemistry for sialyl 6-sulfo LeX and either CD3 and CD20 or CD4 and CD8 was conducted to determine which lymphocyte subset is more closely associated with HEV-like vessels. RESULTS: HEV-like vessels expressing sialyl 6-sulfo LeX were detected in 27 of 41 (65.9%) OSCC cases, and these vessels were more frequently found in early disease (T1/T2 stages) compared with advanced (T3/T4) stages. The number of T cells attached to the inner surface of these HEV-like vessels was significantly greater than that of B cells, while the number of CD4+ helper T cells and CD8+ cytotoxic T cells did not differ significantly. Interestingly, sialyl 6-sulfo LeX was also expressed on the membrane of a fraction of OSCC cells, and CD8+ cytotoxic T cells were almost exclusively found attached to these carcinoma cells. CONCLUSIONS: Sialyl 6-sulfo LeX is displayed not only on HEV-like vessels but also on OSCC cells and may potentially function in antitumor immunity against OSCC.

A potential role for 6-sulfo sialyl Lewis X in metastasis of bladder urothelial carcinoma.

OBJECTIVES: It is widely accepted that sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA, also known as CA 19-9) glycans expressed on cancer cells function in E-selectin-mediated metastasis. Recently, it was reported that 6-sulfo sLeX glycans detected by the MECA-79 monoclonal antibody are expressed in roughly a quarter of gastric adenocarcinoma cases, and that these cases show a poorer prognosis than MECA-79-negative cases do. The present study was undertaken to assess expression of 6-sulfo sLeX glycans in bladder urothelial carcinoma and evaluate potential clinical implications. MATERIALS AND METHODS: We analyzed 78 specimens representing bladder urothelial carcinoma, as well as 4 bladder urothelial carcinoma cell lines, by immunostaining with a battery of anticarbohydrate antibodies. We also undertook an E-selectin·IgM chimera binding assay to assess E-selectin binding to 6-sulfo sLeX expressed on bladder urothelial carcinoma cells and performed reverse transcription polymerase chain reaction and complementary DNA transfection to determine which N-acetylglucosamine-6-O-sulfotransferases function in 6-sulfo sLeX biosynthesis in those cells. Finally, we performed double-immunofluorescence staining for MECA-79 and either CD3 or CD8 to evaluate potential association between high endothelial venule (HEV)-like vessels and tumor-infiltrating T lymphocytes. RESULTS: 6-Sulfo sLeX glycans were expressed in ~20% of bladder urothelial carcinoma cases, particularly in plasmacytoid and micropapillary variants. Positive cells were also bound by E-selectin·IgM chimeras in a calcium-dependent manner. Transcripts encoding N-acetylglucosamine-6-O-sulfotransferase-2 were detected preferentially in HT-1197 bladder urothelial carcinoma cells expressing 6-sulfo sLeX, and transfection of the enzyme complementary DNA into HT-1376 cells, which do not express 6-sulfo sLeX glycans, resulted in cell surface expression of 6-sulfo sLeX. Furthermore, 6-sulfo sLeX glycans were expressed in HEV-like vessels induced in and around lymphocyte aggregates formed near carcinoma cell nests. These HEV-like vessel-associated tumor-infiltrating lymphocytes were composed primarily of CD3(+) T cells, with a fraction of CD8(+) cytotoxic T cells. CONCLUSIONS: Our findings indicate that 6-sulfo sLeX glycans likely play 2 roles in bladder urothelial carcinoma progression: one in lymphocyte recruitment to enhance antitumor immune responses, and the other in E-selectin-mediated tumor cell adhesion to vascular endothelial cells, which is potentially associated with metastasis.

High endothelial venule-like vessels and lymphocyte recruitment in testicular seminoma.

Seminoma, the most common testicular malignant neoplasm, originates from germ cells and is characterized by the presence of numerous tumour-infiltrating lymphocytes (TILs). Although it is widely accepted that TILs function in surveillance and cytotoxicity in various tumours including seminoma, detailed mechanisms governing TIL recruitment are not fully understood. It has been shown that high endothelial venule (HEV)-like vessels are induced in inflamed and neoplastic tissues and contribute to lymphocyte recruitment in a manner similar to the way physiological lymphocyte homing occurs in secondary lymphoid organs. Here, we report that HEV-like vessels, which express MECA-79(+) 6-sulfo sialyl Lewis X-capped structures, are induced in TIL aggregates in seminoma, and that such vessels potentially recruit circulating lymphocytes, as an E-selectin•IgM chimera bound these vessels in a calcium-dependent manner. These HEV-like vessels express intercellular adhesion molecule 1 (ICAM-1), but not vascular cell adhesion molecule 1 (VCAM-1) or mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which likely contributes to lymphocyte firm attachment. We also found that the number of T cells attached to the luminal surface of HEV-like vessels was greater than the number of B cells (p < 0.0001). Interestingly, while CD8(+) cytotoxic T lymphocytes (CTLs) attached to the lumen of HEV-like vessels were scarcely detected, significant numbers of proliferative CTLs were observed outside vessels. These histological findings strongly suggest that TILs, particularly T cells, are recruited to seminoma tissues via HEV-like vessels, and that tumour-infiltrating CTLs then undergo proliferation after transmigration through HEV-like vessels in testicular seminoma.

Significant expression patterns of lewis X-related antigens as a prognostic predictor of low-stage renal cell carcinomas.

