Identification of CD98 as a Novel Biomarker for HIV-1 Permissiveness and Latent Infection.

Human immunodeficiency virus type 1 (HIV-1) can integrate viral DNA into host cell chromosomes to establish a long-term stable latent reservoir, which is a major obstacle to cure HIV-1 infection. The characteristics of the HIV-1 latent reservoir have not been fully understood. Here, we identified 126 upregulated plasma membrane proteins in HIV-1 latently infected cells by a label-free liquid chromatography-tandem mass spectrometry analysis. The higher levels of CD98 expression in multiple HIV-1 latently infected cell lines and primary CD4+ T cells compared to uninfected cells were further confirmed by quantitative reverse transcription PCR (RT-qPCR) and flow cytometry analyses. In addition, CD98high CD4+ T cells displayed hyper-permissiveness to HIV-1 infection and possessed distinct immune phenotypic profiles associated with Th17 and peripheral follicular T helper (pTFH) characteristics. Notably, the CD98high resting memory CD4+ T cells harbored significantly higher cell-associated viral RNA and intact provirus than CD98low counterparts in HIV-1-infected individuals receiving combined antiretroviral therapy. Furthermore, CD98high CD4+ T cells exhibited a robust proliferative capacity and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. Our study demonstrates that CD98 can be used as a novel biomarker of HIV-1 latently infected cells to indicate the effect of various strategies to reduce the viral reservoir. IMPORTANCE Identification of cellular biomarkers is the crucial challenge to eradicate the HIV-1 latent reservoir. In our study, we identified CD98 as a novel plasma membrane biomarker for HIV-1 permissiveness and latent infection. Importantly, CD98high CD4+ T cells exhibited a hyper-permissiveness to HIV-1 infection and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. CD98 could be targeted to develop therapeutic strategies to reduce the HIV-1 latent reservoir in further research.

A global live cell barcoding approach for multiplexed mass cytometry profiling of mouse tumors.

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

Rapid phenotypic fingerprinting of cell products by robust measurement of ubiquitous surface markers.

Flow cytometry is one of the most versatile and powerful approach for the quantitative analysis of cell suspensions. With widespread applications in basic and clinical research, its commonest use is in the detection of cell populations labelled against markers specific for a particular phenotype. In this study, we aimed to expand the potential of flow cytometry by describing a method based on robust automated quantification of ubiquitous cell surface markers. We demonstrate that automatic fluorescence standardization combined with whole cell population analysis yields highly reproducible results and can alleviate many of the difficulties associated with conventional analyses. This new generic approach is very flexible for quantifying the uniqueness of entire cell populations regardless of their composition. It provides quantitative phenotypic fingerprints rapidly, does not incorporate subjective factors, is more amenable to standardization, and is easily transferable across a wide diversity of applications, such as quality control for cell manufacture and authentication of cell products.

CD98 is a promising prognostic biomarker in biliary tract cancer.

CD98 has been described to play a crucial role in tumor progression and survival. However, the role of CD98 in biliary tract cancer remains unclear. We found that 36.7% of all patients with biliary tract cancer had a high CD98 expression. Statistical analysis using Spearman's rank correlation showed that CD98 was significantly correlated with L-type amino acid transporter 1 (LAT1, r=0.562, P<0.001), Ki-67 (r=0.230, P=0.006) and CD34 (r=0.290, P=0.005). Multivariate analysis confirmed that a high CD98 expression was an independent prognostic factor for predicting poor outcome. CD98 is closely associated with tumor growth, biological aggressiveness, and survival of patients. With these data we proposed that CD98 expression is necessary for the development and pathogenesis of biliary tract cancer.

Human spermatozoa antigens in unexplained infertility.

OBJECTIVE: To determine and compare the immunolocalization of functionally important antigens in human spermatozoa in an unexplained infertility (UI) group. MATERIALS AND METHODS: In this study, the sperm samples of 20 patients undergoing evaluation belonging to normozoospermic group, whose primary reason of infertility was under investigation for this purpose, were screened. CD46, CD55 and CD52, CD69, CD98, fMLP, HI307, and 80280 were stained on the spermatozoa through indirect immunofluorescence technique. RESULTS: In addition to CD46, CD55, and CD52 antigens, which are known to be localized on human spermatozoa, significant immunolocalization of several novel antigens including: CD52, CD69, CD98, fMLP, HI307, and 80280 were determined on the spermatozoa of the unexplained infertility group, possibly reflecting important roles in the pathophysiology of such unresolved clinical situations. CONCLUSION: Identification and characterization of antigens present on sperm cells is crucial for understanding of the diagnosis and treatment of unexplained infertility. Further studies were conducted to evaluate a possible correlation between the expression of these antigens and clinical outcomes in different well-defined infertility groups.

