Targeted delivery of the ErbB2/HER2 tumor antigen to professional APCs results in effective antitumor immunity

Activation of T cells by professional APCs that present peptide epitopes of tumor-associated Ags is critical for the induction of cell-mediated immunity against tumors. To facilitate targeted delivery of the ErbB2 (HER2, neu) tumor Ag to APCs in vivo, we have generated chimeric proteins that contain the extracellular domain of CTLA-4 for binding to B7 molecules on the APC surface, which is genetically fused to a human ErbB2 fragment as an antigenic determinant. Bacterially expressed CTLA-4-ErbB2 fusion protein and a similar molecule harboring in addition the translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed specific binding to B7-expressing cells, followed by protein internalization and intracellular degradation. Vaccination of BALB/c mice with the fusion proteins resulted in the induction of ErbB2-specific CD8(+) T cells and CTL-dependent protection from subsequent challenge with ErbB2-expressing but not ErbB2-negative murine renal carcinoma cells. In a therapeutic setting, injection of CTLA-4-ErbB2 protein vaccines caused rejection of established ErbB2-expressing tumors. Thereby, immunological memory was induced, leading to long-term systemic immunity and protection against rechallenge several months later. Our results demonstrate that these chimeric protein vaccines are effective tools for the induction of ErbB2-specific, T cell-mediated immunity.     

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.     

The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine-mediated T-cell responses

Blockade of CTLA-4 by monoclonal antibodies (mAb) can mediate regression of tumors and increase the efficacy of tumor antigen specific vaccines. Blockade of CTLA-4 has also been shown to significantly increase the avidity of antigen-specific T cells after immunization with live recombinant viral vector based vaccine. Here, we demonstrate a biological synergy between CTLA-4 blockade and active vaccine therapy consisting of recombinant vaccinia and avipox viruses expressing carcinoembryonic antigen (CEA) and three T cell costimulatory molecules to enhance antitumor effects. However, this synergy was very much dependent on the temporal relationship of scheduling of the two agents. We evaluated the strategies in both a foreign antigen model using β-galactosidase as immunogen, and in a “self” antigen model using CEA as immunogen. For antitumor activity the model used consisted of mice transgenic for human CEA and a murine carcinoma cell line transfected with CEA. The enhanced antitumor activity after vaccine and CTLA-4 blockade did not result in any signs of autoimmunity. These studies form a rational basis for the use of vector-based vaccines with anti-CTLA-4 and demonstrate that both enhancement of positive costimulatory signals and inhibition of negative costimulatory signals can be simultaneously exploited. These studies also underscore the importance of “drug” scheduling in vaccine combination therapies.     

Protein kinase A-mediated gating of neuregulin-dependent ErbB2-ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody prevents rejection of allogeneic cells

Natural engagement of CTLA-4 on host B7 limits T cell activation. We hypothesized that therapeutic cross-linking of CTLA-4 in vivo may further inhibit T cell function and prevent allograft rejection. However, none of the currently available CTLA-4-binding reagents have ligating properties when injected in vivo. The observation that surface-immobilized anti-CTLA-4 mAb inhibits T cell activation in vitro prompted us to develop a membrane-bound single-chain anti-CTLA-4 Ab (7M). To model whether tissue expression of 7M could suppress allograft rejection, we examined the ability of H-2L(d)-specific TCR-transgenic T cells to reject 7M-expressing allogeneic tumor cells injected s.c. Expression of 7M significantly inhibited allogeneic rejection in mice that received CTLA-4(+/+) but not CTLA-4(-/-) T cells. Furthermore, CTLA-4(+/+) T cells that had encountered 7M-expressing tumors in vivo acquired defects in cytokine production and cytotoxicity. Thus, deliberate ligation of CTLA-4 in vivo potently inhibits allogeneic T cell responses.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.     

ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli

Demyelination is a common pathologic feature in many neurodegenerative diseases including infection with leprosy-causing Mycobacterium leprae. Because of the long incubation time and highly complex disease pathogenesis, the management of nerve damage in leprosy, as in other demyelinating diseases, is extremely difficult. Therefore, an important challenge in therapeutic interventions is to identify the molecular events that occur in the early phase before the progression of the disease. Here we provide evidence that M. leprae-induced demyelination is a result of direct bacterial ligation to and activation of ErbB2 receptor tyrosine kinase (RTK) signaling without ErbB2-ErbB3 heterodimerization, a previously unknown mechanism that bypasses the neuregulin-ErbB3-mediated ErbB2 phosphorylation. MEK-dependent Erk1 and Erk2 (hereafter referred to as Erk1/2) signaling is identified as a downstream target of M. leprae-induced ErbB2 activation that mediates demyelination. Herceptin (trastuzumab), a therapeutic humanized ErbB2-specific antibody, inhibits M. leprae binding to and activation of ErbB2 and Erk1/2 in human primary Schwann cells, and the blockade of ErbB2 activity by the small molecule dual ErbB1-ErbB2 kinase inhibitor PKI-166 (ref. 11) effectively abrogates M. leprae-induced myelin damage in in vitro and in vivo models. These results may have implications for the design of ErbB2 RTK-based therapies for both leprosy nerve damage and other demyelinating neurodegenerative diseases.     

