Adoptive Transfer Studies Research Articles

The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice

BackgroundRecent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.Materials and MethodsFemale NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.ResultsAG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.Scid mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.ConclusionThe use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.

Music Exposure Induced Prolongation of Cardiac Allograft Survival and Generated Regulatory CD4+ Cells in Mice

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2K) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, and CD4+CD25+ cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory cells.

The role of a cx3cr1+/egfp mucosal resident dendritic cell network in mucosal homeostasis (49.10)

Abstract Cxcr1 is expressed in all CD11c+ mucosal dendritic cells (DC). Mucosal DCs are perivascular and are interconnected to form a network. However, the importance of the network in maintaining mucosal homeostasis is poorly understood. We first aimed to gain insight on the structure of the DC network. Using multi-photon intravital microscopy, we demonstrate that the cx3cr1+/egfp DC network interconnects DCs between villi and throughout small and large bowel. The network overlaps a radiation resistant non-hematopoietic cell network. We next aimed to examine the role of DCs in vascular permeability. cx3cr1+/egfp DCs were selectively depleted by targeting depletion with CD11c-diptheria toxin receptor (DTR) transgenic intercross. Preliminary data suggests that DCs are not necessary to maintain vascular integrity, as measured by permeability of 70kDa dextran fluor. Critical for maintaining adaptive immunity is spatial temporal regulation of T cell migration as T cells survey antigen present cells (APC). Since DCs are a critical APC, T cells patrolling was examined following depletion of the mucosal DC network. Adoptive transfer studies demonstrate that CD4 and CD8 T cell migration remains intact despite selective depletion of DCs. In summary, the mucosal DC network represents a higher order of organization of DCs than previously appreciated. Resident DCs may play a role in maintaining vascular permeability but surprisingly do not play a role in regulating mucosal T cell migration.

Induction of Regulatory CD4+ Cells and Prolongation of Survival of Fully Allogeneic Murine Cardiac Grafts by Danazol

Danazol, a modified testosterone, has been used to treat endometriosis and pretreatment before in vitro fertilization and embryo transfer, although its reproductive mechanisms remain unclear. We investigated the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2k) that underwent transplantation of C57BL/6 (B6, H2b) hearts received danazol (0.4 and 4 mg/kg/d) by intraperitoneal injection from the day of transplantation to days 6. We performed an adoptive transfer study to determine regulatory cells as well as cell proliferation, cytokine, and flow cytometry assessments. Danazol-treated (4 mg/kg/d) CBA mice showed prolonged allograft survival (median survival time [MST], 63 days). Moreover, secondary CBA recipients of whole splenocytes and CD4+ cells from primary danazol-treated (4 mg/kg/d) CBA recipients at 30 days after transplantation displayed prolonged allograft survival (MSTs, 29 and 60 days, respectively). Cell proliferation, interleukin (IL)-2, and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed alloproliferation in mixed leukocyte cultures. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population among splenocytes from danazol-treated mice. In conclusion, danazol induced prolonged cardiac allograft survival and generation of regulatory CD4+ cells.

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Auditory stimulation of opera music induced prolongation of murine cardiac allograft survival and maintained generation of regulatory CD4+CD25+ cells

BackgroundInteractions between the immune response and brain functions such as olfactory, auditory, and visual sensations are likely. This study investigated the effect of sounds on alloimmune responses in a murine model of cardiac allograft transplantation.MethodsNaïve CBA mice (H2k) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to one of three types of music--opera (La Traviata), classical (Mozart), and New Age (Enya)--or one of six different single sound frequencies, for 7 days. Additionally, we prepared two groups of CBA recipients with tympanic membrane perforation exposed to opera for 7 days and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment). An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Immunohistochemical, cell-proliferation, cytokine, and flow cytometry assessments were also performed.ResultsCBA recipients of a B6 cardiac graft that were exposed to opera music and Mozart had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to a single sound frequency (100, 500, 1000, 5000, 10,000, or 20,000 Hz) or Enya did not (MSTs, 7.5, 8, 9, 8, 7.5, 8.5 and 11 days, respectively). Untreated, CBA mice with tympanic membrane perforations and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment) rejected B6 cardiac grafts acutely (MSTs, 7, 8 and 8 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, or CD4+CD25+ cells from opera-exposed primary allograft recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and > 100 days, respectively). Proliferation of splenocytes, interleukin (IL)-2 and interferon (IFN)-γ production was suppressed in opera-exposed mice, and production of IL-4 and IL-10 from opera-exposed transplant recipients increased compared to that from splenocytes of untreated recipients. Flow cytometry studies showed an increased CD4+CD25+ Forkhead box P3 (Foxp3)+ cell population in splenocytes from those mice.ConclusionOur findings indicate that exposure to opera music, such as La traviata, could affect such aspects of the peripheral immune response as generation of regulatory CD4+CD25+ cells and up-regulation of anti-inflammatory cytokines, resulting in prolonged allograft survival.

