CXCL1, CXCL10 and CXCL12 Chemokines are Variously Expressed in Acute Myeloid Leukemia Patients Prior and Post Bone Marrow Transplantation.

AIM: The chemokine-receptor axes play parts in development of leukemia, CXCL1, CXCL10 and CXCL12 are involved in immune responses. Thus, we have examined the serum levels of these chemokines in parallel with their related cognate receptors (CXCR1, CXCR3 and CXCR4) in AML (acute myeloid leukemia) patients prior and post BMT (bone marrow transplantation) therapy. MAIN METHODS: Clinical specimens were collected from 46 AML patients (23 M1 and 23 M3 subtypes) before/after BMT. CXCL1, CXCL10 and CXCL12 concentrations were determined by ELISA. The mRNA levels of the related receptors were detected by QRT_PCR. Data were analyzed by T-test, χ2 and ANOVA statistical methods in SPSS software version 18. A difference was regarded significant if P value < 0.05. KEY FINDINGS: Our results indicated that the elevated levels of CXCL12 in AML patients were remained unchanged after transplantation.  The CXCL10 concentration was decreased in patients. All studied chemokines were elevated in BMT patients with history of 9 times PLT transfusion. In patients who received BMT from siblings CXCL1 and CXCL10 have been elevated, whereby they were compared to patients who received BMT from parents while CXCL12 sustained unchanged in groups. Serum measures of CXCL1 and CXCL10 were induced in acute and chronic GVHD patients in compare to these without GVHD. SIGNIFICANCE: According to the results, it can be concluded that these chemokines play fundamental parts in pathogenesis of both AML and BMT. It is worthy to note that chemokines could be used as diagnostic markers alongside with possible promising therapeutic targets.

Circulating CD4+ FoxP3- CXCR5- CXCR3+ PD-1hi cells are elevated in active rheumatoid arthritis and reflect the severity of the disease.

OBJECTIVE: To examine the expression and clinical significance of circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in rheumatoid arthritis (RA). METHODS: CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in peripheral blood of 35 patients with active RA, 17 with RA in stable remission, and 24 healthy controls were analyzed by flow cytometry. Serum IgG and circulating plasmablast percentages were measured and correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were systematically analyzed. Disease Activity Scale 28 (DAS28) scores were also calculated and correlation analysis with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells was conducted. The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were compared before and after disease-modifying anti-rheumatic drug treatment. Cytokine levels in plasma and cytokine secretion in CD4 cells were measured and their correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were further analyzed. RESULTS: The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in the peripheral blood of patients with active RA were significantly increased compared with healthy controls. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA were positively correlated with serum IgG and DAS28 scores. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were significantly decreased in patients after treatment. Plasma interleukin-10 concentrations and interleukin-10-positive CD4 cell percentages were significantly positively correlated with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cell levels. CONCLUSION: Circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA are increased and could reflect the severity of the disease, which may play a potential role in the pathogenesis of RA.

Association of the CXCL9-CXCR3 and CXCL13-CXCR5 axes with B-cell trafficking in giant cell arteritis and polymyalgia rheumatica.

OBJECTIVE: B-cells are present in the inflamed arteries of giant cell arteritis (GCA) patients and a disturbed B-cell homeostasis is reported in peripheral blood of both GCA and the overlapping disease polymyalgia rheumatica (PMR). In this study, we aimed to investigate chemokine-chemokine receptor axes governing the migration of B-cells in GCA and PMR. METHODS: We performed Luminex screening assay for serum levels of B-cell related chemokines in treatment-naïve GCA (n = 41), PMR (n = 31) and age- and sex matched healthy controls (HC, n = 34). Expression of chemokine receptors on circulating B-cell subsets were investigated by flow cytometry. Immunohistochemistry was performed on GCA temporal artery (n = 14) and aorta (n = 10) and on atherosclerosis aorta (n = 10) tissue. RESULTS: The chemokines CXCL9 and CXCL13 were significantly increased in the circulation of treatment-naïve GCA and PMR patients. CXCL13 increased even further after three months of glucocorticoid treatment. At baseline CXCL13 correlated with disease activity markers. Peripheral CXCR3+ and CXCR5+ switched memory B-cells were significantly reduced in both patient groups and correlated inversely with their complementary chemokines CXCL9 and CXCL13. At the arterial lesions in GCA, CXCR3+ and CXCR5+ B-cells were observed in areas with high CXCL9 and CXCL13 expression. CONCLUSION: Changes in systemic and local chemokine and chemokine receptor pathways related to B-cell migration were observed in GCA and PMR mainly in the CXCL9-CXCR3 and CXCL13-CXCR5 axes. These changes can contribute to homing and organization of B-cells in the vessel wall and provide further evidence for an active involvement of B-cells in GCA and PMR.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer.