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.     

Immunization with the gene expressing woodchuck hepatitis virus nucleocapsid protein fused to cytotoxic-T-lymphocyte-associated antigen 4 leads to enhanced specific immune responses in mice and woodchucks

A number of options are available to modify and improve DNA vaccines. An interesting approach to improve DNA vaccines is to fuse bioactive domains, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to an antigen. Such fusion antigens are expressed in vivo and directed to immune cells by the specific bioactive domain and therefore possess great potential to induce and modulate antigen-specific immune responses. In the present study, we tested this new approach for immunomodulation against hepadnavirus infection in the woodchuck model. Plasmids expressing the nucleocapsid protein (WHcAg) and e antigen (WHeAg) of woodchuck hepatitis virus (WHV) alone or in fusion to the extracellular domain of woodchuck CTLA-4 and CD28 were constructed. Immunizations of mice with plasmids expressing WHcAg or WHeAg led to a specific immunoglobulin G2a (IgG2a)-dominant antibody response. In contrast, fusions of WHcAg to CTLA-4 and CD28 induced a specific antibody response with comparable levels of IgG1 and IgG2a. Furthermore, the specific IgG1 response to WHcAg/WHeAg developed immediately after a single immunization with the CTLA-4-WHcAg fusion. Woodchucks were immunized with plasmids expressing WHeAg or the CTLA-4-WHcAg fusion and subsequently challenged with WHV. CTLA-4-WHcAg showed an improved efficacy in induction of protective immune responses to WHV. In particular, the anti-WHsAg antibody response developed earlier after challenge in woodchucks that received immunizations with CTLA-4-WHcAg, consistent with the hypothesis that anti-WHs response is dependent on a Th cell response to WHcAg. In conclusion, the use of fusion genes represents a generally applicable strategy to improve DNA vaccination.     

Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis

Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.     

Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production

Background

The co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.

Methodology/Principal Findings

We find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.

Conclusions/Significance

This study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.     

Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6-8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using beta(2)-microglobulin-knockout, IFN-gamma-knockout, and B cell-deficient mice, CD4(+) and CD8(+) cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8(+) T cells are not necessary at all, and CD4(+) T cells are necessary for only 36-48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcgammaRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.     

The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo

Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-beta could not induce CD4+CD25- cells from CTLA-4(-/-) mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-beta to induce FoxP3-expressing mouse suppressor cells. TGF-beta accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-beta to induce CD4+CD25- cells to express FoxP3 and develop suppressor activity. TGF-beta also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4(-/-) mice, the lack of TGF-beta-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.     

Schizophrenia susceptibility pathway neuregulin 1-ErbB4 suppresses Src upregulation of NMDA receptors

Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β-ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst-induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.     

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.     

Physiological mechanisms of regulating alloimmunity: cytokines,CTLA-4, CD25+ cells,and the alloreactive T cell clone size

The mechanisms underlying physiological regulation of alloimmune responses remain poorly defined. We investigated the roles of cytokines, CTLA-4, CD25(+) T cells, and apoptosis in regulating alloimmune responses in vivo. Two murine cardiac transplant models were used, B10.D2 (minor mismatch) and C57BL/6 (major mismatch), into BALB/c recipients. Recipients were wild type, STAT4(-/-) (Th1 deficient), or STAT6(-/-) (Th2 deficient) mice. Minor mismatched allografts were accepted spontaneously in approximately 70% of wild type and STAT4(-/-) mice. By contrast, there was significantly shorter graft survival in minor mismatched STAT6(-/-) mice. Either the adoptive transfer of STAT4(-/-) splenocytes or the administration of IL-4Fc fusion protein into STAT6(-/-) mice resulted in long term graft survival. Blocking CTLA-4 signaling accelerated the rejection in all recipients, but was more pronounced in the minor combination. This was accompanied by an increased frequency of alloreactive T cells. Furthermore, CTLA-4 blockade regulated CD4(+) or CD8(+) as well as Th1 or Th2 alloreactive T cells. Finally, while anti-CD25 treatment prolonged graft survival in the major mismatched combination, the same treatment accelerated graft rejection in the minor mismatched group. The latter was associated with an increased frequency of alloreactive T cells and inhibition of T cell apoptosis. These data demonstrate that cytokine regulation, CTLA-4 negative signaling, and T cell apoptosis play critical roles in regulating alloimmunity, especially under conditions where the alloreactive T cell clone size is relatively small.