Identification of different functions of CD8+ T cell subpopulations by a novel monoclonal antibody.

The explicit identification of CD8+ T cell subpopulation is important for deciphering the role of CD8+ T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8+ T cells, FE-1H10+ and FE-1H10- CD8+ T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8+ T cell subsets. The functions of FE-1H10- and FE-1H10+ CD8+ T cells were investigated. T cell proliferation assays revealed that FE-1H10- CD8+ T cells exhibited a higher proliferation rate than FE-1H10+ CD8+ T cells, whereas FE-1H10+ CD8+ T cells produced higher levels of IFN-γ and TNF-α than FE-1H10- CD8+ T cells. In T cell cytotoxicity assays, FE-1H10+ CD8+ T cells were able to kill target cells better than FE-1H10- CD8+ T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10+ CD8+ T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10- CD8+ T cells have greater expression of genes related to memory CD8+ T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8+ T cell subpopulations. The FE-1H10+ CD8+ T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8+ T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8+ T cells in cancer treatment.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Comparison for immunophysiological responses of Jeju and Thoroughbred horses after exercise.

OBJECTIVE: The study was conducted to investigate variations in the immunophysiological responses to exercise-induced stress in Jeju and Thoroughbred horses. METHODS: Blood samples were collected from the jugular veins of adult Jeju (n = 5) and Thoroughbred (n = 5) horses before and after 30 min of exercise. The hematological, biochemical, and immunological profiles of the blood samples were analyzed. Blood smears were stained and observed under a microscope. The concentration of cell-free (cf) DNA in the plasma was determined using real time polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells were separated using Polymorphprep, and the expression of various stress-related and chemokine receptor genes was measured using reverse transcriptase (RT) and real-time PCR. RESULTS: After exercise, Jeju and Thoroughbred horses displayed stress responses with significantly increased rectal temperatures, cortisol levels, and muscle catabolism-associated metabolites. Red blood cell indices were significantly higher in Thoroughbred horses than in Jeju horses after exercise. In addition, exercise-induced stress triggered the formation of neutrophil extracellular traps (NETs) and reduced platelet counts in Jeju horses but not in Thoroughbred horses. Heat shock protein 72 and heat shock protein family A (Hsp70) member 6 expression is rapidly modulated in response to exercise-induced stress in the PBMCs of Jeju horses. The expression of CXC chemokine receptor 4 in PBMCs was higher in Thoroughbred horses than in Jeju horses after exercise. CONCLUSION: In summary, the different immunophysiological responses of Jeju and Thoroughbred horses explain the differences in the physiological and anatomical properties of the two breeds. The physiology of Thoroughbred horses makes them suitable for racing as they are less sensitive to exercise-induced stress compared to that of Jeju horses. This study provides a basis for investigating the link between exercise-induced stresses and the physiological alteration of horses. Hence, our findings show that some of assessed parameters could be used to determine the endurance performance of horses.

Triggering of CD99 on monocytes by a specific monoclonal antibody regulates T cell activation.

CD99, a leukocyte surface glycoprotein, has been implicated in many cellular processes including cell adhesion, cell migration and T cell activation. Our previous study demonstrated the anti-CD99 monoclonal antibody (mAb) clone MT99/3 inhibited T cell activation; however, the mechanism is unclear. In this study, we demonstrated that CD99 expressed on monocytes played a role in the inhibition of T cell activation. Anti-CD99 mAb MT99/3 downregulated the expression of costimulatory molecule CD86, but upregulated IL-6, IL-10 and TNF-α production by monocytes. The inhibitory effect of mAb MT99/3 required cell to cell contact between monocytes and lymphocytes. The soluble mediators produced by monocytes alone were insufficient to induce hypo-function of T lymphocytes. In summary, we demonstrated that ligation of CD99 on monocytes by anti-CD99 mAb MT99/3 could mediate T cell hypo-responsiveness. These findings provide the first evidence of the role of CD99 on monocytes that contributes to T cell activation.

The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse.

OBJECTIVE: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme 1α (IRE1α)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates IRE1α signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. METHODS: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. RESULTS: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. CONCLUSION: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

Antibody biosensors for the measurement and characterization of soluble CD147 molecules.

BACKGROUND: Soluble CD147 (sCD147) is the shed form of membrane-bound CD147, which is involved in the regulation of cellular functions. The presence of sCD147 in body fluids is associated with several diseases. OBJECTIVE: In this study, we aimed to establish antibody (Ab) biosensors for the simultaneous differential detection of the general and truncated forms of sCD147. METHOD: By combining biolayer interferometry technology (BLItz) and different anti-CD147 monoclonal antibodies (mAbs) specific to different extracellular domains of the CD147 molecule, Ab-based biosensors were established to rapidly measure and characterize sCD147 isoforms. RESULTS: Two types of Ab-biosensors, desginated the single Ab-biosensor and double Ab-biosensor, were established for the measurment and characterization of sCD147 isoforms. For the single Ab-biosensor system, monoclonal antibodies specific for CD147 domain 1 (D1) or domain 2 (D2) were immobilized on the sensor tips and used for the quantification of sCD147 using a BLItz optical interferometric biosensor. For the double Ab-biosensor system, following the single Ab-biosensor step, secondary anti-CD147 mAbs specific for each domain of the CD147 molecule were added and monitored by a BLItz biosensor. By combining the results obtained from the single Ab- and double Ab-biosensors, sCD147 isoforms including the general form (D1 linked to D2) and the truncated forms (sCD147 containing D1 or D2) could be determined. CONCLUSIONS: This method may be a beneficial tool for the determination of sCD147 isoforms for disease diagnosis and prognosis as well as for the definition of the cellular mechanisms of the immune system.

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva.

BACKGROUND: Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva. RESULTS: With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured. CONCLUSIONS: A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

A simple and highly sensitive ELISA for screening of the α-thalassemia-1 Southeast Asian-type deletion.

Couples in which both partners carry the α-thalassemia-1 trait have a 25% risk of hemoglobin Bart's hydrops fetalis in each pregnancy. Identification of α-thalassemia-1 trait is, therefore, necessary in order to control this severe form of α-thalassemia. We have generated monoclonal antibodies specific to the ζ-globin chain without cross reaction with other globin chains. A simple and sensitive ELISA was developed by using poly-l-lysine to increase the protein binding to the ELISA plate. The developed poly-l-lysine pre-coated ELISA has a very high sensitivity (100%) and specificity (97%) for detection of carriers of α-thalassemia-1 with Southeast Asian-type deletion.

Accumulation of versican facilitates wound healing: Implication of its initial ADAMTS-cleavage site.

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSß domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFß-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.

Ligation of Na, K ATPase ß3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase ß3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase ß3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.