The allopurinol metabolite, oxypurinol, drives oligoclonal expansions of drug-reactive T cells in resolved hypersensitivity cases and drug-naïve healthy donors.

Allopurinol (ALP) is a successful drug used in the treatment of gout. However, this drug has been implicated in hypersensitivity reactions that can cause severe to life-threatening reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Individuals who carry the human leukocyte antigen (HLA)-B*58:01 allotype are at higher risk of experiencing a hypersensitivity reaction (odds ratios ranging from 5.62 to 580.3 for mild to severe reactions, respectively). In addition to the parent drug, the metabolite oxypurinol (OXP) is implicated in triggering T cell-mediated immunopathology via a labile interaction with HLA-B*58:01. To date, there has been limited information regarding the T-cell receptor (TCR) repertoire usage of reactive T cells in patients with ALP-induced SJS or TEN and, in particular, there are no reports examining paired αßTCRs. Here, using in vitro drug-treated PBMCs isolated from both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors, we show that OXP is the driver of CD8+ T cell-mediated responses and that drug-exposed memory T cells can exhibit a proinflammatory immunophenotype similar to T cells described during active disease. Furthermore, this response supported the pharmacological interaction with immune receptors (p-i) concept by showcasing (i) the labile metabolite interaction with peptide/HLA complexes, (ii) immunogenic complex formation at the cell surface, and (iii) lack of requirement for antigen processing to elicit drug-induced T cell responsiveness. Examination of paired OXP-induced αßTCR repertoires highlighted an oligoclonal and private clonotypic profile in both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors.

Exosomal microRNAs from PBMCs stimulated with culprit drugs enhanced keratinocyte cell death in Stevens-Johnson syndrome/toxic epidermal necrolysis.

BACKGROUND: Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and systemic symptoms (DRESS) are both severe cutaneous adverse reactions. Keratinocyte death is much more prominent in SJS/TEN compared to DRESS. OBJECTIVE: This study aimed to investigate the role of exosomal miRNAs on keratinocyte death in SJS/TEN. METHODS: Peripheral blood mononuclear cells (PBMCs) from SJS/TEN and DRESS patients were stimulated with the culprit drugs. The exosomes released in cell supernatants were co-incubated with HaCaT cells to study the cytotoxic effects on keratinocytes. Exosomal miRNA sequencing analysis was performed to compare the expression patterns between SJS/TEN and DRESS subjects. HaCaT cells were then transfected with miRNA mimics and inhibitors to explore the functions of miRNAs on keratinocyte cell death. RESULTS: Cytotoxic effects of PBMC-derived exosomes on keratinocytes were demonstrated in SJS/TEN and could be neutralized with exosome inhibitors. Cytotoxic effects of PBMC-derived exosomes from SJS/TEN subjects were higher after incubating PBMCs with the culprit drugs than those incubating with irrelevant drugs and unstimulated controls. The sequencing data revealed differential expressions of 61 exosomal miRNAs between SJS/TEN and DRESS. Exosomal miR-4488 was upregulated while miR-486-5p, miR-96-5p and miR-132-3p were downregulated in SJS/TEN compared to DRESS as determined by quantitative real-time PCR. The increased percentage of apoptotic cells upon transfection of HaCat cells was 36.3% and 34.9% with miR-4488 mimic and miR-96-5p inhibitor, respectively. CONCLUSION: This study illustrated the regulatory functions of exosomal miRNAs in controlling keratinocyte death in SJS/TEN. Exosome inhibitors might have a therapeutic role in SJS/TEN.

Differential antigenic requirements by diverse MR1-restricted T cells.

