Adoptive Transfer

Resolution of acute hepatitis B virus (HBV) infection re-Adoptive immunity transfer has been reported to be quires adequate B-and T-cell responses, which lead to the effective in clearing chronic hepatitis B virus (HBV) inproduction of protective antibodies, as well as a broad-based fection. Two hundred twenty-six patients who received T-cell response against multiple HBV antigenic determinants allogeneic bone marrow transplantation (BMT) between located on the viral envelope, nucleocapsid, and polymerase May 1990 and September 1995 were screened for hepatigene products. 6 Similar responses have been found in chronitis B markers. Twenty-one patients were hepatitis B surcally infected patients during spontaneous or interferon-inface antigen (HBsAg) positive before BMT. The median duced HBeAg seroconversion, but the cytotoxic T lymphocyte follow-up period was 20 months (range, 2-59 months). response is absent or weak in other chronically infected pa-Two of these patients had sustained clearance of HBV tients. 7 infection after transplantation. Both patients were hep-Recently, it has been shown that adoptive transfer of imatitis B e antigen (HBeAg)-negative, hepatitis B e antimunity to HBV can be accomplished by bone marrow transbody (anti-HBe)-positive, and serum HBV DNA-negaplantation (BMT) from donors who are immune because of tive (by dot-blot hybridization) before BMT. Both had past infection or active immunization. 8-11 Furthermore, cleara flare in the serum alanine transaminase (ALT) level ance of chronic HBV infection has been reported after BMT around the time of HBsAg clearance. Sustained clearfrom immune donors, possibly through the transfer of both ance of HBsAg was observed in 2 of the 5 patients who humoral and cellular immunity. 12,13 This has led to the sugreceived hepatitis B surface antibody (anti-HBs)-posigestion that adoptive immunity transfer may be employed to tive marrow but in none of the 16 patients who received treat chronic HBV infection. However, it is not clear how anti-HBs-negative marrow (P õ .05). One additional pafrequent clearance of chronic HBV infection occurs after BMT tient who received anti-HBs-positive marrow had tranfrom immune donors. In addition, there is concern that sucsient HBsAg seroconversion. Among the 18 patients who cessful transfer of the cytotoxic T lymphocyte response may remained persistently HBsAg-positive after BMT, 3 had lead to massive lysis of infected hepatocytes, resulting in HBeAg seroconversion and 3 had reversion to HBeAg acute liver failure. 8 Clearly, the major obstacle to the use of positivity. In this study, we found a significant associaadoptive immunity transfer as a treatment for chronic HBV tion between clearance of HBV infection and anti-HBsinfection is the high morbidity and mortality associated with positive bone marrow donors. Adoptive immunity trans-BMT. The aim of this study was to determine the serological fer is effective in clearing HBV from patients with and clinical outcome of HBsAg-positive BMT recipients in chronic HBV infection. (HEPATOLOGY 1997;25:1497-1501.) relation to the HBV status (carrier/immune/seronegative) of the donors. Chronic hepatitis B infection is a serious global health problem affecting approximately 300 million individuals. Of these, many will die of cirrhosis or hepatocellular carcinoma.

Effector cells derived from central memory CD8 + T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with thi...

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8 + T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8 + T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8 + T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy. Nonstandard abbreviations used: ACT, adoptive cell transfer; PMB, polymyxin B; rFPhgp100, recombinant fowlpox virus encoding human gp100; rhIL-2, recombinant human IL-2; TBI, total body irradiation.

Graft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8+ pmel-1 or CD4+ TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma–bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persi...

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA− and CD45RO+ after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA+ and CD45RO− phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited...

Many tissues including blood, skin, gut and germ cells are continuously maintained by tissue stem cells 1-2 . Under certain conditions, however, other organs can undergo repair using stem-cell-like progenitors generated by cell de-differentiation 3 . Cell fates have been broadened experimentally [4][5][6][7] , but mechanisms allowing de-differentiation to a stem cell state are poorly known. Germline stem cells begin to differentiate by forming interconnected germ cell cysts (cystocytes), and under certain letters to nature

