anti-CD4 mAb Treatment Research Articles

Agonistic anti-CD137 antibody treatment leads to antitumor response in mice with liver cancer.

Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40-60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti-CD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.

NOD2 Regulates CXCR3-Dependent CD8+ T Cell Accumulation in Intestinal Tissues with Acute Injury

Polymorphisms in NOD2 confer risk for Crohn's disease, characterized by intestinal inflammation. How NOD2 regulates both inflammatory and regulatory intestinal T cells, which are critical to intestinal immune homeostasis, is not well understood. Anti-CD3 mAb administration is used as therapy in human autoimmune diseases, as well as a model of transient intestinal injury. The stages of T cell activation, intestinal injury, and subsequent T tolerance are dependent on migration of T cells into the small intestinal (SI) lamina propria. Upon anti-CD3 mAb treatment of mice, we found that NOD2 was required for optimal small intestinal IL-10 production, in particular from CD8(+) T cells. This requirement was associated with a critical role for NOD2 in SI CD8(+) T cell accumulation and induction of the CXCR3 ligands CXCL9 and CXCL10, which regulate T cell migration. NOD2 was required in both the hematopoietic and nonhematopoietic compartments for optimal expression of CXCR3 ligands in intestinal tissues. NOD2 synergized with IFN-γ to induce CXCL9 and CXCL10 secretion in dendritic cells, macrophages, and intestinal stromal cells in vitro. Consistent with the in vitro studies, during anti-CD3 mAb treatment in vivo, CXCR3 blockade, CD8(+) T cell depletion, or IFN-γ neutralization each inhibited SI CD8(+) T cell recruitment, and reduced chemokine expression and IL-10 expression. Thus, NOD2 synergizes with IFN-γ to promote CXCL9 and CXCL10 expression, thereby amplifying CXCR3-dependent SI CD8(+) T cell migration during T cell activation, which, in turn, contributes to induction of both inflammatory and regulatory T cell outcomes in the intestinal environment.

Depletion of Host CCR7+ Dendritic Cells Prevented Donor T Cell Tissue Tropism in Anti-CD3–Conditioned Recipients

We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103+ dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103+ DCs and peripheral lymph node (PLN) DCs include CCR7+ and CCR7− subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7+, but not CCR7−, DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7+ but not CCR7− DCs by inducing sequential expansion and apoptosis of CCR7+ DCs in MLN and PLN. Apoptosis of CCR7+ DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7+ host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.

Teplizumab (Anti-CD3 mAb) Treatment Preserves C-Peptide Responses in Patients With New-Onset Type 1 Diabetes in a Randomized Controlled Trial

Trials of immune therapies in new-onset type 1 diabetes (T1D) have shown success, but not all subjects respond, and the duration of response is limited. Our aim was to determine whether two courses of teplizumab, an Fc receptor–nonbinding anti-CD3 monoclonal antibody, reduces the decline in C-peptide levels in patients with T1D 2 years after disease onset. We also set out to identify characteristics of responders. We treated 52 subjects with new-onset T1D with teplizumab for 2 weeks at diagnosis and after 1 year in an open-label, randomized, controlled trial. In the intent to treat analysis of the primary end point, patients treated with teplizumab had a reduced decline in C-peptide at 2 years (mean −0.28 nmol/L [95% CI −0.36 to −0.20]) versus control (mean −0.46 nmol/L [95% CI −0.57 to −0.35]; P = 0.002), a 75% improvement. The most common adverse events were rash, transient upper respiratory infections, headache, and nausea. In a post hoc analysis we characterized clinical responders and found that metabolic (HbA1c and insulin use) and immunologic features distinguished this group from those who did not respond to teplizumab. We conclude that teplizumab treatment preserves insulin production and reduces the use of exogenous insulin in some patients with new-onset T1D. Metabolic and immunologic features at baseline can identify a subgroup with robust responses to immune therapy.