INTRODUCTION: NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. METHODS: Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. RESULTS: The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3-CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3- CD56dim) and regulatory (CD3- CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27-), regulatory (CD3-CD56+ CD11b+/- CD27+) and tolerant (CD3-CD56+ CD27- CD11b-) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3-CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN- patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN- patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. CONCLUSION: Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN- patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.

Reduced CD4+T Cell CXCR3 Expression in Patients With Allergic Rhinitis.

While T cells are considered to play a primary role in IgE-mediated atopic diseases, little is known about the systemic variations of T cell subsets from patients with allergic rhinitis (AR). To elucidate the characteristics of peripheral T cells, we analyzed natural killer, B cell, and T cell populations, performed T cell subset construction, and assessed chemokine receptor and associated serum cytokine expression in 25 AR patients and 20 healthy controls. Our results revealed increased levels of CD4+T cells, serum interleukin (IL)-10, IL-6, and interferon (IFN)-γ, and reduced Th1 and Th17 subsets, identified by their chemokine receptors, in AR patients. These results suggest a systemic activation of T cell responses in AR. We further demonstrated that AR patients exhibit significantly reduced CD4+T cell CXCR3 expression, especially in patients with moderate-severe disease severity, demonstrating that CXCR3 is a potential key molecule that hinders the Th1/Th2 balance in AR pathology. Overall, systemic T cell activation occurred in AR patients and CXCR3 dramatically decreased in CD4+T cells, which may ultimately be used as a potential disease and/or therapeutic target.

HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo.

The lungs are relatively unexplored anatomical human immunodeficiency virus (HIV) reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T cells are a subset of T cells that lack expression of CD4 and CD8 (CD4- CD8-) and may have both regulatory and effector functions during HIV infection. Notably, circulating DN T cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from 35 PLWH on ART and 16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV-negative (HIV-) adults displayed higher frequencies of DN T cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) than blood. We also observed that DN T cells were less senescent (CD28- CD57+) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B, and perforin in the BAL fluid than in the blood of PLWH. Importantly, fluorescence-activated cell sorter (FACS)-sorted DN T cells from the BAL fluid of PLWH under suppressive ART harbored HIV DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T cells than those of the blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART.IMPORTANCE Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections, suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4- CD8- DN T cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute to the development of targeted HIV eradication strategies.

Tebentafusp, A TCR/Anti-CD3 Bispecific Fusion Protein Targeting gp100, Potently Activated Antitumor Immune Responses in Patients with Metastatic Melanoma.

PURPOSE: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. PATIENTS AND METHODS: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment. RESULTS: Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway-related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival. CONCLUSIONS: These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

CD52-negative T cells predict acute graft-versus-host disease after an alemtuzumab-based conditioning regimen.