Interleukin-7 (IL-7) plays a key role in maturation and function of both T and B cells. We investigate the potential use of recombinant human IL-7 for facilitation of graft-versus-leukemia (GVL) effects mediated by T cells following transplantation in a murine model. Administration of IL-7 in vivo to allogeneic-transplanted mice improved disease-free survival: 67% of mice treated with IL-7 remained alive and disease free for more than 60 days, in comparison to 17% of the controls (Po0.05). Similar results were obtained when C57BL/6 spleen cells sensitized against irradiated B-cell leukemia (BCL 1 ) cells in the presence of IL-7 were transplanted to F 1 mice, followed by IL-7 treatment in vivo. Of the BALB/c mice that received spleen cells from F 1 mice treated with IL-7 following transplantation of C57BL/6 spleen cells sensitized with irradiated BCL 1 in the presence of IL-7, only 29% developed leukemia, as compared to 79% in the control group (Po0.05). Mice treated with IL-7 showed increased splenic and thymic cellularity and improved T cell-dependent proliferative responses compared to the controls (Po0.05). IL-7 may provide a novel tool to enhance immune reconstitution following transplantation of mismatched stem cells and for enhancement of GVL effects mediated by alloreactive lymphocytes.

Deposition of immune complexes (IC) triggers FcγR-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif−/− mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif−/− mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT control...

Purpose: LTX-315 is a first-in-class, 9-mer membranolytic peptide that has shown potent immunomodulatory properties in preclinical models. We conducted a phase I dose-escalating study of intratumoral LTX-315 administration in patients with advanced solid tumors. Patients and Methods: Thirty-nine patients were enrolled, receiving LTX-315 injections into accessible tumors. The primary objective was to assess the safety and tolerability of this approach, with antitumor and immunomodulatory activity as secondary objectives. Tumor biopsies were collected at baseline and posttreatment for analysis of immunologic parameters. Results: The most common treatment-related grade 1–2 adverse events were vascular disorders including transient hypotension (18 patients, 46%), flushing (11 patients, 28%), and injection site reactions in 38% of patients. The most common grade 3 LTX-315–related toxicities were hypersensitivity or anaphylaxis (4 patients, 10%). Analysis of immune endpoints in serial bio...

To define the role of IFN-gamma in the control of acute infection with a noncytopathogenic virus, mice with targeted defects of the genes encoding IFN-gamma, perforin, or both were infected i.v. with two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in wild-type mice. Our results reveal that IFN-gamma is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus-specific TCR are adoptively transferred before virus challenge, indicating that the disease is the result of an unfortunate balance between virus replication in internal organs, e.g., liver and spleen, and the host response; resetting this balance by increasing host responsiveness will again lead to a rapidly controlled infection and limited tissue damage. Thus, the presence or absence of IFN-gamma determines whether CTLs will clear infection with this noncytopathogenic virus or induce severe immunopathology.

Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-γ transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts wi...

Chilakamarti V. Ramana,* Matthew P. DeBerge,* Aseem Kumar, Christopher S. Alia, Joan E. Durbin, and Richard I. Enelow Department of Medicine, Geisel School of Medicine at Dartmouth College, Lebanon, New Hampshire; Department of Pathology, Feinberg School of Medicine at Northwestern University, Chicago, Illinois; Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada; Department of Medicine, Yale University School of Medicine, New Haven, Connecticut; Department of Pathology, Rutgers-New Jersey Medical School of Medicine, Newark, New Jersey; and Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth College, Lebanon, New Hampshire

Immunosurveillance of mucosal sites presents immune cells with challenges not encountered in the periphery. T cells in the gut must distinguish enteric pathogens from innocuous non-self Ag derived from food or commensal bacteria. The mechanisms that regulate T cells in the gut remain incompletely understood. We assessed the effect of the Peyer’s patch microenvironment on T cell responses to chemokines. Chemokines are believed to play an important role during T cell priming by facilitating T cell migration into and within lymphoid tissues as well as T cell encounter and interaction with APCs. We found a profound suppression of chemokine-stimulated T cell chemotaxis and actin polymerization in Peyer’s patch relative to lymph node. Chemokine hyporesponsiveness is imposed upon T cells within hours of their entry into Peyer’s patches and is reversed following their removal. Suppression was not restricted to chemokine stimulation, as T cell responses to Con A and PMA were also suppressed....