Evaluation of thoracic tumors with (18)F-FMT and (18)F-FDG PET-CT: a clinicopathological study.

L-[3-(18)F]-alpha-methyltyrosine ((18)F-FMT) is an aminoacid tracer for positron emission tomography (PET). The aim of this study was to determine whether PET-CT with (18)F-FMT provides additional information for the preoperative diagnostic workup as compared with (18)F-FDG PET. PET-CT studies with (18)F-FMT and (18)F-FDG were performed as a part of the preoperative workup in 36 patients with histologically confirmed bronchial carcinoma, 6 patients with benign lesions and a patient with atypical carcinoid. Expression of L-type amino acid transporter 1 (LAT1), CD98, Ki-67 labeling index, VEGF, CD31 and CD34 of the resected tumors were analyzed by immunohistochemical staining, and correlated with the uptake of PET tracers. For the detection of pulmonary malignant tumors, (18)F-FMT PET exhibited a sensitivity of 84% whereas the sensitivity for (18)F-FDG PET was 89% (p = 0.736). (18)F-FMT PET-CT and (18)F-FDG PET-CT agreed with pathological staging in 85 and 68%, respectively (p = 0.151). (18)F-FMT uptake was closely correlated with LAT1, CD98, cell proliferation and angiogenesis. The specificity of (18)F-FMT PET for diagnosing thoracic tumors was higher than that of (18)F-FDG PET. Our results suggest that coexpression of LAT1 and CD98 in addition to cell proliferation and angiogenesis is relavant for the progression and metastasis of lung cancer.

Expression of LAT1 predicts risk of progression of transitional cell carcinoma of the upper urinary tract.

L-type amino acid transporter 1 (LAT1), a neutral amino acid transporter, requires covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional form. We investigated the importance of LAT1 and 4F2hc expressions to progression in upper urinary tract cancer. We examined their expressions and their relationships to clinicopathologic parameters and clinical outcome in 124 cases. Positive expressions of LAT1 (protein and messenger ribonucleic acid) and 4F2hc (protein) were recognized in 79.8, 89.5, and 87.9% of tumor samples, respectively. In tumor cells, LAT1 protein was detected either as nodular granules within the cytoplasm or diffusely within the cytoplasm and/or on plasma membrane. In the normal urothelium, its expression was detected as nodular granules within the cytoplasm. A correlation with stage was shown for LAT1 protein expression and for a cooperative expression of LAT1 protein with 4F2hc protein (active form of LAT1 protein). Further, in all tumors, a cooperative expression of LAT1 protein and 4F2hc protein was significantly correlated with both overall and disease-free survival rates in the univariate analysis but not in the multivariate analysis. In conclusion, the detection of the active form of LAT1 protein would appear to be of value in informing the risk of progression in transitional cell carcinoma of the upper urinary tract.

Activation antigens during the proliferative and secretory phases of endometrium and early-pregnancy decidua.

BACKGROUND: Clarifying the normal distribution of activation antigens will contribute to database construction studies of monoclonal-antibody-based therapies in endometrial disorders. METHODS: In this study, endometrial tissue samples obtained during proliferative and secretory phases and decidual samples of early pregnancies were immunostained by the monoclonal antibodies anti-CD26, anti-CD30, anti-CD70, anti-CD71, and anti-CD98 using the indirect immunoperoxidase method. RESULTS: CD26 is expressed on the glandular epithelium in the endometrium and decidua. Endothelial CD26 is expressed less in the decidua when compared to the endometrium. CD30 is strongly expressed by decidual cells. It is only weakly expressed on endometrial and decidual vessels. Glandular and endothelial CD70 expression is mainly seen in the proliferative phase of the menstrual cycle. Glandular CD71 expression is less in the decidua when compared to the endometrium. Its expression on stromal cells is more in the secretory phase of the menstrual cycle and in early pregnancy deciduae. It is expressed on endometrial vessels but not on decidual vessels. Glandular CD98 is expressed more in the decidua when compared to the endometrium. This antigen exists on endometrial lymphocytes. It is strongly expressed on the endothelium in the endometrium and decidua. CONCLUSION: It seems that CD26 and CD70 are not involved in the functions of endometrial and decidual stromal cells. CD30 and CD71 are thought to be involved in decidualization. Absence of activation antigens other than CD98 on lymphocytes indicated an antigenic profile for large granular lymphocytes that is different from regular lymphocytes.

CD98, a novel marker of transient amplifying human keratinocytes.