Allogeneic haematopoietic stem cell transplantation (HSCT) after a reduced-intensity conditioning (RIC) regimen with fludarabine, melphalan and alemtuzmab is an effective therapy for haematological malignancies. Alemtuzumab, a monoclonal antibody against CD52, a glycosylphosphatidylinositol-anchor-bound surface protein on lymphocytes, depletes T cells to prevent graft-versus-host disease (GVHD). Despite this, acute and chronic GVHD (a/cGVHD) remain life-threatening complications after HSCT. The aim of the present study was to identify parameters to predict GVHD. In 69 patients after HSCT, T-cell subsets were functionally analysed. Reconstitution of CD52neg T cells and CD52neg regulatory T cells (Tregs) correlated with onset, severity and clinical course of aGVHD. Patients with aGVHD showed significantly lower levels of CD52pos T cells compared to patients with cGVHD or without GVHD (P < 0·001). Analysis of T-cell reconstitution revealed a percentage of <40% of CD52pos CD4pos T cells or CD52pos Tregs at day +50 as a risk factor for the development of aGVHD. In contrast, CD52neg Tregs showed significant decreased levels of glycoprotein A repetitions predominant (GARP; P < 0·001), glucocorticoid-induced TNFR-related protein (GITR; P < 0·001), chemokine receptor (CXCR3; P = 0·023), C-C chemokine receptor type 5 (CCR5; P = 0·004), but increased levels of immunoglobulin-like transcript 3 (ILT3; P = 0·001), as well as a reduced suppressive capacity. We conclude that reconstitution of CD52neg T cells and CD52neg Tregs is a risk factor for development of aGVHD.

Soluble PD1 levels are increased with disease activity in paediatric onset autoimmune hepatitis and inflammatory bowel disease.

Introduction: Immune mediated liver diseases entail a broad category which are associated with increased morbidity and mortality amongst the paediatric population. Programmed Death 1 (PD1) is an inhibitory receptor mainly expressed by T cells, and when activated shed into plasma as soluble PD1(sPD1). The AIM of this study was to evaluate sPD1 levels in plasma of paediatric patients with Autoimmune Hepatitis (AIH), Primary Sclerosing Cholangitis (PSC), AIH and PSC overlap, Inflammatory Bowel Disease (IBD) alone, and concurrent PSC/IBD and AIH/IBD in order to identify a biomarker to response or predict relapse verses remission.Methods: Plasma samples were collected from 41 paediatric patients. AIH patients were further categorized into active, incomplete responders and responders, based on response to standard therapy. sPD1 levels were measured and compared between PSC, PSC/AIH, IBD alone, PSC/IBD and AIH/IBD patients and between active AIH, incomplete responders and responders. Flow cytometry was performed to further analyze CD45RA+, CD3CD4, CD8, CCR7, CXCR3, CD38 and PD1.Results: In the AIH group, those with active disease demonstrated a significantly higher sPD1 levels in comparison to responders (*p > .001). However, the incomplete responders didn't show a reduction in sPD1 in comparison to active AIH and patients with IBD alone. Interestingly, patients with PSC showed significantly lower level of sPD1 compared to active AIH (*p < .002), whereas, patients with PSC in conjunction with AIH (*p < .006) or IBD (*p < .02) demonstrated a significant increase in sPD1. In addition, we have observed increased levels of circulating CD4 and CD8 bound PD1 in active AIH but not in PSC or responders suggesting T cells activation. CD4+ PD1 double positive cells demonstrated increased expression of CXCR3. Thus, suggesting the activation of PD1 + T cells is mediating through CXCR3 in Autoimmune hepatitis.Conclusions: Our study demonstrates that sPD1 levels correlate with active disease state of AIH and IBD. sPD1 levels did not correlate with PSC. However, PSC in conjunction with AIH or IBD showed higher levels of sPD1. This suggests that T cell activation plays a critical role in active AIH and IBD but not in PSC. Soluble PDI levels could be used as a clinical biomarker to assess response in patients with AIH and for prospectively monitoring PSC patients for development of IBD or AIH.

Circulating CXCR3+ Tfh cells positively correlate with neutralizing antibody responses in HCV-infected patients.