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4 ؉ and CD8 ؉ T cells are markedly different. Gads ؊/؊ CD4 ؉ T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads ؊/؊ CD4 ؉ T cells continued to proliferate at a higher rate than wild-type CD4 ؉ T cells, demonstrating a defect in homeostatic proliferation. Gads ؊/؊ CD8 ؉ T cells had a memory-like phenotype, produced IFN-␥ in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads ؊/؊ T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads ؊/؊ CD4 ؉ T cells, but not CD8 ؉ T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads ؊/؊ CD4 ؉ T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4 ؉ and CD8 ؉ T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4 ؉ T cells.

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4+ and CD8+ T cells are markedly different. Gads−/− CD4+ T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads−/− CD4+ T cells continued to proliferate at a higher rate than wild-type CD4+ T cells, demonstrating a defect in homeostatic proliferation. Gads−/− CD8+ T cells had a memory-like phenotype, produced IFN-γ in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads−/− T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads−/− CD4+ T cells, but not CD8+ T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid tu...

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Conventional dendritic cells (cDC) are necessary and sufficient to drive mixed maladaptive Th2/Th17 immune responses toward aeroallergens in experimental allergy models. Previous studies suggest that the anaphylatoxin C3a promotes, whereas C5a protects from the development of maladaptive immunity during allergen sensitization. However, only limited evidence exists that such effects are directly mediated through anaphylatoxin-receptor signaling in cDCs. In this study, we assessed the impact of C3a and C5a on cDC-mediated induction pulmonary allergy by adoptively transferring house dust mite (HDM)-pulsed bone marrow-derived DCs (BMDC) from wild-type (WT) C3aR 2/2 , C5aR1 2/2 , or C3aR 2/2 /C5aR1 2/2 into WT mice. Transfer of HDM-pulsed WT BMDCs promoted a strong asthmatic phenotype characterized by marked airway resistance, strong Th2 cytokine, and mucus production, as well as mixed eosinophilic and neurophilic airway inflammation. Surprisingly, C3aR 2/2 cDCs induced a strong allergic phenotype, but no IL-17A production, whereas HDM-pulsed C5aR1 2/2 cDCs failed to drive pulmonary allergy. Transfer of C3aR 2/2 /C5aR1 2/2 cDCs resulted in a slightly reduced allergic phenotype associated with increased IFN-g production. Mechanistically, C3aR and C5aR1 signaling is required for IL-23 production from HDM-pulsed BMDCs in vitro. Furthermore, C3aR 2/2 BMDCs produced less IL-1b. The mechanisms underlying the failure of C5aR1 2/2 BMDCs to induce experimental allergy include a reduced capability to migrate into the lung tissue and a decreased potency to direct pulmonary homing of effector T cells. Thus, we uncovered a crucial role for C5a, but only a minor role for C3a in BMDC-mediated pulmonary allergy, suggesting that BMDCs inappropriately reflect the impact of complement on lung cDC-mediated allergic asthma development.

There has been interest in generating T cells expressing chimeric artificial receptors (CARs) targeting CD19/CD20 antigens to treat B-cell lymphomas. If successful, however, this approach would likely impair humoral immunity because T cells may persist long-term. Most low-grade lymphoma and chronic lymphocytic leukemia (B-CLL) cells express monoclonal immunoglobulins carrying either κ or λ light chains. We, therefore, explored whether T lymphocytes could be genetically modified to target the tumor-associated light chain, sparing B lymphocytes expressing the reciprocal light chain, and consequently reduce impairment of humoral immunity. We found that T lymphocytes expressing the anti-κ light chain CAR showed cytotoxic activity against Igκ+ tumor cell lines and B-CLL cells both in vitro and in vivo. We also found that the incorporation of the CD28 endodomain within the CAR enhanced the in vitro and in vivo expansion of transgenic T cells after tumor-associated antigen stimulation. Fre...