Trafficking of High Avidity HER-2/neu-Specific T Cells into HER-2/neu-Expressing Tumors after Depletion of Effector/Memory-Like Regulatory T Cells

BackgroundCancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.Methodology/Principal FindingsHigh and low avidity CD8+ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4+Foxp3+CD25low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.Conclusion/SignificanceDepletion of CD25low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.

Animal models for Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan of worldwide distribution. This unit describes murine models of acute T. gondii infection, toxoplasmic encephalitis, and Toxoplasma retinochoroiditis. T. gondii infection in SCID mice allows the study of T cell-independent mechanisms of defense. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii allow studies of immunization and adoptive transfer. In vivo study of parasite host-interaction is possible with the use of parasites that express fluorescent proteins and model antigens, plus the use of transgenic mice that express the appropriate T cell receptor and fluorescently labeled leukocytes. Parasites that express bioluminescent markers make it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens.

Danazol induces prolonged survival of fully allogeneic cardiac grafts and maintains the generation of regulatory CD4+ cells in mice

Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2(k) ) underwent transplantation of C57BL/6 male (H2(b) ) hearts and received a single dose of danazol (0.4, 1.2 or 4mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4mg/kg/day) mice was 28 and 63days, respectively, compared with 7days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4(+) cells from primary danazol-treated (4mg/kg/day) CBA recipients 30days after transplantation had prolonged allograft survival (MSTs, 29 and 60days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4(+) CD25(+) Foxp3(+) cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4(+) cells.

Interleukin-7 receptor blockade suppresses adaptive and innate inflammatory responses in experimental colitis

BackgroundInterleukin-7 (IL-7) acts primarily on T cells to promote their differentiation, survival, and homeostasis. Under disease conditions, IL-7 mediates inflammation through several mechanisms and cell types. In humans, IL-7 and its receptor (IL-7R) are increased in diseases characterized by inflammation such as atherosclerosis, rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel disease. In mice, overexpression of IL-7 results in chronic colitis, and T-cell adoptive transfer studies suggest that memory T cells expressing high amounts of IL-7R drive colitis and are maintained and expanded with IL-7. The studies presented here were undertaken to better understand the contribution of IL-7R in inflammatory bowel disease in which colitis was induced with a bacterial trigger rather than with adoptive transfer.MethodsWe examined the contribution of IL-7R on inflammation and disease development in two models of experimental colitis: Helicobacter bilis (Hb)-induced colitis in immune-sufficient Mdr1a−/− mice and in T- and B-cell-deficient Rag2−/− mice. We used pharmacological blockade of IL-7R to understand the mechanisms involved in IL-7R-mediated inflammatory bowel disease by analyzing immune cell profiles, circulating and colon proteins, and colon gene expression.ResultsTreatment of mice with an anti-IL-7R antibody was effective in reducing colitis in Hb-infected Mdr1a−/− mice by reducing T-cell numbers as well as T-cell function. Down regulation of the innate immune response was also detected in Hb-infected Mdr1a−/− mice treated with an anti-IL-7R antibody. In Rag2−/− mice where colitis was triggered by Hb-infection, treatment with an anti-IL-7R antibody controlled innate inflammatory responses by reducing macrophage and dendritic cell numbers and their activity.ConclusionsResults from our studies showed that inhibition of IL-7R successfully ameliorated inflammation and disease development in Hb-infected mice by controlling the expansion of multiple leukocyte populations, as well as the activity of these immune cells. Our findings demonstrate an important function of IL-7R-driven immunity in experimental colitis and indicate that the therapeutic efficacy of IL-7R blockade involves affecting both adaptive and innate immunity.