Circulating T follicular helper (cTfh) cells have been identified as counterparts of germinal center Tfh (GC Tfh) cells in humans and can support T-dependent B cell maturation and antibody production in vitro. However, the role of cTfh cells in neutralizing antibody (nAb) responses in HCV infection remains unclear. Here, we characterized the phenotype and function of cTfh cells and demonstrated the associations of cTfh cells and their subsets with nAb responses in HCV infection. A total of 38 HCV-infected individuals and 28 healthy controls were enrolled from a pool of injection drug users. The frequency and function of blood Tfh cells were analyzed by flow cytometry. The titers and breadths of serum nAbs were measured using HCV pseudo-particle neutralization assays. Herein, we report several key observations. First, HCV infection skewed cTfh toward CXCR3+ cTfh cell differentiation. Second, the frequency of CXCR3+ cTfh cells positively correlated with HCV nAb titers and breadths. Third, CXCR3+ cTfh cells showed higher expression of Tfh-associated molecules (PD-1, ICOS, IL-21, Bcl-6) compared with CXCR3- cTfh cells from individuals with HCV infection. Coculture of cTfh cells and autologous memory B cells in vitro indicated that CXCR3+ cTfh cells show a superior ability to support HCV E2-specific B cell expansion compared with CXCR3- cTfh cells from individuals with HCV infection. HCV infection skews cTfh cells toward CXCR3-biased Tfh cell differentiation, which positively correlates with the magnitude and breadth of the HCV nAb response. It is our hope that these findings will provide insights for the rational design of a nAb-based HCV vaccine.

Mig chemokine in primary biliary cirrhosis.

Different studies investigated about the role of T-helper 1 cytokines and chemokines in primary biliary cirrhosis (PBC). Animal models with autoimmune cholangitis have been used to investigate the involvement of (C-X-C motif) receptor (CXCR)3 and its ligand (C-X-C motif) ligand (CXCL)9/monokine induced by interferon (IFN)-γ (MIG) in the pathogenesis of PBC, suggesting a contribution of MIG in the development of PBC. In patients with PBC, in particular at the level of the portal areas of diseased livers, MIG expression and CXCR3+ cells have been found. MIG is positively associated with the severity of liver fibrosis. In PBC, circulating MIG levels and CXCR3+ cells are related with the progression of the disease; in fact, their expression increases significantly in PBC patients with respect to controls. Furthermore, it has been shown a significant reduction of these chemokines in the serum of PBC patients after treatment with ursodeoxycholic acid.

Impaired Th17 cell proliferation and decreased pro-inflammatory cytokine production in CXCR3/CXCR4 double-deficient mice of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is a common observed infection, affecting approximately 75% of women of reproductive age. Drug resistance represents a troublesome stumbling block associated with VVC therapy. Thus the aim of the present study was to provide information regarding the selection of potential drug targets for VVC. CXCR3-, CXCR4-, or CXCR/CXCR4 double-deficient mouse models of VVC were subsequently established, with changes to the load of Candida Albicans evaluated accordingly. The biological behaviors of the vaginal epithelial cells were characterized in response to the CXCR3-, CXCR4-, or CXCR3/CXCR4 double-knockout in vivo. Our initial observations revealed that in mice with VVC, CXCR3-, CXCR4-, or CXCR3 - CXCR4 double-knockout resulted in a decreased load of C. Albicans as well as reduced levels and proportion of Th17 cells. Proinflammatory cytokine production was found to be inhibited by CXCR3-, CXCR4-, or CXCR3/CXCR4 double-knockout whereby the mRNA and protein expressions CXCR3, CXCR4, IL-17, IL-6, and TNF-α exhibited decreased levels. CXCR3-, CXCR4-, or CXCR3/CXCR4 double-knockout appeared to function as positive proliferation factors, while playing a negative role in the processes of apoptosis and the cell cycle of vaginal epithelial cells. Taken together, the key findings of the study suggested that CXCR3/CXCR4 double-knockout could act to hinder the progression of VVC, highlighting its promise as a novel therapeutic target in the treatment of VVC. CXCR3 and CXCR4 genes may regulate Th17/IL-17 immune inflammatory pathways to participate in antifungal immunity.

MIG in psoriatic arthritis.

