Antibody-Suppressor CXCR5+CD8+ T Cells Are More Potent Regulators of Humoral Alloimmunity after Kidney Transplant in Mice Compared to CD4+ Regulatory T Cells.

Adoptive cell therapy (ACT), especially with CD4+ regulatory T cells (CD4+ Tregs), is an emerging therapeutic strategy to minimize immunosuppression and promote long-term allograft acceptance, although much research remains to realize its potential. In this study, we investigated the potency of novel Ab-suppressor CXCR5+CD8+ T cells (CD8+ TAb-supp) in comparison with conventional CD25highFoxp3+CD4+ Tregs for suppression of humoral alloimmunity in a murine kidney transplant (KTx) model of Ab-mediated rejection (AMR). We examined quantity of peripheral blood, splenic and graft-infiltrating CD8+ TAb-supp, and CD4+ Tregs in KTx recipients and found that high alloantibody-producing CCR5 knockout KTx recipients have significantly fewer post-transplant peripheral blood and splenic CD8+ TAb-supp, as well as fewer splenic and graft-infiltrating CD4+ Tregs compared with wild-type KTx recipients.

CXCR5 + CD8 + T Cell-mediated Suppression of Humoral Alloimmunity and AMR in Mice Is Optimized With mTOR and Impaired With Calcineurin Inhibition.

Background: Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT.

Methods: We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant.

Invariant NKT Cells Promote the Development of Highly Cytotoxic Multipotent CXCR3CCR4CD8 T Cells That Mediate Rapid Hepatocyte Allograft Rejection.

Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8 T cells, with or without CD4 T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4 T cells.

CXCR5CD8 T cells: A Review of their Antibody Regulatory Functions and Clinical Correlations.

CD8 T cells have conventionally been studied in relationship to pathogen or tumor clearance. Recent reports have identified novel functions of CXCR5CD8 T cells that can home to lymphoid follicles, a key site of antibody production. In this review we provide an in-depth analysis of conflicting reports regarding the impact of CXCR5CD8 T cells on antibody production and examine the data supporting a role for antibody-enhancement (B cell "helper") and antibody-downregulation (antibody-suppressor) by CXCR5CD8 T cell subsets.

Inverse Association Between the Quantity of Human Peripheral Blood CXCR5+IFN-γ+CD8+ T Cells With De Novo DSA Production in the First Year After Kidney Transplant.

Background: We recently reported that a novel CXCR5IFN-γCD8 T-cell subset significantly inhibits posttransplant alloantibody production in a murine transplant model. These findings prompted the current study to investigate the association of human CD8 T cells with the same phenotype with the development of de novo donor-specific antibody (DSA) after kidney transplantation.

Methods: In the current studies, we prospectively and serially analyzed peripheral blood CD8 and CD4 T-cell subsets and monitored for the development of de novo DSA in kidney transplant recipients during the first-year posttransplant.

Antibody-suppressor CD8+ T Cells Require CXCR5.

Background: We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function.

Methods: C57BL/6 mice were transplanted with FVB/N hepatocytes.

Critical Role of Macrophage FcγR Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known.

Unique CD8+ T Cell-Mediated Immune Responses Primed in the Liver.

Background: The liver immune environment is tightly regulated to balance immune activation with immune tolerance. Understanding the dominant immune pathways initiated in the liver is important because the liver is a site for cell transplantation, such as for islet and hepatocyte transplantation. The purpose of this study is to examine the consequences of alloimmune stimulation when allogeneic cells are transplanted to the liver in comparison to a different immune locale, such as the kidney.

mTOR Inhibition Suppresses Posttransplant Alloantibody Production Through Direct Inhibition of Alloprimed B Cells and Sparing of CD8+ Antibody-Suppressing T cells.

Background: De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity.

Methods: Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes.

Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells.

Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1.

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta.

Introduction: Although IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.

Methods: Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture.