Reply to the Editor—Atrial Fibrillation: An Inflammatory and Autoimmune Disorder

Many factors contribute to the pathogenesis of atrial fibrillation, including electrical, structural, neurohumoral, and inflammatory mechanisms. 1 Schotten U. Verheule S. Kirchhof P. Goette A. Pathophysiological mechanisms of atrial fibrillation: a translational appraisal. Physiol Rev. 2011; 91: 265-325 Crossref PubMed Scopus (854) Google Scholar Our review indicated that various autoantibodies that may play a role in the development and maintenance of atrial fibrillation have been identified. 2 Lee H.C. Huang K.T. Wang X.L. Shen W.K. Autoantibodies and cardiac arrhythmias. Heart Rhythm. 2011; 8: 1788-1795 Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Indeed, mounting evidence demonstrates correlations between diseases with autoimmune mechanisms, including Graves' disease, 3 Stavrakis S. Yu X. Patterson E. et al. Activating autoantibodies to the beta-1 adrenergic and m2 muscarinic receptors facilitate atrial fibrillation in patients with Graves' hyperthyroidism. J Am Coll Cardiol. 2009; 54: 1309-1316 Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar celiac disease, 4 Emilsson L. Smith J.G. West J. Melander O. Ludvigsson J.F. Increased risk of atrial fibrillation in patients with coeliac disease: a nationwide cohort study. Eur Heart J. 2011; 32: 2430-2437 Crossref PubMed Scopus (69) Google Scholar and psoriasis, 5 Ahlehoff O. Gislason G.H. Jorgensen C.H. et al. Psoriasis and risk of atrial fibrillation and ischaemic stroke: a Danish nationwide cohort study. Eur Heart J. 2011; (Epub ahead of print) PubMed Google Scholar and an increased risk of atrial fibrillation. Schairer and Levis suggested that T-cell–mediated autoimmune disorders should be considered in the pathogenesis of atrial fibrillation. Such mechanisms in atrial fibrillation are currently unknown and unexplored. They proposed the intriguing hypothesis that atrial fibrillation is a T-cell autoimmune disorder involving cytotoxic T helper cells acting on His-Purkinje cells, which express contactin-2, a target of TH17 in patients with multiple sclerosis. 6 Derfuss T. Parikh K. Velhin S. et al. Contactin-2/TAG-1-directed autoimmunity is identified in multiple sclerosis patients and mediates gray matter pathology in animals. Proc Natl Acad Sci USA. 2009; 106: 8302-8307 Crossref PubMed Scopus (172) Google Scholar It is not known whether patients with multiple sclerosis have a higher incidence of atrial fibrillation, but paroxysmal atrial fibrillation associated with attacks of multiple sclerosis has been reported. 7 Chagnac Y. Martinovits G. Tadmor R. Goldhammer Y. Paroxysmal atrial fibrillation associated with an attack of multiple sclerosis. Postgrad Med J. 1986; 62: 385-387 Crossref PubMed Scopus (24) Google Scholar , 8 Schroth W.S. Tenner S.M. Rappaport B.A. Mani R. Multiple sclerosis as a cause of atrial fibrillation and electrocardiographic changes. Arch Neurol. 1992; 49: 422-424 Crossref PubMed Scopus (37) Google Scholar Most of the His-Purkinje cells are in the ventricles, and Purkinje fibers are implicated in the development of idiopathic ventricular fibrillation. 9 Haissaguerre M. Shoda M. Jais P. et al. Mapping and ablation of idiopathic ventricular fibrillation. Circulation. 2002; 106: 962-967 Crossref PubMed Scopus (550) Google Scholar It is thought-provoking that atrial Purkinje cells may similarly precipitate fibrillation in the atria. The recently reported association as well as potential immune-mediated mechanisms leading to atrial fibrillation in psoriasis needs to be validated and further explored. Although TH1–TH17 cells are critical to induce the skin pathology of psoriasis, the potential for disease-linked autoantibody, cytokine, or other autoinflammatory-mediated mechanisms will need to be examined in the subpopulation of patients with psoriasis who develop or are at risk for atrial fibrillation. Proteomic, immune, and molecular screening technologies to identify and characterize these pathogenic mechanisms can be leveraged to enhance the yield in this search. Adoptive transfer studies reported by Derfuss et al 6 Derfuss T. Parikh K. Velhin S. et al. Contactin-2/TAG-1-directed autoimmunity is identified in multiple sclerosis patients and mediates gray matter pathology in animals. Proc Natl Acad Sci USA. 2009; 106: 8302-8307 Crossref PubMed Scopus (172) Google Scholar may be useful but have limitations to more broadly characterize novel disease pathomechanisms. We fully agree that further studies to investigate atrial fibrillation in autoimmune disorders as well as to delineate autoimmune mechanisms in idiopathic atrial fibrillation are warranted. Connexin43 and the regulation of intercalated disc functionHeart RhythmVol. 9Issue 5PreviewGap junctions mediate the passage of ions and small molecules between cells. In the adult working cardiac ventricles, gap junctions are formed predominantly by the oligomerization of the 43-kDa protein Connexin43 (Cx43). It is generally accepted that gap junction–mediated intercellular communication is modulated by changes in the intracellular environment. The activity of various kinases, the concentration of protons or calcium, and the association of Cx43 with other intracellular components all converge to determine the filtering capabilities of intercellular channels. Full-Text PDF