Monokine induced by interferon (IFN)-γ (MIG) / chemokine (C-X-C motif) ligand 9 (CXCL)9 is involved in the pathogenesis of psoriatic arthritis (PsA). It was demonstrated that both blood plasma-derived dendritic cells (pDCs) and pDCs isolated from rheumatoid arthritis (RA) and PsA synovial fluid (SF), expressed CXC receptor (R) 3 and CXCR4, and that the chemotaxis of blood-derived pDCs is stimulated by MIG, (IFN)-γ-inducible protein 10 (IP-10)/CXCL10, IFN-inducible T-cell α chemoattractant (I-TAC) )/CXCL11 and stromal cell-derived factor 1 (SDF-1)/ CXCL12, present in RA and PsA SF. In PsA patients have been found a Th1 immune predominance at early stage of disease, while a reduction of these chemokines has been observed in long lasting PsA, with a significant increase of monocyte chemoattractant protein-1/IP-10 ratio. This suggest a shift from Th1 to the Th2 immune response in long lasting PsA. High levels of MIG has been found in patients with PsA and autoimmune thyroiditis too. This chemokine has been proposed as a useful marker to monitor the activity as well the progression of PsA. Efforts have been made to modulate or prevent the production of MIG in PsA aiming to alter the course of the disease.

CXCR3+ Monocytes Increase Significantly in Graft Blood Compared to Peripheral Blood in Patients With Stable Kidney Graft Function.

INTRODUCTION: We have recently reported that some lymphocyte populations do not maintain the same proportion in kidney graft blood as in peripheral blood, despite a stable function of the transplanted kidney. These results suggest that a comparative study between leukocyte cells from graft blood and those obtained from peripheral blood could provide information about the inflammatory state of the transplanted organ. In this work we selected the population of CD4+ lymphocytes and monocytes expressing CXCR3 to test this hypothesis. MATERIAL AND METHODS: The study was performed by flow cytometry during month 3, 6, and 12 after transplantation in 58 patients who received an isolated kidney transplant and the same immunosuppressive regimen. The peripheral blood sample was obtained by venipuncture and the graft blood by fine needle aspiration. RESULTS: We found a significant percentage decrease in CXCR3+ monocytes throughout the first year of transplantation in peripheral blood (15.9 ± 20.7 vs. 12.6 ± 12.4 vs. 6.3 ± 9.0, at 3, 6, and 12 months, respectively; P = .001), whereas the percentage of CXCR3+ monocytes in graft blood did not change over this period. This situation resulted in a significant percentage difference between the CXCR3+ monocytes from the graft blood and those from the peripheral blood at the sixth (15.8 ± 8.1 vs. 12.6 ± 12.4, respectively; P = .008) and 12th months (12.9 ± 8.1 vs. 6.3 ± 9.0, respectively; P < .001). CONCLUSIONS: Therefore, we can conclude that the significant percentage increase of CXCR3+ monocytes in graft blood with respect to peripheral blood suggests the presence of inflammatory activity despite renal function being stable during the second half of the first year post-transplantation.

Antibodies against chemokine receptors CXCR3 and CXCR4 predict progressive deterioration of lung function in patients with systemic sclerosis.

BACKGROUND: The chemokine receptors CXCR3 and CXCR4 are involved in the pathogenesis of fibrosis, a key feature of systemic sclerosis (SSc). It is hypothesized that immunoglobulin (Ig)G antibodies (abs) against these two receptors are present in patients with SSc and are associated with clinical findings. METHODS: Anti-CXCR3 and anti-CXCR4 ab levels were measured in 449 sera from 327 SSc patients and in 234 sera from healthy donors (HD) by enzyme-linked immunosorbent assay (ELISA). In SSc, ab levels were compared with clinical data in a cross-sectional and longitudinal setting. Protein expression of CXCR3 and CXCR4 on peripheral blood mononuclear cells (PBMCs) was analyzed in 17 SSc patients and 8 HD by flow cytometry. RESULTS: Anti-CXCR3 and anti-CXCR4 ab levels were different among SSc subgroups compared with HD and were highest in diffuse SSc patients. The ab levels strongly correlated with each other (r = 0.85). Patients with SSc-related interstitial lung disease (SSc-ILD) exhibited higher ab levels which negatively correlated with lung function parameters (e.g., r = -0.5 and r = -0.43 for predicted vital capacity, respectively). However, patients with deterioration of lung function showed lower anti-CXCR3/4 ab levels compared with those with stable disease. Frequencies and median fluorescence intensities (MFI) of CXCR3+ and CXCR4+ PBMCs were lower in SSc patients compared with HD and correlated with the severity of skin and lung fibrosis. They correlated with the severity of skin and lung fibrosis. CONCLUSIONS: Anti-CXCR3/4 abs and their corresponding receptors are linked with the severity of SSc-ILD. Antibody levels discriminate patients with stable or decreasing lung function and could be used for risk stratification.