The control of CD8+ T cell responses is preserved in perforin-deficient mice and released by depletion of CD4+CD25+ regulatory T cells

Immune suppression by Treg has been demonstrated in a number of models, but the mechanisms of this suppression are only partly understood. Recent work has suggested that Tregs may suppress by directly killing immune cell populations in vivo in a perforin- and granzyme B-dependent manner. To establish whether perforin is necessary for the regulation of immune responses in vivo, we examined OVA-specific CD8(+) T cell responses in WT and PKO mice immunized with OVA and α-GalCer and the expansion of WT OT-I CD8(+) T cells adoptively transferred into WT or PKO mice immunized with DC-OVA. We observed similar expansion, phenotype, and effector function of CD8(+) T cells in WT and PKO mice, suggesting that CD8(+) T cells were subjected to a similar amount of regulation in the two mouse strains. In addition, when WT and PKO mice were depleted of Tregs by anti-CD25 mAb treatment before DC-OVA immunization, CD8(+) T cell proliferation, cytotoxicity, and cytokine production were increased similarly, suggesting a comparable involvement of CD25(+) Tregs in controlling T cell proliferation and effector function in these two mouse strains. These data suggest that perforin expression is not required for normal immune regulation in these models of in vivo CD8(+) T cell responses induced by immunization with OVA and α-GalCer or DC-OVA.

Mechanisms and Rescue Strategies of Calcineurin Inhibitor Mediated Tolerance Abrogation Induced by Anti-CD4 mAb Treatment

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.

Bacillus Calmette-Guérin Suppresses Asthmatic Responses via CD4(+)CD25(+) Regulatory T Cells and Dendritic Cells.

PurposeBacillus Calmette-Guérin (BCG) is known to suppress the asthmatic responses in a murine model of asthma and to induce dendritic cells (DCs) maturation. Mature DCs play a crucial role in the differentiation of regulatory T cells (Tregs), which are known to regulate allergic inflammatory responses. To investigate whether BCG regulates Tregs in a DCs-mediated manner, we analyzed in a murine model of asthma.MethodsBALB/c mice were injected intraperitoneally with BCG or intravenously with BCG-stimulated DCs and then sensitized and challenged with ovalbumin (OVA). Mice were analysed for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. To identify the mechanisms, IgE, IgG1 and IgG2a in the serum were analysed and the CD25+ Tregs in the mice were depleted with anti-CD25 monoclonal antibody (mAb).ResultsBCG and the transfer of BCG-stimulated DCs both suppressed all aspects of the asthmatic responses, namely, BHR, the production of total IgE and OVA-specific IgE and IgGs, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment reversed these effects.ConclusionsBCG can attenuate the allergic inflammation in a mouse model of asthma by a Tregs-related mechanism that is mediated by DCs.

A Novel Synthetic Mycolic Acid Inhibits Bronchial Hyperresponsiveness and Allergic Inflammation in a Mouse Model of Asthma

PurposeRecognition of microbes is important to trigger the innate immune system. Mycolic acid (MA) is a component of the cell walls of mycobacteria such as Mycobacterium bovis Bacillus Calmette-Guerin. MA has immunogenic properties, which may modulate the innate and adaptive immune response. This study aimed to investigate whether a novel synthetic MA (sMA) inhibits allergic inflammatory responses in a mouse model of asthma.MethodsBALB/c mice were injected intraperitoneally with sMA followed by sensitization and challenge with ovalbumin (OVA). Mice were examined for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells into the lung tissues, histopathological changes in the lungs and CD4+CD25+Foxp3+ T cells in the spleen, and examined the response after the depleting regulatory T cells (Tregs) with an anti-CD25mAb.ResultsTreatment of mice with sMA suppressed the asthmatic response, including BHR, bronchoalveolar inflammation, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment abrogated the suppressive effects of sMA in this mouse model of asthma and totally depleted CD4+CD25+Foxp3+ T cells in the spleen.ConclusionssMA attenuated allergic inflammation in a mouse model of asthma, which might be related with CD4+CD25+Foxp3+ T cell.