graft-versus host disease (GVHD) prophylaxis. The primary objective was to test the non-inferiority of overall survival after IV BU compared to TBI. From March 2009 to February 2011,1,483 eligible patients were enrolled (IV BU, n¼1025, TBI, n¼458) from 120 transplant centers. The 2 cohorts were similar with respect to median age (45 years), gender (50% female), race (88% Caucasian), performance status (68% !90), and HCT-CI. Most patients had acute myeloid leukemia (68% BU, 78% TBI). Disease status was early (51%), intermediate (18%) and advanced (31%) and were balanced in both cohorts. Grafts were primarily PB (77%) from matched sibling (40%) or well-matched unrelated donors (48%), balanced in both cohorts. IV BU regimens were CY (59%) or Flu (41%) based. 2-yr probabilities of overall survival (95% CI), were 56% (53-60%) and 48% (43-54%) for IV BU and TBI, respectively (p¼0.02). Corresponding probabilities of progression-free survival (PFS) were 49% (45-52%) and 44% (40-49%), (p¼0.17). The incidence of hepatic veno-occlusive disease (VOD)/sinusoidal obstruction syndrome (SOS) was 5% for BU and 1% for TBI; (p < 0.001). Outcomes were the same for BUþCY and BUþFlu regimens. There were no differences in engraftment of neutrophils or platelets, disease relapse, grade 2-4 acute or chronic GVHD. Multivariate Analysis (MVA) (adjusting for HCT-CI, disease and status, donor type, age, PS, and race) of the main outcomes is summarized in Table . Compared to TBI, IV BU was associated with superior survival and no increased risk of relapse or TRM. These results support the use of myeloablative BU-over TBI-based conditioning regimens for treatment of AML, MDS, and CML.

More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that inSH2D1A-/-mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent inSH2D1A-/-mice upon primary immunization, and because SH2D1A was detectable inwtgerminal center B cells, we examined whetherSH2D1A-/-B cell functions were impaired. Using the adoptive cotransfer of B lymphocytes from hapten-primedSH2D1A-/-mice with CD4+T cells from primedwtmice into irradiatedwtmice provided evidence that signal transduction events controlled by SH2D1A are essential for B cell activities resulting in antigen specific IgG production. Defects in naïveSH2D1A-/-B cells became ...

An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiv...

Aldosterone mediates actions of the renin-angiotensin-aldosterone system inducing hypertension, oxidative stress, and vascular inflammation. Recently, we showed that angiotensin II–induced hypertension and vascular damage are mediated at least in part by macrophages and T-helper effector lymphocytes. Adoptive transfer of suppressor T-regulatory lymphocytes (Tregs) prevented angiotensin II action. We hypothesized that Treg adoptive transfer would blunt aldosterone-induced hypertension and vascular damage. Thirteen to 15-week–old male C57BL/6 mice were injected intravenously at 1-week intervals with 3×10 5 CD4 + CD25 + cells (representing Treg) or control CD4 + CD25 − cells and then infused or not for 14 days with aldosterone (600 μg/kg per day, SC) while receiving 1% saline to drink. Aldosterone induced a small but sustained increase in blood pressure ( P &lt;0.001), decreased vasodilatory responses to acetylcholine by 66% ( P &lt;0.001), increased both media:lumen ratio ( P &lt;0.00...

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like

ABSTRACTPrevious studies of mice have demonstrated that an orchestrated sequence of innate and adaptive immune responses is required to control West Nile virus (WNV) infection in peripheral and central nervous system (CNS) tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also known as CD253) has been reported to inhibit infection with dengue virus, a closely related flavivirus, in cell culture. To determine the physiological function of TRAIL in the context of flavivirus infection, we compared the pathogenesis of WNV in wild-type andTRAIL−/−mice. Mice lacking TRAIL showed increased vulnerability and death after subcutaneous WNV infection. Although no difference in viral burden was detected in peripheral tissues, greater viral infection was detected in the brain and spinal cord at late times after infection, and this was associated with delayed viral clearance in the few survivingTRAIL−/−mice. While priming of adaptive B and T cell responses and trafficking of...

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2 b ) mice, were transplanted with MHC incompatible BALB/c (H-2 d ) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGF ␤ . Anti-TGF ␤ completely abrogated the protective effects of the activated donor NK cells indicating that TGF ␤ plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tu-mor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena. (

Objective— Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Methods and Results— Sixteen CAD patients had reduced CD34 + /CD133 + (0.0224±0.0063% versus 0.121±0.038% mononuclear cells [MNCs], P &lt;0.01) and CD133 + /VEGFR-2 + cells, consistent with EPC phenotype (0.00033±0.00015% versus 0.0017±0.0006% MNCs, P &lt;0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2±1/well) compared with 16 healthy subjects at high risk (13±4/well, P &lt;0.05) or 14 at low risk (22±3/well, P &lt;0.001) for CAD. G-CSF 10 μg/kg per day for 5 days increased CD34 + /CD133 + cells from 0.5±0.2/μL to 59.5±10.6/μL and CD133 + / VEGFR-2 + cells from 0.007...