CXCL10/CXCR3 axis is associated with disease activity and the development of mucocutaneous lesions in patients with Behçet's disease.

The objective of this study was to investigate CXC chemokines and its receptor in patients with Behcet's disease (BD) and their associations with disease activity. Blood samples were collected from 109 BD patients and 36 age- and sex-matched healthy controls (HCs). Twenty-two follow-up blood samples were collected in BD patients. Serum CXC chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13 and CXCL16) and cell surface marker expression (CD3, CD4 and CXCR3) in peripheral blood mononuclear cells (PBMCs) were assayed. Clinical features including disease activity were evaluated at the time of blood collection. CXCR3 expression in skin and intestinal lesions from BD patients and HCs was assessed via immunohistochemistry. Serum CXCL10 levels were correlated with disease activity in terms of Behçet's Disease Current Activity Form (BDCAF) (p < 0.001). In follow-up BD patients, changes in serum CXCL10 levels tended to be correlated with those of BDCAF. The percentage of CXCR3 expression in CD3-positive cells in PBMCs was inversely correlated with serum CXCL10 levels in BD patients (p = 0.022). By immunohistochemistry, the number of CXCR3-positive mononuclear cells was higher in skin and intestinal lesions of BD patients than in those of HCs. These results suggest that the CXCL10/CXCR3 axis may contribute to the pathogenesis of BD.

High frequencies of circulating Tfh-Th17 cells in myasthenia gravis patients.

Recent studies show that the frequencies of circulating follicullar helper T (cTfh) cells are significantly higher in myasthenia gravis (MG) patients compared with healthy controls (HC). And, they are positively correlated with levels of serum anti-acetylcholine receptor antibody (anti-AchR Ab). It is unclear whether cTfh cell subset frequencies are altered and what role they play in MG patients. In order to clarify this, we examined the frequencies of cTfh cell counterparts, their subsets, and circulating plasmablasts in MG patients by flow cytometry. We determined the concentrations of serum anti-AChR Ab by enzyme-linked immunosorbent assay (ELISA). We assayed the function of cTfh cell subsets by flow cytometry and real-time polymerase chain reaction (RT-PCR). We found higher frequencies of cTfh cell counterparts, cTfh-Th17 cells, and plasmablasts in MG patients compared with HC. The frequencies of cTfh cell counterparts and cTfh-Th17 cells were positively correlated with the frequencies of plasmablasts and the concentrations of anti-AChR Ab in MG patients. Functional assays showed that activated cTfh-Th17 cells highly expressed key molecular features of Tfh cells including ICOS, PD-1, and IL-21. Results indicate that, just like cTfh cell counterparts, cTfh-Th17 cells may play a role in the immunopathogenesis and the production of anti-AChR Ab of MG.

CXCL10 Is a Circulating Inflammatory Marker in Patients with Advanced Heart Failure: a Pilot Study.

Chemokines are involved in the remodeling of the heart; however, their significance as biomarkers in heart failure is unknown. We observed that circulating CXCR3 receptor chemokines CXCL9 and CXCL10 in a rat model of heart failure were increased 1 week after myocardial infarction. CXCL10 was also increased in both remote and infarcted regions of the heart and remained elevated at 16 weeks; CXCL9 was elevated in the remote area at 1 week. In humans, hierarchical clustering and principal component analysis revealed that circulating CXCL10, MIP-1α, and CD40 ligand were the best indicators for differentiating healthy and heart failure subjects. Serum CXCL10 levels were increased in patients with symptomatic heart failure as indexed by NYHA classification II through IV. The presence of CXCL10, MIP-1α, and CD40 ligand appears to be dominant in patients with advanced heart failure. These findings identify a distinct profile of inflammatory mediators in heart failure patients.