Cytotoxic effector function of CD4-independent, CD8(+) T cells is mediated by TNF-α/TNFR.

Background: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms.

Inhibition of recall responses through complementary therapies targeting CD8+ T-cell- and alloantibody-dependent allocytotoxicity in sensitized transplant recipients.

Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies.

CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production.

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production.

Modulation of SOCS protein expression influences the interferon responsiveness of human melanoma cells.

Background: Endogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells.

Methods: Basal and interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells.

Interleukin-29 binds to melanoma cells inducing Jak-STAT signal transduction and apoptosis.

Interleukin-29 (IL-29) is a member of the type III IFN family that has been shown to have antiviral activity and to inhibit cell growth. Melanoma cell lines were tested for expression of the IL-29 receptor (IL-29R) and their response to IL-29. Expression of IL-28R1 and IL-10R2, components of IL-29R, was evaluated using reverse transcription-PCR.

Gene expression profiling reveals similarities between the in vitro and in vivo responses of immune effector cells to IFN-alpha.

Purpose: The precise molecular targets of IFN-alpha therapy in the context of malignant melanoma are unknown but seem to involve signal transducers and activators of transcription 1 signal transduction within host immune effector cells. We hypothesized that the in vitro transcriptional response of patient peripheral blood mononuclear cells (PBMC) to IFN-alpha would be similar to the in vivo response to treatment with high-dose IFN-alpha.

Experimental Design: The gene expression profiles of PBMCs and immune cell subsets treated in vitro with IFN-alpha were evaluated, as were PBMCs obtained from melanoma patients receiving adjuvant IFN-alpha.

Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells.

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection.

IFN-alpha-2b-induced signal transduction and gene regulation in patient peripheral blood mononuclear cells is not enhanced by a dose increase from 5 to 10 megaunits/m2.

Purpose: The precise molecular targets of IFN-alpha therapy of melanoma are unknown but likely involve signal transducer and activator of transcription (STAT) 1 signal transduction within host immune effector cells. We hypothesized that intermediate and high doses of IFN-alpha would be equally effective in activating patient immune cells.

Experimental Design: Eleven metastatic melanoma patients who were enrolled in a clinical trial of bevacizumab in combination with escalating doses of IFN-alpha-2b (5 megaunits/m(2) and then 10 megaunits/m(2)) were included in the study.

Melanoma cells exhibit variable signal transducer and activator of transcription 1 phosphorylation and a reduced response to IFN-alpha compared with immune effector cells.

Purpose: IFN-alpha is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1 activation allows melanoma cells to evade the direct actions of IFN-alpha.

Experimental Design: Tyr(701)-phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-alpha-stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC).

STAT1-dependent and STAT1-independent gene expression in murine immune cells following stimulation with interferon-alpha.

Purpose: The precise molecular targets of interferon-alpha (IFN-alpha) therapy of melanoma are unknown but likely involve signal transducer and activator of transcription 1 (STAT1) signal transduction within host immune effector cells. We hypothesized that microarray analysis could be utilized to identify candidate molecular targets important for mediating the anti-tumor effect of exogenously administered IFN-alpha.

Experimental Methods: To identify the STAT1-dependent genes regulated by IFN-alpha, the gene expression profile of splenocytes from wild type (WT) and STAT1(-/-) mice was characterized.

IFN-alpha-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins.

Proteins belonging to the suppressors of cytokine signaling (SOCS) family have been shown to regulate cytokine signal transduction in various cell types but their role in modulating the response of immune cells to IFN-alpha has not been fully explored. We hypothesized that SOCS proteins would inhibit the antitumor activity of IFN-alpha-stimulated immune cells. Transcripts for SOCS1, SOCS2, SOCS3, and cytokine-inducible Src homology 2-containing protein were identified in total human PBMC (PBMCs, NK cells, and T cells) within 1-2 h of stimulation with IFN-alpha (10(3)-10(5) U/ml).