Induction of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts by administration of Tokishakuyaku-san in mice

The Japanese herbal medicine Tokishakuyaku-san (TJ-23) has been used to treat neurodegenerative, immune, and respiratory tract diseases, as well as many gynecologic disorders, with few adverse effects. This study investigated the effect of TJ-23 on alloimmune responses in a murine model of cardiac allograft transplantation. CBA mice underwent transplantation of a C57BL/6 heart and received oral administration of 2 g/kg per day of TJ-23 or 1 of 16 other commonly used Japanese herbal medicines from the day of transplantation until 7 days afterward. An adoptive transfer study was conducted to determine whether regulatory cells were generated. Histologic and cell proliferation studies, cytokine measurements, and flow cytometry analyses were also performed. Of the 17 herbal medicines studied, only TJ-23, given in a dose of 2 g/kg per day, induced significantly prolonged allograft survival (median survival time [MST], >100 days). TJ-23 also suppressed proliferation of splenocytes and production of interleukin-2, interleukin-6, and interferon-γ. Adoptive transfer of either whole splenocytes or CD4(+) or CD4(+) CD25(+) cells from TJ-23-treated allograft recipients resulted in indefinite survival of allografts in naive secondary recipients (MST >100 days). Flow cytometry studies showed that the CD4(+) CD25(+) forkhead/winged-helix (FOXP3)(+) regulatory cell population was increased in transplant recipients given TJ-23. TJ-23 induced hyporesponsiveness to fully allogeneic cardiac allografts and generated CD4(+) CD25(+) regulatory cells in our model.

Peripheral education of the immune system by colonic commensal microbiota

SummaryThe instruction of the immune system to be tolerant of self, thereby preventing autoimmunity, is facilitated by the education of T cells in a specialized organ, the thymus, where self-reactive cells are either eliminated or differentiated into tolerogenic Foxp3+ regulatory T(Treg) cells1. However, it is unknown whether T cells are also educated to be tolerant of foreign antigens, such as those from commensal bacteria, in order to prevent immunopathology such as inflammatory bowel disease2–4. Here, we show that encounter with commensal microbiota results in the peripheral generation of Treg cells, rather than pathogenic effectors. We observed that colonic Treg cells utilized T cell antigen receptors (TCRs)different from those used by Treg cells in other locations, implying an important role for local antigens in shaping the colonic Treg cell population. Many of the local antigens appeared to be derived from commensal bacteria based on the in vitro reactivity of common colon Treg TCRs. Interestingly, these TCRs did not facilitate thymic Treg cell development, implying that manycolonic Treg cells arise instead via antigen-driven peripheral Treg cell development. Further analysis of two of these TCRs by the creation of retroviral bone marrow chimeras and a TCR transgenic linerevealed that microbiota indigenous to our mouse colony was required for the generation of colonic Treg cells from otherwise naive T cells. If T cells expressing these TCRs fail to undergo Treg cell development and instead become effector cells, they have the potential to induce colitis, as evidenced by adoptive transfer studies. These results suggest that the efficient peripheral generation of antigen-specific populations of Treg cells in response to an individual’s microbiota provides important post-thymic education of the immune system to foreign antigens, thereby providing tolerance to commensal microbiota.

Maximal T Cell–Mediated Antitumor Responses Rely upon CCR5 Expression in Both CD4+ and CD8+ T Cells

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.