MHC-related protein 1 (MR1) presents microbial riboflavin metabolites to mucosal-associated invariant T (MAIT) cells for surveillance of microbial presence. MAIT cells express a semi-invariant T-cell receptor (TCR), which recognizes MR1-antigen complexes in a pattern-recognition-like manner. Recently, diverse populations of MR1-restricted T cells have been described that exhibit broad recognition of tumor cells and appear to recognize MR1 in association with tumor-derived self-antigens, though the identity of these antigens remains unclear. Here, we have used TCR gene transfer and engineered MR1-expressing antigen-presenting cells to probe the MR1 restriction and antigen reactivity of a range of MR1-restricted TCRs, including model tumor-reactive TCRs. We confirm MR1 reactivity by these TCRs, show differential dependence on lysine at position 43 of MR1 (K43) and demonstrate competitive inhibition by the MR1 ligand 6-formylpterin. TCR-expressing reporter lines, however, failed to recapitulate the robust tumor specificity previously reported, suggesting an importance of accessory molecules for MR1-dependent tumor reactivity. Finally, MR1-mutant cell lines showed that distinct residues on the α1/α2 helices were required for TCR binding by different MR1-restricted T cells and suggested central but distinct docking modes by the broad family of MR1-restricted αß TCRs. Collectively, these data are consistent with recognition of distinct antigens by diverse MR1-restricted T cells.

Upregulation of antigen presentation function and inflammation in chondrocytes by induction of proteoglycan aggrecan peptides (P16-31 and P263-280).

OBJECTIVES: A central hallmark of osteoarthritis (OA) is cartilage destruction. Chondrocytes not only control cartilage metabolism, but are capable of immunogenic responses. The role of chondrocytes in the pathogenesis of OA is still unclear. In this study, we aimed to determine the immunological role of chondrocytes in response to proteoglycan aggrecan (PG) peptides. METHODS: Human chondrocytes were isolated from cartilage of knee OA patients undergoing knee arthroplasty and stimulated with proteoglycan aggrecan peptides in the presence of IFNγ. Antigen presentation markers, co-stimulatory molecules, cytokine production, gene expression and antigen presentation to T cells were evaluated. RESULTS: Our results show that IFNγ was required for the expression of MHC class I and II. However, stimulation with PG peptides P16-31 and P263-280, but not P2379-2394, increased expression level of co-stimulatory molecules (CD80 and CD86) and IL-6, IL-8 and TNFα production. This upregulation was seen in chondrocytes to nearly comparable levels of professional antigen-presenting cells. A similar pattern of gene expression was observed between P16-31 and P263-280 peptide stimulation on chondrocytes and this was different from P2379-2394 peptide treatment. Co-culture with autologous T cells revealed signi cant proliferation of cells when stimulating with the P263-280 peptides. CONCLUSIONS: Our study shows that human chondrocytes display unique features of antigen presentation. Their ability to process certain proteoglycan aggrecan peptides, in which these molecules are synthesised by the cartilage themselves render the possibility of a role for "self-antigens" in the immunopathogenesis of OA.

Cytokine Profiling and Intra-Articular Injection of Autologous Platelet-Rich Plasma in Knee Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease leading to joint pain and stiffness. Due to lack of effective treatments, physical and psychological disabilities caused by OA have a detrimental impact on the patient's quality of life. Emerging evidence suggests that intra-articular injection of platelet-rich plasma (PRP) may provide favorable results since PRP comprises not only a high level of platelets but also a huge amount of cytokines, chemokines, and growth factors. However, the precise mechanism and standardization method remain uncertain. This study aimed to examine cytokine profiling in both PRP and platelet-poor plasma (PPP) of knee OA patients and to determine the effects of PRP on OA chondrocytes and knee OA patients. PRP contained a wide variety of cytokines, chemokines, growth factors, and autologous intra-articular PRP injection resulted in favorable outcomes in knee OA patients. Significant increases in levels of IL-1, IL-2, IL-7, IL-8, IL-9, IL-12, TNF-α, IL-17, PDGF-BB, bFGF, and MIP-1ß were detected in PRP compared to PPP (p < 0.001). An in vitro study showed a marked increase in proliferation in OA chondrocytes cultured with PRP, compared to PPP and fetal bovine serum (p < 0.001). In a clinical study, knee OA patients treated with PRP showed improvement of physical function and pain, assessed by physical performance, Western Ontario and McMaster Universities Arthritis Index and visual analog scale. Our findings from both in vitro and clinical studies suggest that intra-articular PRP injection in knee OA patients may be a potential therapeutic strategy for alleviating knee pain and delaying the need for surgery.