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti–CTLA-4 antibody are not anergized, and behave identically to T cells which have e...

This report describes a microtiter system that should be useful for making preliminary determinations of which components of plasmodia may be useful as vaccines. The system is based on antigenmediated blockage of inhibition of plasmodial growth in culture by immune serum. The necessity for a quantitative approach to the analysis is emphasized if reproducible results are to be obtained.

Humoral responses to nonproteinaceous Ags (i.e., T cell independent [TI]) are a key component of the early response to bacterial and viral infection and a critical driver of systemic autoimmunity. However, mechanisms that regulate TI humoral immunity are poorly defined. In this study, we report that B cell-intrinsic induction of the tryptophan-catabolizing enzyme IDO1 is a key mechanism limiting TI Ab responses. When Ido1(-/-) mice were immunized with TI Ags, there was a significant increase in Ab titers and formation of extrafollicular Ab-secreting cells compared with controls. This effect was specific to TI Ags, as Ido1 disruption did not affect Ig production after immunization with protein Ags. The effect of IDO1 abrogation was confined to the B cell compartment, as adoptive transfer of Ido1(-/-) B cells to B cell-deficient mice was sufficient to replicate increased TI responses observed in Ido1(-/-) mice. Moreover, in vitro activation with TLR ligands or BCR crosslinking rapidly...

Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient&#39;s own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer. Experimental Design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+ ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD–targeted T cells in SCID-Beige mice bearin...

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4+ T cells in fed animals compared with controls, but there was no change in CD8+ T cell numbers. The CD8+ GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients...

Regulatory T cells (T reg cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b + myeloid cells in recombination-activating gene 1-deficient (Rag1 -/-) recipient mice was needed to prevent the colitis induced by transferred CD4 + CD45RB hi T cells. In Il10 -/-Rag1 -/-mice, T reg cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb -/-) T reg cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T reg cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T reg cells to maintain Foxp3 expression. CD4 + regulatory T cells (T reg cells) express the transcription factor Foxp3 (A002750), which is required for their suppressive function. A T cell-transfer model of colitis has been widely used to study the function of T reg cells in vivo. When CD4 + CD45RB hi T cells are transferred into immunodeficient mice, some of the transferred T cells secrete proinflammatory cytokines and induce an inflammatory bowel disease-like syndrome 1,2 . Cotransfer of sufficient numbers of T reg cells can prevent or even cure this disease . The transferred T reg cell populations expand considerably in vivo, and most maintain Foxp3 expression . Mice deficient in interleukin 10 (IL-10 (A001243); Il10 -/-mice) or the IL-10 receptor βchain (IL-10Rβ (A001245); Il10rb -/-mice) develop spontaneous inflammation of the large intestine, a process dominated by a T helper type 1 immune response7 ,8 . Many cell types

Mammalian target of rapamycin (mTOR) plays a crucial role in the control of T cell fate determination; however, the precise regulatory mechanism of the mTOR pathway is not fully understood. We found that T cell-specific deletion of the gene encoding tuberous sclerosis 1 (TSC1), an upstream negative regulator of mTOR, resulted in augmented Th1 and Th17 differentiation and led to severe intestinal inflammation in a colitis model. Conditional Tsc1 deletion in Tregs impaired their suppressive activity and expression of the Treg marker Foxp3 and resulted in increased IL-17 production under inflammatory conditions. A fate-mapping study revealed that Tsc1-null Tregs that lost Foxp3 expression gained a stronger effector-like phenotype compared with Tsc1 -/-Foxp3 + Tregs. Elevated IL-17 production in Tsc1 -/-Treg cells was reversed by in vivo knockdown of the mTOR target S6K1. Moreover, IL-17 production was enhanced by Treg-specific double deletion of Tsc1 and Foxo3a. Collectively, these studies suggest that TSC1 acts as an important checkpoint for maintaining immune homeostasis by regulating cell fate determination.

exposure to Ag can act as immunotherapeutic agents to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2 and TGF-␤1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require innate CD25 ؉ regulatory cells for generation or function, nor do they share some characteristics typically associated with them, including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate Th1 cells in lymphoid tissues, where Fas/FasL interactions potentially play a role because Th1 cells persist when this pathway is blocked. The results suggest that adaptive regulatory CD4 cells may control diabetes in part by impairing the survival of islet-specific Th1 cells, and thereby inhibiting the localization and response of autoaggressive T cells in the pancreatic islets.