Identification of plasma membrane markers of basal keratinocytes is essential for sorting basal cells and, subsequently, adult epidermal stem cells. In this study, we isolated caveolin-1-enriched microdomains from human HaCaT keratinocytes and identified proteins representing potential cell surface markers of the epidermis by a proteomic approach. The purification of this caveolae domain allowed us to characterize 53 proteins of which 26% were transmembrane and 32% associated-membrane proteins. One of them, CD98, was found to be co-localized with beta1 integrin at the plasma membrane of the basal keratinocytes of healthy human epidermis. We then isolated CD98-positive keratinocytes from fresh skin biopsies. Using clonogenic assays, we demonstrate that CD98 may be considered as a marker of transient amplifying human keratinocytes.

System L: heteromeric exchangers of large, neutral amino acids involved in directional transport.

The plasma membrane transport system L is in many cells the only (efficient) pathway for the import of large branched and aromatic neutral amino acids. The corresponding transporters are hetero(di)mers composed of a catalytic subunit (LAT1 or LAT2=light chain=glycoprotein-associated amino acid transporter) associated covalently with the glycoprotein 4F2hc/CD98 (heavy chain). The tissue distribution of LAT1 suggests that it is involved mainly in transporting amino acids into growing cells and across some endothelial/epithelial secretory barriers, whereas the localization of LAT2 indicates that it is mainly involved in the basolateral efflux step of transepithelial (re)absorptive amino acid transport. However, system L transporters are obligatory amino acid exchangers with 1:1 stoichiometry, with similar (but not identical) intra- and extracellular substrate selectivities and with highly asymmetrical apparent affinities (low affinity inside). Therefore, net directional transport of large, neutral amino acids by system L depends on the parallel expression of a unidirectional transporter with overlapping selectivity (for instance systems A or N) that provides/recycles amino acids that drive system L exchange function. By mediating the regulated flux of these exchange substrates, unidirectional transporters control the activity of system L.

The presence and prognostic importance of glomerular macrophage infiltration in renal allografts.

AIM: The purpose of this study was to analyze the role of intraglomerular macrophage infiltration in human renal allografts by examining biopsies from kidney grafts that were dysfunctional after transplantation. METHODS: Eighty-three patients (58 men, 25 women) of a mean age of 30.2 +/- 1.4 years were evaluated. In all cases, biopsy specimens were examined for the presence of macrophage infiltration in the glomeruli. The infiltration of these cells was evaluated immunohistochemically using monoclonal antibody CD68, which labels macrophage cytoplasm. 10 renal allograft biopsies with normal histopathology were used as control group. The CD68-positive macrophages in all glomeruli were counted and the glomerular macrophage index (GMI) was calculated. RESULTS: Of the 83 patients, 40 showed acute rejection (AR), 33 showed chronic rejection (CR) and 10 showed cyclosporin A (CsA) toxicity. Only the biopsies of 28 patients stained positive for CD68 in the glomeruli. Neither patients with CsA toxicity nor controls showed intraglomerular macrophages. The CD68-positive group consisted of 7/33 CR and 21/40 AR patients. We observed intraglomerular macrophages in only 6 of the 20 AR cases that responded to steroid therapy (mean GMI 0.3 +/- 0.1) and in 15 of the 20 steroid-resistant AR cases (mean GMI 1.7 +/- 1.2; p < 0.01). The outcome of grafts that contained intraglomerular macrophages was significantly worse than the outcomes of other grafts noticed during the follow-up. CONCLUSION: We conclude that the presence of glomerular macrophages can be considered a marker for rejection and is a valuable additional criterion of rejection in the histological examination of renal allograft biopsies. The presence of intraglomerular macrophages indicates that the outcome of the graft will be significantly worse than that of grafts without intraglomerular macrophage infiltration.

Beta1 integrins show specific association with CD98 protein in low density membranes.

BACKGROUND: The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear. RESULTS: We demonstrate that CD98 constitutively and specifically associates with beta1 integrins (alpha2beta1,alpha3beta1, alpha5beta1 and alpha6beta1), but minimally with alpha4beta1. Integrin-CD98 association was established by reciprocal immunoprecipitation experiments, and confirmed by CD98-induced clustering of alpha3beta1 but not alpha4beta1 on the surface of rhabdomyosarcoma cells. Integrin-CD98 association is independent of the alpha subunit cytoplasmic tail, is maintained in alpha3beta1 ligand-interaction deficient mutants, and is not inhibited by EDTA. Within the CD98 heavy chain, a C109S mutation (but not a C330S mutation) caused a loss of beta1 integrin association. The same C109S mutation also caused a loss of CD98 light chain association. Importantly, CD98 associated selectively with beta1 integrins present in low density "light membrane" fractions on a sucrose gradient. CD98 was not present in dense fractions that contained the majority of beta1 integrins. Notably, the C109S mutant of CD98, that did not associate with beta1 integrins, showed also a reduced localization into light membrane fractions. CONCLUSIONS: We demonstrate that CD98 association with beta1 integrins is specific, occurs in the context of low density membranes, and may require the CD98 light chain.