Notch signaling regulates function of human mucosal-associated invariant T (MAIT) cells.

BACKGROUND: Notch signaling plays an important role in the development of T lymphocytes and regulates their effector functions. The regulatory roles of Notch signaling on T cells have been intensely investigated, but whether it involves in effector functions of mucosal-associated invariant T (MAIT) cells has never been reported. OBJECTIVE: To elucidate the expression profiles of Notch receptors/ligands and to investigate their roles in human MAIT cell function. METHODS: Peripheral blood mononuclear cells (PBMCs) from health donors were stimulated with or without anti-CD3/ CD28-coupled beads, recombinant IL-12/IL-18 cytokines, riboflavin- or non-riboflavin-synthesizing bacterial cultured supernatant for 24 hours. The expression profiles of Notch receptors and ligands on MAIT cells were detected by flow cytometry. PBMCs were treated with a Notch signaling inhibitor, gamma secretase inhibitor (GSI), before stimulation to investigate the impact of interfering with Notch signaling on activation and function of MAIT cells. RESULTS: Resting MAIT cells predominantly expressed Notch2 receptor and the ligand, Jagged 2, on their surface. Upon stimulation, MAIT cells further upregulated Notch2 and also Notch1 with its cleaved form, indicating active Notch signaling. Cytokines and cytotoxic molecules which are secreted by activated MAIT cells, were suppressed by treatment with GSI. Moreover, both TCR-dependent MAIT cell activation by microbial-derived riboflavin intermediates and TCR-independent MAIT cell activation driven by IL-18 in synergy with IL-12, were blocked by GSI treatment. CONCLUSIONS: Notch signaling is operating in MAIT cells and is involved in their activation both in a TCR-independent and -dependent manners.

Notch 2 receptor expression and reduced cytotoxicity in MAIT cells of active pulmonary TB patients.

BACKGROUND: Knowledge of the prevalence of common sensitizing allergens may aid in overall management of allergic disease in a specified area. OBJECTIVE: The aim of this study was to identify and analyse the prevalence of common inhaled and food sensitizing allergens in Beijing. METHODS: This was a retrospective study, analysing demographic data and serum sIgE antibody test results from 59057 outpatients who presented to Beijing TongRen Hospital, from January 2013 to December 2019. RESULTS: 28879 patients (48.9%) showed positive sIgE test results; with significantly more males aged under 16 years sensitized to at least one allergen than females, and most patients (53.62%) were sensitized to multiple allergens. The first inhaled sensitizing allergens was Artemisia grass (11910 (41.24%)); and the first food allergens was crab (3547 (12.28%)). For Artemisia sensitized patients, sIgE levels were mostly at level 5. The number of patients with ragweed allergy is increasing year by year. The detection rates for sIgE to Artemisia, common ragweed, and Humulus grass allergens were significantly higher in August and September. R package ggplot2 analysis, demonstrated strong correlations between tree allergens and common ragweed and Humulus grass allergens (phi coefficients = 0.50 and 0.46, respectively; both P < 0.01). CONCLUSIONS: The prevalence of sensitization to different allergens in Beijing showed Artemisia grass was the most commonly inhaled sensitizing allergen, and the number of patients with ragweed grass allergy was increasing by year.