Curing type 1 diabetes (T1D) will require lasting control of the autoimmune response that destroys insulin-producing islet β-cells. Re-establishing tolerance by restoring/replacing Tregs has significant potential for treatment of T1D but will require strategies to augment and maintain their efficacy. We previously showed that polyclonal in vitro-induced Tregs can reverse recent onset of T1D in ~50% of NOD mice. Here we report that treatment of newly hyperglycemic animals with a short course of anti-CD44 at the time of Treg transfer improved diabetes reversal to >90%. Anti-CD44 treatment alone delayed diabetes onset and increased the frequencies of pancreatic CD4 + T cells producing IL-2 or TGF-β, cytokines that support Treg function and survival, without altering production of IFN-γ. These anti-CD44 effects on endogenous T cells were also observed in the context of polyclonal Treg transfer, and the combination treatment also reduced pancreatic infiltrates. The results provide compelling evidence that approaches to modulate the pancreatic milieu to support Treg function and counteract inflammation in the pancreas can greatly enhance the efficacy of adoptively transferred Tregs, and suggest that approaches achieving these outcomes hold promise for long-term control of autoimmunity in T1D.

Migration of CD4 cells into the pancreas represents a hallmark event in the development of insulin-dependent diabetes mellitus. Th1, but not Th2, cells are associated with pathogenesis leading to destruction of islet β-cells and disease onset. Lymphocyte extravasation from blood into tissue is regulated by multiple adhesion receptor/counter-receptor pairs and chemokines. To identify events that regulate entry of CD4 cells into the pancreas, we transferred Th1 or Th2 cells induced in vitro from islet-specific TCR transgenic CD4 cells into immunodeficient (NOD.scid) recipients. Although both subsets infiltrated the pancreas and elicited multiple adhesion receptors (peripheral lymph node addressin, mucosal addressin cell adhesion molecule-1, LFA-1, ICAM-1, and VCAM-1) on vascular endothelium, entry/accumulation of Th1 cells was more rapid than that of Th2 cells, and only Th1 cells induced diabetes. In vitro, Th1 cells were also distinguished from Th2 cells by the capacity to synthesize...

Migration of CD4 cells into the pancreas represents a hallmark event in the development of insulin-dependent diabetes mellitus. Th1, but not Th2, cells are associated with pathogenesis leading to destruction of islet ␤-cells and disease onset. Lymphocyte extravasation from blood into tissue is regulated by multiple adhesion receptor/counter-receptor pairs and chemokines. To identify events that regulate entry of CD4 cells into the pancreas, we transferred Th1 or Th2 cells induced in vitro from islet-specific TCR transgenic CD4 cells into immunodeficient (NOD. scid) recipients. Although both subsets infiltrated the pancreas and elicited multiple adhesion receptors (peripheral lymph node addressin, mucosal addressin cell adhesion molecule-1, LFA-1, ICAM-1, and VCAM-1) on vascular endothelium, entry/accumulation of Th1 cells was more rapid than that of Th2 cells, and only Th1 cells induced diabetes. In vitro, Th1 cells were also distinguished from Th2 cells by the capacity to synthesize several chemokines that included lymphotactin, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1␣, whereas both subsets produced macrophage inflammatory protein-1␤. Some of these chemokines as well as RANTES, MCP-3, MCP-5, and cytokine-response gene-2 (CRG-2)/IFN-inducible protein-10 (IP-10) were associated with Th1, but not Th2, pancreatic infiltrates. The data demonstrate polarization of chemokine expression by Th1 vs Th2 cells, which, within the microenvironment of the pancreas, accounts for distinctive inflammatory infiltrates that determine whether insulin-producing ␤-cells are protected or destroyed.