Thymic Damage, Impaired Negative Selection, and Development of Chronic Graft-versus-Host Disease Caused by Donor CD4+ and CD8+ T Cells

Prevention of chronic graft-versus-host disease (cGVHD) remains a major challenge in allogeneic hematopoietic cell transplantation (HCT) owing to limited understanding of cGVHD pathogenesis and lack of appropriate animal models. In this study, we report that, in classical acute GVHD models with C57BL/6 donors and MHC-mismatched BALB/c recipients and with C3H.SW donors and MHC-matched C57BL/6 recipients, GVHD recipients surviving for >60 d after HCT developed cGVHD characterized by cutaneous fibrosis, tissue damage in the salivary gland, and the presence of serum autoantibodies. Donor CD8(+) T cells were more potent than CD4(+) T cells for inducing cGVHD. The recipient thymus and de novo-generated, donor-derived CD4(+) T cells were required for induction of cGVHD by donor CD8(+) T cells but not by donor CD4(+) T cells. Donor CD8(+) T cells preferentially damaged recipient medullary thymic epithelial cells and impaired negative selection, resulting in production of autoreactive CD4(+) T cells that perpetuated damage to the thymus and augmented the development of cGVHD. Short-term anti-CD4 mAb treatment early after HCT enabled recovery from thymic damage and prevented cGVHD. These results demonstrate that donor CD8(+) T cells cause cGVHD solely through thymic-dependent mechanisms, whereas CD4(+) T cells can cause cGVHD through either thymic-dependent or independent mechanisms.

Low dose intradermal infection with Trypanosoma congolense leads to expansion of regulatory T cells and enhanced susceptibility to reinfection (P1055)

Abstract BALB/c mice are highly susceptible to experimental T. congolense infection. Interestingly, a recent report showed that these mice are relatively resistant to primary intradermal low dose infection. Paradoxically, repeated low dose intradermal infections predispose to enhanced susceptibility to an otherwise non-infectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low dose reinfection. We found that akin to intraperitoneal infection, low dose intradermal infection leads to production of IL-10, IL-6, IL-12, TGF-β and IFN-γ by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low dose intradermal infection led to expansion of CD4+CD24+Foxp3+ cells (Tregs) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 mAb treatment during primary or before reinfection following repeated low dose infection completely abolished the low dose-induced enhanced susceptibility to rechallenge infection. In addition, Tregs depletion was associated with dramatic reduction in serum levels of TGF-β and IL-10. Collectively, these findings show that low dose intradermal infection leads to rapid expansion of Tregs and these cells mediate enhanced susceptibility to subsequent infection.

CD4 + CD25 + regulatory T cells-induced tolerance of islet transplantation by targeting OX40/ OX40L costimulation pathway