Down-regulation of miR-155 after treatment with narrow-band UVB and methotrexate associates with apoptosis of keratinocytes in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease arising from a complex interaction between genetics, epigenetics, the host's immune system and the environment. Recent accumulated data revealed the dysregulation of various microRNAs (miRNAs) in several diseases including psoriasis. OBJECTIVE: We explored the functional role and regulation of hsa-miR-155-5p (miR-155) in an immortalized keratinocyte cell line (HaCaT), in relation to the pathogenesis and treatment of psoriasis. METHODS: miR-155 expression in normal skin and psoriatic skin lesion before and after treatment with methotrexate (MTX) and narrow-band ultraviolet B phototherapy (NB-UVB) were analyzed using quantitative reverse transcription PCR (qRT-PCR). Apoptotic activity, cell cycle and viable cells of miR-155 transfected HaCaT were measured using flow cytometry and MTS assay. Since, caspase-3 (CASP3) gene was predicted as a target gene of miR-155, the expression of CASP3 was detected in transfected HaCaT using western blot. RESULTS: We discovered that both MTX and NB-UVB significantly down-regulated miR-155 expression in psoriatic skin lesions. We also found that overexpression of miR-155 in HaCaT led to suppression of cell apoptosis and induced cell arrest at G0/G1 phase. Moreover, CASP3 expression was down-regulated in miR-155 transfected HaCaT. CONCLUSIONS: This study demonstrates down-regulation of miR155 after treatment with MTX and NB-UVB in psoriatic skin lesion. miR155 plays significant role in apoptosis on HaCaT via CASP3. This finding provides a better understanding of the pathogenesis of psoriasis and might aid on developing the new monitoring tool or therapy for psoriasis in the future.

T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Monitoring Anti-Pythium insidiosum IgG Antibodies and (1→3)-ß-d-Glucan in Vascular Pythiosis.

Despite aggressive treatment, vascular pythiosis has a mortality rate of 40%. This is due to delays in diagnosis and a lack of effective monitoring tools. To overcome this drawback, serum beta-d-glucan (BG) and P. insidiosum-specific antibody (Pi-Ab) were examined as potential monitoring markers in vascular pythiosis. A prospective cohort study of vascular pythiosis patients was carried out from January 2010 to July 2016. Clinical information and blood samples were collected and evaluated by the BG and Pi-Ab assays. Linear mixed-effect models were used to compare BG and Pi-Ab levels. The in vitro susceptibility test was performed with all P. insidiosum isolates from culture-positive cases. A total of 50 patients were enrolled: 45 survived and 5 died during follow-up. The survivors had a significantly shorter time to medical care (P < 0.0001) and a significantly shorter waiting time to the first surgery (P < 0.0001). There were no differences in BG levels among the groups at diagnosis (P = 0.33); however, BG levels among survivors were significantly lower than those of the deceased group at 0.5 months (P < 0.0001) and became undetectable after 3 months. Survivors were able to maintain an enzyme-linked immunosorbent assay (ELISA) value (EV) of Pi-Ab above 8, whereas the EV among deceased patients was less than 4. In vitro susceptibility results revealed no synergistic effects between itraconazole and terbinafine. This study showed that BG and Pi-Ab are potentially valuable markers to monitor the disease after treatment initiation. An unchanged BG level at 2 weeks after surgery should prompt an evaluation for residual disease.

MR1 presents microbial vitamin B metabolites to MAIT cells.

Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.

The frequency of mucosal-associated invariant T cells is selectively increased in dermatitis herpetiformis.

BACKGROUND/OBJECTIVES: Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like T-cells that are enriched in mucosal tissues. Their presence in human skin has only recently been recognised. We describe the expression of skin-tropic molecules on human skin MAIT cells at steady state and investigate their contribution to various dermatoses with known T-cell involvement. METHODS: To examine the expression of skin-tropic molecules by MAIT cells at steady state, we performed a flow cytometric analysis of blood and skin samples from healthy donors. To investigate any potential wider contribution of MAIT cells to skin disease, we examined psoriasis, alopecia areata and dermatitis herpetiformis biopsies using immunofluorescent staining to identify the proportion of T-cells expressing MAIT cell surface markers. RESULTS: We found that MAIT cells constituted a small population of T-cells in normal human skin, similar to the percentage found in peripheral blood. Like other skin T-cells, skin MAIT cells expressed high levels of the skin-associated markers, cutaneous lymphocyte antigen and CD103. In psoriasis and alopecia areata the proportion of MAIT cells was similar to that found in normal skin, but in dermatitis herpetiformis it was significantly elevated. CONCLUSIONS: The expression of skin-tropic molecules by skin MAIT cells is consistent with their resident status in normal human skin. Our results suggest that MAIT cells may play a role in the pathogenesis of dermatitis herpetiformis.