AIM: To examine expression profiles of LewisX(Le(x))-related antigens, which have a possible role in hematogenous metastatic spread in various malignancies, in renal cell cancer (RCC) tissue in order to evaluate their prognostic value for patients with low-stage RCC. PATIENTS AND METHODS: Fifty-two patients with pT1-2N0M0 disease were evaluated for their expression patterns of Le(x)-related antigens using FH6 and CSLEX1 antibodies for sLe(x) and AG223 for 6-sulfo Le(x), immunohistochemically. RESULTS: None of the expression levels of Le(x)-related antigens directed by the three antibodies related to the clinical outcomes of patients with low-stage RCC. However, combined use of FH6 and CSLEX1 antibodies enabled prognostic prediction, namely that patients with low intensity with FH6 and high intensity with CSLEX1 had a higher chance of disease progression and poor survival. CONCLUSION: Expression levels Le(x)-related antigens determined by combined use of FH6 and CSLEX1 antibodies could be of value as a prognostic indicator for patients with low-stage RCC.

Roles of gastric mucin-type O-glycans in the pathogenesis of Helicobacter pylori infection.

Helicobacter pylori is a Gram-negative bacterium that infects over 50% of the world's population. This organism causes various gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer. H. pylori possesses lipopolysaccharides that share structural similarity to Lewis blood group antigens in gastric mucosa. Such antigenic mimicry could result in immune tolerance against antigens of this pathogen. On the other hand, H. pylori colonizes gastric mucosa by utilizing adhesins that bind Lewis blood group antigen-related carbohydrates expressed on gastric epithelial cells. After colonization, H. pylori induces acute inflammatory responses mainly by neutrophils. This acute phase is gradually replaced by a chronic inflammatory response. In chronic gastritis, lymphocytes infiltrate the lamina propria, and such infiltration is facilitated by the interaction between L-selectin on lymphocytes and peripheral lymph node addressin (PNAd), which contains 6-sulfo sialyl Lewis X-capped O-glycans, on high endothelial venule (HEV)-like vessels. H. pylori barely colonizes gland mucous cell-derived mucin where alpha1,4-GlcNAc-capped O-glycans exist. In vitro experiments show that alpha1,4-GlcNAc-capped O-glycans function as a natural antibiotic to inhibit H. pylori growth. These findings show that distinct sets of carbohydrates expressed in the stomach are closely associated with pathogenesis and prevention of H. pylori-related diseases, providing therapeutic potentialities based on specific carbohydrate modulation.

Inflammation-induced transcriptional regulation of Golgi transporters required for the synthesis of sulfo sLex glycan epitopes.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

CHST1 and CHST2 sulfotransferase expression by vascular endothelial cells regulates shear-resistant leukocyte rolling via L-selectin.

Sulfation is an essential component of the selectin ligands, potentially mediated by members of a new family of carbohydrate sulfotransferases. In this study, we assessed the contributions of CHST1, CHST2, CHST3, and CHST4 in producing functional L-selectin ligands. Human umbilical vein endothelial cells predominantly expressed CHST1 and CHST2 transcripts with low levels of CHST3 mRNA, while cytokine activation up-regulated CHST2 expression and induced low-level CHST4 expression. A human umbilical vein endothelial cell line, EA.hy926, displayed functional L-selectin ligands that correlated with CHST1 and CHST2 expression in the absence of CHST4 expression. Increased CHST1 or CHST2 expression by a cell line expressing low-level L-selectin ligand activity during in vitro flow chamber assays increased rolling leukocyte numbers, reduced rolling velocities, and enhanced leukocyte rolling under higher shear stresses. These results suggest that CHST1 and CHST2 contribute to the generation of optimal L-selectin ligands in vascular endothelial cells at sites of inflammation.

The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes.

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form "cyclic sialic acid." The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral-alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-gammadelta T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines and O-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called "sialic acid cyclase," and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.

Human N-acetylglucosamine-6-O-sulfotransferase involved in the biosynthesis of 6-sulfo sialyl Lewis X: molecular cloning, chromosomal mapping, and expression in various organs and tumor cells.

N-Acetylglucosamine-6-O-sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of a non-reducing N-acetylglucosamine (GlcNAc) residue. We have cloned human GlcNAc-6-O-sulfotransferase cDNA, based on the sequence homology to cloned cDNA of mouse GlcNAc-6-O-sulfotransferase. The predicted protein sequence of the human enzyme was highly homologous to that of the mouse enzyme; in the 363 amino acid stretch of the catalytic region, the two proteins were nearly identical except for conservative changes in 3 amino acid residues. The expressed enzyme transferred sulfate to GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc. Co-transfection of the enzyme cDNA and fucosyltransferase VII cDNA into COS-7 cells resulted in cell surface expression of 6-sulfo sialyl Lewis X. Fluorescence in situ hybridization analysis revealed that the GlcNAc-6-O-sulfotransferase gene is located on human chromosome 7q31. mRNA of the human enzyme was strongly expressed in the bone marrow, peripheral blood leukocytes, spleen, brain, spinal cord, ovary, and placenta, and moderate levels of expression were observed in many organs including lymph nodes and thymus. In situ hybridization with the mouse system showed that the transcript was localized in specific regions of the brain, i.e. pyramidal cells in the CA3 subregion of the hippocampus, cerebellar nucleus and Purkinje cells. Among human tumor cells, strong expression of the mRNA was found in MOLT-4 and Jarkat lymphoblastic leukemia cells, Raji lymphoma cells, K-562 chronic myelogeneous leukemia cells, U251 glioma cells, and G361 melanoma cells. Carbohydrate structures synthesized by the sulfotransferase may be involved in various aspects of the differentiation and behavior of blood cells, their progenitor cells, and neurons in the central nervous system.