C-fms blockade reverses glomerular macrophage infiltration and halts development of crescentic anti-GBM glomerulonephritis in the rat

Depletion and adoptive transfer studies have demonstrated that macrophages induce glomerular lesions in experimental anti-glomerular basement membrane (anti-GBM) glomerulonephritis. However, there is no current therapeutic strategy that can rapidly and selectively remove these cells from the glomerulus in order to halt disease development. This study examined whether inhibition of the receptor for macrophage colony-stimulating factor (known as c-fms), which is selectively expressed by monocyte/macrophages, can eliminate the macrophage infiltrate in a rat model of crescentic anti-GBM glomerulonephritis. Wistar–Kyoto rats were treated with 10 or 30 mg/kg bid of fms-I (a selective c-fms kinase inhibitor) from the time of anti-GBM serum injection until being killed 1, 5 or 14 days later. fms-I treatment had only a minor effect upon the glomerular macrophage infiltrate on day 1 and did not prevent the subsequent induction of proteinuria. However, fms-I treatment reduced the glomerular macrophage infiltrate by 60% at day 5 and completely reversed the macrophage infiltrate by day 14. In addition, fms-I treatment downregulated the glomerular expression of pro-inflammatory molecules (TNF-α, NOS2, MMP-12, CCL2 and IL-12) on days 1 and 5, suggesting a suppression of the macrophage M1-type response. Despite a significant early loss of glomerular podocytes, ongoing proteinuria and glomerular tuft adhesions to Bowman's capsule, the reversal of the macrophage infiltrate prevented the development of glomerulosclerosis, crescent formation, tubulointerstitial damage and renal dysfunction. In conclusion, this study has identified c-fms kinase inhibition as a selective approach to target infiltrating macrophages in acute glomerular injury, which may have therapeutic potential in rapidly progressive crescentic glomerulonephritis.

A potential role for CD25+ regulatory T-cells in the protection against casein allergy by dietary non-digestible carbohydrates

Dietary non-digestible carbohydrates reduce the development of cows' milk allergy in mice. In the present study, the contribution of CD25+ regulatory T-cells (Treg) was investigated using in vivo Treg depletion and adoptive transfer studies. Mice were orally sensitised with casein and fed a diet containing 2% short-chain galacto-, long-chain fructo- and acidic oligosaccharides (GFA) or a control diet. Donor splenocytes of mice sensitised with casein and fed the GFA or control diet were adoptively transferred to naive recipient mice, which were casein- or sham-sensitised and fed the control diet. In addition, in vivo or ex vivo CD25+ Treg depletion was performed using anti-CD25 (PC61). The acute allergic skin response upon intradermal casein challenge and casein-specific Ig were determined. Furthermore, T-helper (TH) 1 and TH2 cell numbers were analysed in the mesenteric lymph nodes. The oligosaccharide diet strongly reduced the development of the acute allergic skin response, which was abrogated by the in vivo anti-CD25 treatment. The diet enhanced the percentage of TH1 cells and tended to reduce the percentage of TH2 cells in casein-sensitised mice. Recipient mice were protected against the development of an acute allergic skin response when transferred with splenocytes from casein-sensitised GFA-fed donor mice before sensitisation. Ex vivo depletion of CD25+ Treg abrogated this transfer of tolerance. Splenocytes from sham-sensitised GFA-fed donor mice did not suppress the allergic response in recipient mice. In conclusion, CD25+ Treg contribute to the suppression of the allergic effector response in casein-sensitised mice induced by dietary intervention with non-digestible carbohydrates.

LAG-3, TGF-β, and cell-intrinsic PD-1 inhibitory pathways contribute to CD8 but not CD4 T-cell tolerance induced by allogeneic BMT with anti-CD40L

AbstractAdministration of a single dose of anti-CD40L mAb at the time of allogeneic BM transplantation tolerizes peripheral alloreactive T cells and permits establishment of mixed hematopoietic chimerism in mice. Once engrafted, mixed chimeras are systemically tolerant to donor Ags through a central deletion mechanism and will accept any donor organ indefinitely. We previously found that the PD-1/PD-L1 pathway is required for CD8 T-cell tolerance in this model. However, the cell population that must express PD-1 and the role of other inhibitory molecules were unknown. Here, we report that LAG-3 is required for long-term peripheral CD8 but not CD4 T-cell tolerance and that this requirement is CD8 cell-extrinsic. In contrast, adoptive transfer studies revealed a CD8 T cell–intrinsic requirement for CTLA4/B7.1/B7.2 and for PD-1 for CD8 T-cell tolerance induction. We also observed that both PD-L1 and PD-L2 are independently required on donor cells to achieve T-cell tolerance. Finally, we uncovered a requirement for TGF-β signaling into T cells to achieve peripheral CD8 but not CD4 T-cell tolerance in this in vivo system.