Curing type 1 diabetes (T1D) will require lasting control of the autoimmune response that destroys insulin-producing islet β-cells. Re-establishing tolerance by restoring/replacing Tregs has significant potential for treatment of T1D but will require strategies to augment and maintain their efficacy. We previously showed that polyclonal in vitro-induced Tregs can reverse recent onset of T1D in ∼ 50% of NOD mice. Here we report that treatment of newly hyperglycemic animals with a short course of anti-CD44 at the time of Treg transfer improved diabetes reversal to &gt;90%. Anti-CD44 treatment alone delayed diabetes onset and increased the frequencies of pancreatic CD4(+) T cells producing IL-2 or TGF-β, cytokines that support Treg function and survival, without altering production of IFN-γ. These anti-CD44 effects on endogenous T cells were also observed in the context of polyclonal Treg transfer, and the combination treatment also reduced pancreatic infiltrates. The results provide c...

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing β-cells in the pancreatic islets. There is an immediate need to restore both β-cell function and immune tolerance to control disease progression and ultimately cure T1D. Currently, there is no effective treatment strategy to restore glucose regulation in patients with T1D. FoxP3-expressing CD4(+) regulatory T cells (Tregs) are potential candidates to control autoimmunity because they play a central role in maintaining self-tolerance. However, deficiencies in either naturally occurring Tregs (nTregs) themselves and/or their ability to control pathogenic effector T cells have been associated with T1D. Here, we hypothesize that nTregs can be replaced by FoxP3(+) adaptive Tregs (aTregs), which are uniquely equipped to combat autoreactivity in T1D. Unlike nTregs, aTregs are stable and provide long-lived protection. In this review, we summarize the current understanding of aTregs and their potential for use as ...

T cell migration is crucial for an effective adaptive immune response to invading pathogens. Naive and memory T cells encounter pathogen antigens, become activated, and differentiate into effector cells in secondary lymphoid tissues, and then migrate to the site(s) of infection where they exert effector activities that control and eliminate pathogens. To achieve activation, efficient effector function, and good memory formation,T cells must traffic between lymphoid and non-lymphoid tissues within the body. This complex process is facilitated by chemokine receptors, selectins, CD44, and integrins that mediate the interactions of T cells with the environment. The expression patterns of these migration receptors (MR) dictate the tissues into which the effector T cells migrate and enable them to occupy specific niches within the tissue. While MR have been considered primarily to facilitate cell movement, we highlight how the heterogeneity of signaling through these receptors influences the function and fate of T cells in situ. We explore what drives MR expression heterogeneity, how this affects migration, and how this impacts T cell effector function and memory formation.

Type 1 diabetes is a CD4 cell-dependent disease that results from destruction of insulin-producing β cells in pancreatic islets. An ideal therapy would reverse diabetes shortly after onset when islet function in not yet fully ablated, and also prevent re-emergence of disease through the generation of memory cells that control the autoimmune response. In this study, we show that adaptive/induced polyclonal regulatory (TR) cells, which contain islet-reactive cells, fulfill these criteria in the NOD mouse model. CD4 cells induced to express FoxP3, IL-10, and TGF-β1 in response to TCR signaling and TGF-β1 can reverse diabetes with clinical restoration of prediabetic serum levels of IL-10. Unlike naturally occurring TR cells, these adaptive TR cells persist indefinitely (&gt;1 year) as FoxP3+, CD25− memory cells that self-renew. Establishment of memory is accompanied by narrowing of the T cell repertoire to usage of a single TCR β-chain, Vβ11, implying selection by Ag. With islet-specifi...

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β-cells. Although Th1 cells are key orchestrators of T1D, the function(s) of the more recently identified Th17 subset are unclear due to inherent plasticity. In this study, we analyzed Th17 cells for stability and diabetogenicity in NOD mice. We found that like Th1 cells, Th17 are a distinct population throughout the prediabetic phase. At diabetes onset, there were marked increases in IL-17–producing Th17 cells and IFN-γ–producing Th1 cells in the pancreas as well as in the serum levels of these cytokines, indicating that these proinflammatory mediators serve as biomarkers of advanced autoimmunity. Although naturally occurring Th17 cells in diabetic mice did not contribute to diabetes development in transfer models, islet-specific Th17 cells were diabetogenic independently of IL-17 and displayed inflammation-induced Th17-to-Th1 reprogramming that could be elicited by Th1 cells. However, an inability to generate ...