Objective To observe the role of OX40/OX40L costimulation pathway in the CD4+CD25 + regulatory T (Treg) cells-induced tolerance of islet transplantation.Methods Diabetes mellitus was induced in C57BL/6 mice as recipients,and islets from DBA/2 mice were transplanted into the C57BL/6 mice.The recipients were divided into three groups (n =8):treated with IgG as controls,antiOX40L mAb (RM134L) + anti-CD154 mAb (MR1),and RM134L + MR1 + anti-CD25 mAb.The mean survival time (MST) of allograft was observed.The expression of OX40 in T effector cells,Treg cells of wide type and CD154 knock-out mice was detected.The expression of Foxp3 gene in allograft in the absence and absence of anti-OX40L mAb and anti-CD154 mAb was detected.T effector cells and Treg cells were co-cultured at different ratio,and the cell proliferation was assayed.Results The MST in RM134L +MR1 group was ＞ 150 days (n =8),significantly longer than in IgG group ( MST + 19 days,n =5 ) and RM134L + MR1 + anti-CD25 mAb (23 days,n =4) (P ＜0.05).Anti-OX40L mAb and anti-CD154 mAb treatment could successfully induce tolerance,and also induce donor specific tolerance,but the tolerance was blocked by anti-CD25 mAb treatment (P ＜ 0.05 ).OX40 was expressed on the Treg cells surface,whereas the T effector cells had no OX40 expression.The expression level of Foxp3 gene in the allograft treated with anti-OX40L mAb and anti-CD154 mAb was 123 AU,significantly higher than without treatment (21 AU) and syngenic control ( 8 AU) ( P ＜ 0.05 ).After Treg cells were co-cultured with T effector cells at different ratio ( 1∶2 and 1∶ 4),the mean count per minute (CPM) of T effector cells was 63 x 103and 41 × 103 respectively (P ＜0.05),and after addition of OX40,the CPM was 128 × 103 and 135 × 103respectively ( P ＞ 0.05 ).Conclusion CD4 + CD25 + Treg cells were critical to tolerance induction in mice islet transplant recipients through targeting OX40/OX40L costimulation pathway. Key words: Islet transplantation; Regulatory T cells; Tolerance; OX40

Regulatory T Cells Enhance Susceptibility to Experimental Trypanosoma Congolense Infection Independent of Mouse Genetic Background

BackgroundBALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines.Methodology/FindingsBALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 103 T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25+ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4+CD25+Foxp3+ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25+ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25+ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines.ConclusionCollectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice.

177 The Roles of TNFR Signaling and NOX1 in Epithelial ß-Catenin Activation During Colitis

TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.

180 The Extracellular Calcium Sensing Receptor Mediates Local Inhibitory Reflexes Evoked by Aromatic L-Amino Acids in Guinea Pig Jejunum

TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.

Transient Attenuated Foxp3 Expression on CD4+ T cells Treated with 7D4 mAb Contributes to the Control of Parasite Burden in DBA / 2 Mice Infected with Lethal Plasmodium chabaudi chabaudi AS

CD4(+) CD25(+) regulatory T (Treg) cells expressing Foxp3(+) play a critical role in maintaining immune homoeostasis and controlling excessive immune responses. However, controversy about the immunoregulatory role of Treg cells exists in malaria studies. Given the role of maintenance of Foxp3 expression in Treg cells' activities, we investigated whether anti-CD25 mAb (7D4 clone) treatment affects Foxp3 expression in CD4(+) T cells in DBA/2 mice infected with Plasmodium chabaudi chabaudi AS (P.c. chabaudi AS). We found that DBA/2 mice succumbed to P.c. chabaudi AS infection, which was accompanied by increased expression of Foxp3 in CD4(+) T cells at the peak parasitemia. In contrast, Foxp3 expression was impaired in CD25-depleted mice with 7D4 mAb treatment, leading to delayed parasitemia and extended survival of infected mice. Production of IFN-γ, TNF-α and IL-6, as well as NO was significantly enhanced in CD25-depleted mice. The majority of CD4(+) CTLA-4(+) cells expressed high levels of Foxp3 (Foxp3(hi) cells) in control mice with P.c. chabaudi AS infection. However, the number of CD4(+) Foxp3(hi) CTLA-4(+) cells was reduced in CD25-depleted mice. Together, these data suggest that CD4(+) Foxp3(hi) CTLA-4(+) cells may be involved in regulating the intensity of pro-inflammatory responses via CTLA-4.