Enhancement of T-cell–Mediated Antitumor Response: Angiostatic Adjuvant to Immunotherapy against Cancer

Tumor-released proangiogenic factors suppress endothelial adhesion molecule (EAM) expression and prevent leukocyte extravasation into the tumor. This is one reason why immunotherapy has met with limited success in the clinic. We hypothesized that overcoming EAM suppression with angiogenesis inhibitors would increase leukocyte extravasation and subsequently enhance the effectiveness of cellular immunotherapy. Intravital microscopy, multiple color flow cytometry, immunohistochemistry, and various tumor mouse (normal and T-cell deficient) models were used to investigate the temporal dynamics of cellular and molecular events that occur in the tumor microenvironment during tumor progression and angiostatic intervention. We report that while EAM levels and T-cell infiltration are highly attenuated early on in tumor growth, angiostatic therapy modulates these effects. In tumor models with normal and T-cell-deficient mice, we show the active involvement of the adaptive immune system in cancer and differentiate antiangiogenic effects from antiangiogenic mediated enhancement of immunoextravasation. Our results indicate that a compromised immune response in tumors can be obviated by the use of antiangiogenic agents. Finally, with adoptive transfer studies in mice, we show that a phased combination of angiostatic therapy and T-cell transfer significantly (P < 0.0013) improves tumor growth inhibition. This research contributes to understand the cellular mechanism of action of angiostatic agents and the immune response within the tumor microenvironment, in particular as a consequence of the temporal dynamics of EAM levels. Moreover, our results suggest that adjuvant therapy with angiogenesis inhibitors holds promise for cellular immunotherapy in the clinic.

Phase I/II study of adoptive transfer of γδ T cells in combination with zoledronic acid and IL-2 to patients with advanced renal cell carcinoma

Human Vγ2Vδ2-bearing T cells have recently received much attention in cancer immunotherapy. In this study, we conducted a phase I/II clinical trial of the adoptive transfer of γδ T cells to patients with advanced renal cell carcinoma. Eleven patients who had undergone nephrectomy and had lung metastasis were enrolled. Peripheral blood γδ T cells obtained from the patients were stimulated ex vivo with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), a synthetic pyrophosphomonoester antigen, and transferred in combination with zoledronic acid (Zol) and teceleukin (recombinant human interleukin-2). Expanded γδ T cells exhibited potent cytotoxic activity against tumor cells in vitro, and the proportion of peripheral blood γδ T cells among CD3(+) cells typically peaked three to 5days after transfer. Tumor doubling time was prolonged in all 11 patients, and the best overall responses were 1 CR, 5 SD, and 5 PD, as defined based on Response Evaluation Criteria in Solid Tumors (RECIST). Although ten patients developed adverse reactions of grade ≥3, they were likely to have been the result of the concomitant infusion of Zol and IL-2, and most symptoms swiftly reverted to normal during the course of treatment. In conclusion, this clinical trial demonstrated that our regimen for the adoptive transfer of γδ T cells in combination with Zol and IL-2 was well tolerated and that objective clinical responses could be achieved in some patients with advanced renal cell carcinoma.

Chemokine receptor expression on T and B cells in a murine model of ovalbumin-induced allergic airway disease (149.17)

Abstract In our biphasic Ovalbumin (OVA)-induced Allergic Airway Disease model, short-term exposure to OVA aerosols results in Allergic Airway Disease (AAD) whereas continued exposure leads to the resolution of disease and the development of Local Inhalational Tolerance (LIT). We have shown in adoptive transfer studies that Hilar Lymph Node (HLN) B cells of LIT mice suppress development of AAD. To understand trafficking of B cells within HLN, we assessed their expression of CXCR4 and CXCR5 chemokine receptors via flow cytometry in mice at various stages of the model: naïve, sensitized, AAD and LIT. B cell populations were divided into CD5+ and CD5- cells. Our data demonstrates that there was a significant increase in the number and percentage of total B cells expressing CXCR4 in the HLN of mice at AAD and LIT compared to naïve and sensitized mice. We also observed a significant increase in CXCR4 expression on CD5+ B cells compared to CD5- B cells. We also studied the chemokine receptor expression on T cells and observed that Foxp3+ T regulatory cells significantly increased the expression of CXCR4 at AAD and LIT. CXCR4 expression on B cells is required for germinal center development in lymphoid organs. It is unusual to find CD5+ B cells in lymph nodes. We hypothesize that these CD5+ B cells may be directly interacting with effector T cells in a regulatory manner converting them into regulatory T cells within germinal centers and hence contributing to the resolution of AAD.