The Usefulness of Serum CXCR3 Ligands for Evaluating the Early Treatment Response in Tuberculosis: A Longitudinal Cohort Study.

Cell-mediated immunity plays an important role in the pathobiology of tuberculosis (TB). The ligands for CXC chemokine receptor 3 (CXCR3) activate the T-helper type 1 lymphocyte pathway. The CXCR3 ligands are reportedly useful clinical markers for the diagnosis and follow-up of TB. The objective of this study was to assess the utility of CXCR3 ligands for evaluating early treatment responses in TB.We recruited 88 patients who underwent antituberculous chemotherapy. The serum levels of interferon (IFN)-γ and the CXCR3 ligands CXCL9 (monokine induced by IFN-γ [MIG]), CXCL10 (IFN-γ-inducible 10-kDa protein [IP-10]), and CXCL11 (IFN-inducible T-cell α chemoattractant [I-TAC]) were measured before and 2 months after the start of treatment. Treatment responses were divided into "fast" and "slow" based on the clinical, radiological, and bacteriological improvement at 2 months. A change in level of 20% or more at 2 months was defined as "significant."In patients with treatment success, 58 patients exhibited a fast response and 20 patients exhibited a slow response. Treatment failure occurred in 5 patients, and the diagnoses were changed to non-TB diseases in 5 patients. The levels of all CXCR3 ligands significantly decreased in the fast-response group (P < 0.01) but did not decrease in the other groups. IFN-γ levels showed no significant changes. The ability of significant decreases in marker levels to predict a fast response was evaluated. CXCL9 showed a sensitivity of 83%, and CXCL10 showed a specificity of 100%. Use of various combinations of CXCR3 ligands resulted in improvements in sensitivity (88%-93%), while specificity (92%-96%) was similar to that using single CXCR3 ligands. The decreases in CXCR3 ligand levels were less marked in the 2-month Mycobacterium tuberculosis culture-positive group than in the culture-negative group. There were significant differences in treatment outcomes in terms of 2-month culture positivity (P < 0.001), the significance of CXCL9 decreases (P < 0.01), and the significance of CXCL11 decreases (P < 0.05).In conclusion, CXCR3 ligands may be useful surrogate markers for the evaluation of early treatment response and showed utility as indicators of possible treatment failure in TB.

Virus-Specific CD4+ T Cells Have Functional and Phenotypic Characteristics of Follicular T-Helper Cells in Patients With Acute and Chronic HCV Infections.

BACKGROUND & AIMS: Follicular T-helper (Tfh) cells contribute to pathogen-specific antibody responses by providing maturation signals to B cells. In mice with viral infections, virus-specific Tfh cells expand and are required to contain the infection. However, less is known about human virus-specific Tfh cells or their functions during infection. We investigated whether virus-specific CD4+ T cells from patients with hepatitis C virus (HCV) infection had phenotypic or functional features of Tfh cells and contribute to the production of HCV-specific antibodies. METHODS: We collected blood samples from patients with acute and chronic HCV infection and healthy individuals (controls). We performed MHC class II tetramer analyses, assays to detect intracellular cytokines in response to HCV exposure, and analyses to quantify HCV-specific antibodies. In addition, we collected liver tissues from patients with chronic HCV infection or nonviral liver disease to analyze markers of Tfh cells. RESULTS: HCV-specific CD4+ T cells from patients with acute HCV infection expressed markers of Tfh cells and secreted interleukin 21 in response to HCV exposure. Longitudinal analyses of HCV-specific T-cell responses and antibody responses showed an association between expression of inducible T-cell co-stimulator and induction of virus-specific antibodies in patients with acute HCV infection. Markers of Tfh cells were barely detectable in the peripheral blood samples from patients with chronic HCV infection, but were detected in liver tissues. CONCLUSIONS: Virus-specific Tfh cells can be detected in blood samples from patients with acute HCV infection; inducible T-cell co-stimulator expression correlates with production of HCV-specific antibodies. In patients with chronic infection, Tfh cells seem to disappear from the blood but are detectable in the liver.