Type 1 diabetes (T1D) develops as a consequence of a progressive autoimmune response that destroys insulin-producing β-cells in pancreatic islets. Because of their role(s) in controlling immune responses, considerable effort has been directed towards resolving whether regulatory T cells (Tregs) offer a clinical treatment to restore tolerance in T1D. We previously reported that in vitro induced adaptive Treg cells (aTregs) can reverse T1D and persist as protective memory cells in the NOD mouse model. In the current study, we investigated mechanisms that regulate aTregs. We found that these FoxP3 + aTregs expressed high levels of the IL-7 receptor, IL-7Rα, without the high affinity receptor for IL-2, CD25, which is found on natural Treg cells (nTregs). IL-7Rα expression was mirrored by the dependency of aTregs on IL-7 for persistence. IL-10 and TGF-β, effector cytokines of aTregs, were not essential for their maintenance at the level of systemic antibody blocking. Nevertheless, IL-10 modulated cytokine production by aTregs and TGF-β was critical for protection. aTregs were found to infiltrate islets and the expression of integrin-β7 was required for their localization in the pancreas. Furthermore, blocking aTreg entry into the pancreas prevented their control of diabetogenic effector T cells, implying the need for local control of the autoimmune response. The distinct homeostatic regulation of aTregs independently of a response to IL-2, which is defective in T1D patients, suggests that these cells represent a translatable candidate to control the autoimmune response.

Adaptive regulatory T cells that develop from naive CD4 cells in response to exposure to Ag can act as immunotherapeutic agents to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2 and TGF-β1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require innate CD25+ regulatory cells for generation or function, nor do they share some characteristics typically associated with them, including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate T...

IFN-γ is generally believed to be important in the autoimmune pathogenesis of type 1 diabetes (T1D). However, the development of spontaneous β cell autoimmunity is unaffected in NOD mice lacking expression of IFN-γ or the IFN-γ receptor (IFNγR), bringing into question the role IFN-γ has in T1D. In the current study an adoptive transfer model was employed to define the contribution of IFN-γ in CD4 + versus CD8 + T cell-mediated β cell autoimmunity. NOD.scid mice lacking expression of the IFNγR β chain (NOD.scid.IFNγRB null ) developed diabetes following transfer of β cell-specific CD8 + T cells alone. In contrast, β cell-specific CD4 + T cells alone failed to induce diabetes despite significant infiltration of the islets in NOD.scid.IFNγRB null recipients. The lack of pathogenicity of CD4 + T cell effectors was due to the resistance of IFNγR-deficient β cells to inflammatory cytokine-induced cell death. On the other hand, CD4 + T cells indirectly promoted β cell destruction by providing help to CD8 + T cells in NOD.scid.IFNγRB null recipients. These results demonstrate that IFN-γR may play a key role in CD4 + T cell-mediated β cell destruction.

The aim of the study is a cross-sectional assessment of the levels of Th cytokines in the jaundice-free post Kasai procedure patients. : There were 40 jaundice-free patients with BA and 28 normal controls enrolled. Patients were divided into 3 groups, including normal liver function, impaired liver function, and portal hypertension. Plasma concentration of Th1 [interferon-Ȗ (INF-Ȗ), interleukin (IL)-2], Th2 (IL-4, IL-10), Th3 [transforming growth factor-ȕ1 (TGF-ȕ1)], Th17 (IL-17) cytokines, and stromal cell-derived factor-1Į (SDF-1Į) were investigated. The IFN-Ȗ level was significantly higher in the BA patients with impaired liver function and portal hypertension than controls (P<0.0001 and P<0.0001, respectively). There was a signi¿ cantly increase of TGF-ȕ1 in all BA groups compared with controls (P=0.003). The reduction of SDF-1Į expression was found in BA groups (P<0.0001). IL-10 levels significantly correlated with aspartate aminotransferase to platelet ratio index (r=0.496, P=0.001). For the cytokine correlations, there were no correlations of Th1, Th2 and Th17 cytokine with the other measured cytokines, but TGF-ȕ1 was negatively correlated with SDF-1Į levels (r=-0.327, P=0.039). Conclusions: IFN-Ȗ and IL-10 are likely to be involved in the disease progression in BA. Besides, TGF-ȕ1 is found to be a suppression marker associated with SDF-1Į levels and reduced production of TGF-ȕ1 may be associated with the disease progression.

Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-γ transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts wi...