Mucosal-associated invariant T cells in clinical diseases.

UNLABELLED: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial infection via vitamin metabolites. The discovery of MAIT cells in the past two decades and the recent discovery of MR1 ligands has opened a new field and potential area for cellular immunotherapy using these unique cells. Their evolutionary conservation in mammals underscore their biological role in the host. In the past two years, we have been involved in the generation of MR1 tetramers as a tool for identification of these cells. Many groups have studied the role of these cells in clinical diseases. OBJECTIVE: Here, we provide an up-to-date comprehensive review of clinical disease that have been studied with regards to MAIT cells. RESULTS: Original articles and review articles under the topic of MAIT cells and their relation to clinical diseases, both in human and animal models were included in the review. CONCLUSION: MAIT cells are potential candidates for future cellular immunotherapy. However, more understanding of the biological role of MAIT cells need to be elucidated first.

Infrapatellar fat pad adipose tissue-derived macrophages display a predominant CD11c+CD206+ phenotype and express genotypes attributable to key features of OA pathogenesis.

Objectives: In knee osteoarthritis (OA), macrophages are the most predominant immune cells that infiltrate synovial tissues and infrapatellar fat pads (IPFPs). Both M1 and M2 macrophages have been described, but their role in OA has not been fully investigated. Therefore, we investigated macrophage subpopulations in IPFPs and synovial tissues of knee OA patients and their correlation with disease severity, examined their transcriptomics, and tested for factors that influenced their polarization. Methods: Synovial tissues and IPFPs were obtained from knee OA patients undergoing total knee arthroplasty. Macrophages isolated from these joint tissues were characterized via flow cytometry. Transcriptomic profiling of each macrophage subpopulations was performed using NanoString technology. Peripheral blood monocyte-derived macrophages (MDMs) were treated with synovial fluid and synovial tissue- and IPFP-conditioned media. Synovial fluid-treated MDMs were treated with platelet-rich plasma (PRP) and its effects on macrophage polarization were observed. Results: Our findings show that CD11c+CD206+ macrophages were predominant in IPFPs and synovial tissues compared to other macrophage subpopulations (CD11c+CD206-, CD11c-CD206+, and CD11c-CD206- macrophages) of knee OA patients. The abundance of macrophages in IPFPs reflected those in synovial tissues but did not correlate with disease severity as determined from Mankin scoring of cartilage destruction. Our transcriptomics data demonstrated highly expressed genes that were related to OA pathogenesis in CD11c+CD206+ macrophages than CD11c+CD206-, CD11c-CD206+, and CD11c-CD206- macrophages. In addition, MDMs treated with synovial fluid, synovial tissue-conditioned media, or IPFP-conditioned media resulted in different polarization profiles of MDMs. IPFP-conditioned media induced increases in CD86+CD206+ MDMs, whereas synovial tissue-conditioned media induced increases in CD86+CD206- MDMs. Synovial fluid treatment (at 1:8 dilution) induced a very subtle polarization in each macrophage subpopulation. PRP was able to shift macrophage subpopulations and partially reverse the profiles of synovial fluid-treated MDMs. Conclusion: Our study provides an insight on the phenotypes and genotypes of macrophages found in IPFPs and synovial tissues of knee OA patients. We also show that the microenvironment plays a role in driving macrophages to polarize differently and shifting macrophage profiles can be reversed by PRP.