Anti-CD3 Therapy Promotes Tolerance by Selectively Depleting Pathogenic Cells while Preserving Regulatory T Cells

Monoclonal anti-CD3 Abs have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR-nonbinding (FNB) anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FNB anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of naive, effector, and regulatory T cells in vivo. Anti-CD3 treatment induced a transient systemic rise in the percentage but not absolute number of CD4(+)Foxp3(+) Tregs due to selective depletion of CD4(+)Foxp3(-) conventional T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of the proapoptotic proteins Fas, caspase-3, and Bim and was not inhibited by overexpression of the anti-apoptotic protein, Bcl-2. Tregs were not preferentially expanded and we found no evidence of conversion of conventional T cells into Tregs, suggesting that the pre-existing Tregs are resistant to anti-CD3-induced cell death. Interestingly, expression of the transcription factor Helios, which is expressed by thymus-derived natural Tregs, was increased in Tregs after FNB anti-CD3 mAb treatment, suggesting that the anti-CD3 treatment can alter, and potentially stabilize, Treg function. Taken together, the results suggest that FNB anti-CD3 therapy promotes tolerance by restoring the balance between pathogenic and regulatory T cells.

Regulatory T-cell depletion synergizes with gp96-mediated cellular responses and antitumor activity.

Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.

T regulatory cells participate in the control of germinal centre reactions

Germinal centre (GC) reactions are central features of T-cell-driven B-cell responses, and the site where antibody-producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti-glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) monoclonal antibody (mAb) to disrupt Treg-cell activity. In anti-GITR-treated mice, the GC B-cell pool was significantly larger compared with control-treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti-CD25 mAb treatment. Within the spleens of immunized mice, CXCR5(+) and CCR7(-) Treg cells were documented by flow cytometry and Foxp3(+) cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti-transforming growth factor-β or anti-interleukin-10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

Important role of neutrophils in the late asthmatic response in mice

Aims Neutrophils have been found increasingly in the lungs of patients with severe asthma; however, it is unclear whether the neutrophils contribute to the induction of the airway obstruction. We determined using a murine model whether neutrophils are involved in the late asthmatic response (LAR), and analyzed mechanisms underlying the antigen-induced airway neutrophilia. Main methods BALB/c mice sensitized by ovalbumin (OVA) + Al(OH) 3 were challenged 4 times by intratracheal administration of OVA. Airway mechanics were measured as specific airway resistance. Key findings Induction of the LAR after the 4th challenge coincided with airway neutrophilia. In contrast, eosinophil infiltration was established prior to the 4th challenge. A treatment with an anti-Gr-1 monoclonal antibody (mAb) before the 4th challenge selectively suppressed increases in the neutrophil number and myeloperoxidase (MPO) level in bronchoalveolar lavage fluid (BALF), and attenuated the magnitude of LAR by 60–70%. Selective suppression of eosinophilia by anti-IL-5 mAb had little effect on the LAR. The increases in neutrophil number and MPO level were partially inhibited by an anti-CD4 mAb treatment. The CD4 + cell depletion also significantly inhibited increases in neutrophil chemoattractants, IL-17A, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in BALF. However, blockade of FcγRII/III failed to suppress the neutrophilia. Significance These data suggest that neutrophils are key inducers of the LAR, and that the antigen-induced neutrophilia is partially dependent on activated CD4 + cells that are involved in the production of IL-17A, KC and MIP-2.

Anti-CD3 mAb therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells (107.12)

Abstract Monoclonal anti-CD3 antibodies have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR non-binding anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FcR non-binding (FNB) anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of CD4+CD25+Foxp3+ Treg cells in vivo. Anti-CD3 mAb treatment induced a transient systemic rise in the percentage but not absolute number of Tregs due to selective deletion of CD4+CD25- naïve and effector T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of pro-apoptotic proteins Fas or Bim and was caspase 3 independent. Tregs were not preferentially expanded and we found no evidence of convertion of conventional T cells into Tregs suggesting that Tregs are resistant to anti-CD3-induced cell death. Interestingly, we found increased Helios expression on Tregs further showing that adaptive Tregs are not induced by anti-CD3 treatment. Taken together, our results suggest that FNB anti-CD3 mAb therapy selectively induce death of potentially pathogenic CD25- T cells and stabilized, although did not induce, Tregs, thus promoting tolerance by restoring the balance between pathogenic T cells and regulatory T cells.