The Immunological Facets Of Chondrocytes In Osteoarthritis: A Narrative Review.

Osteoarthritis (OA) is a disease in which the pathogenesis affects the joint and its surrounding tissues. Cartilage degeneration is the main hallmark of OA and chondrocytes within the cartilage regulate matrix production and degradation. In OA patients and animal models of OA, the pathology of the disease relates to disequilibrium between anabolic and catabolic states of the cartilage. Moreover, chondrocyte phenotype and function are also immunologically altered. Under inflammatory conditions, chondrocytes increase production levels of inflammatory cytokines and cartilage-degrading enzymes, which further drive cartilage destruction. Chondrocytes also have an innate immune function and respond to DAMPs and cartilage fragments via innate immune receptors. In addition, chondrocytes play a role in adaptive immune responses by acting as antigen presenting cells and presenting cartilaginous antigens to T cells. Indirectly, chondrocytes are stimulated by pathogen-associated molecular patterns (PAMPs) present in the joints, a result of the microbiota in the host. Chondrocytes have both direct and indirect relationships with immune cells and the immune compartment of OA patients. Therefore, chondrocytes serve as a target for immunotherapeutic approaches in OA. In this narrative review, we cover the aforementioned immune-related aspects of chondrocytes in OA.

Human mucosal Vα7.2+ CD161hi T cell distribution at physiologic state and in Helicobacter pylori infection.

Mucosal-associated invariant T (MAIT) cells are innate-like, unconventional T cells that are present in peripheral blood and mucosal surfaces. A clear understanding of how MAIT cells in the mucosae function and their role in host immunity is still lacking. Therefore, our aim was to investigate MAIT cell distribution and their characteristics in the gastrointestinal (GI) mucosal tissue based on Vα7.2+ CD161hi identification. We showed that Vα7.2+ CD161hi T cells are present in both intraepithelial layer and lamina propriae of the GI mucosa, but have different abundance at each GI site. Vα7.2+ CD161hi T cells were most abundant in the duodenum, but had the lowest reactivity to MR1-5-OP-RU tetramers when compared with Vα7.2+ CD161hi T cells at other GI tissue sites. Striking discrepancies between MR1-5-OP-RU tetramer reactive cells and Vα7.2+ CD161hi T cells were observed along each GI tissue sites. Vα7.2+ CD161hi TCR repertoire was most diverse in the ileum. Similar dominant profiles of TRBV usage were observed among peripheral blood, duodenum, ileum, and colon. Some TRBV chains were detected at certain intestinal sites and not elsewhere. The frequency of peripheral blood Vα7.2+ CD161hi T cells correlated with mucosal Vα7.2+ CD161hi T cells in lamina propriae ileum and lamina propriae colon. The frequency of peripheral blood Vα7.2+ CD161hi T cells in Helicobacter pylori-infected individuals was significantly lower than uninfected individuals, but this was not observed with gastric Vα7.2+ CD161hi T cells. This study illustrates the biology of Vα7.2+ CD161hi T cells in the GI mucosa and provides a basis for understanding MAIT cells in the mucosa and MAIT-related GI diseases.

TIL expansion with high dose IL-2 or low dose IL-2 with anti-CD3/anti-CD28 stimulation provides different quality of TIL-expanded T cell clones.

Tumor infiltrating lymphocytes (TILs) are cells that are present inside the tumor environment, of which include T cells, B cells and natural killer (NK) cells. At present, TILs are used for immunotherapy in various cancers. Knowledge on adoptive transfer of TILs in ovarian cancer is still limited, especially regarding TIL expansion methods. Therefore, the aim of our study was to compare the quality of T cell clones between two expansion methods for ovarian cancer TILs. We show that TILs stimulated with the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28) and the standard stimulation method (high dose IL-2 only) both increased total number of T cells. TCR repertoire analyses revealed different TCR repertoire patterns between TIL-expanded T cells that were stimulated with the standard stimulation method (high dose IL-2 only) and the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28). Regardless, when TILs were expanded using the standard stimulation method (high dose IL-2 only), the predominant T cell receptor beta variable (TRBV) chains that were used in both TIL-expanded clones of the CD4+ and CD8+ subpopulations were similar. In addition, there were also TIL-expanded CD4+ and CD8+ T cell clones that were dominant in only one or the other subpopulations. These results reveal the bias in TIL quality after being stimulated with different protocols. Further studies are required to understand the selection of TIL expansion, in order for a more efficacy adoptive transfer treatment.

Impact of SARS-CoV-2 infection on the profiles and responses of innate immune cells after recovery.

BACKGROUNDS: SARS-CoV-2 infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic to severe symptoms and death. Most COVID-19 pathogenesis is associated with hyperinflammatory conditions driven primarily by myeloid cell lineages. The long-term effects of SARS-CoV-2 infection post recovery include various symptoms. METHODS: We performed a longitudinal study of the innate immune profiles 1 and 3 months after recovery in the Thai cohort by comparing patients with mild, moderate, and severe clinical symptoms using peripheral blood mononuclear cells (n = 62). RESULTS: Significant increases in the frequencies of monocytes compared to controls and NK cells compared to mild and moderate patients were observed in severe patients 1-3 months post recovery. Increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were observed in all recovered patients, even after 3 months. Increased IL-6 and TNFα levels in monocytes were observed 1 month after recovery in response to lipopolysaccharide (LPS) stimulation, while decreased CD86 and HLA-DR levels were observed regardless of stimulation. A multiplex analysis of serum cytokines performed at 1 month revealed that most innate cytokines, except for TNFα, IL4/IL-13 (Th2) and IFNγ (Th1), were elevated in recovered patients in a severity-dependent manner. Finally, the myelopoiesis cytokines G-CSF and GM-CSF were higher in all patient groups. Increased monocytes and IL-6- and TNFα-producing cells were significantly associated with long COVID-19 symptoms. CONCLUSIONS: These results reveal that COVID-19 infection influences the frequencies and functions of innate immune cells for up to 3 months after recovery, which may potentially lead to some of the long COVID symptoms.

The infrapatellar fat pad produces interleukin-6-secreting T cells in response to a proteoglycan aggrecan peptide and provides dominant soluble mediators different from that present in synovial fluid.

OBJECTIVE: The purpose of this study was to investigate the effects of osteoarthritis (OA) peripheral blood mononuclear cell (PBMC) -stimulating proteoglycan aggrecan peptides on T cells present in infrapatellar fat pads (IPFPs) and synovial tissues, and to correlate these findings with mediators present in synovial fluid of OA patients. METHODS: We tested for interleukin-6 (IL-6) -producing T cells in IPFPs of patients with knee OA using ELISPOT. Cytokine and cytotoxic mediator production from OA PBMCs, IPFPs, synovial tissues, and synovial fluids in response to proteoglycan aggrecan peptides were quantified by cytometric bead array. Patterns of cytokine and cytotoxic mediator production were analyzed and compared. RESULTS: T cells from IPFPs elicited strong responses towards the p263-280 peptide by secreting IL-6. In addition, there was a trend that the p263-280 peptide stimulated higher production of cytokines/cytotoxic mediators than other proteoglycan aggrecan peptides, although this was not statistically significant. In patients with knee OA, a group of cytotoxic mediators (sFas, perforin, granzyme A, and granulysin) and IL-6 were detectable at high levels from the synovial fluid. In addition, inflammation in patients with knee OA was more pronounced in joint-surrounding tissues than levels in circulating peripheral blood. CONCLUSION: Our data suggest that T cells responding to the p263-280 peptide contribute to the secretion of various soluble mediators that are found within the synovial fluid. We also identified potential new candidates that may serve as biomarkers of knee OA.