Nonresponsive State Research Articles

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

Abstract A26: Potent Th cell epitope modified HER2/neu dendritc cell vaccine magnify antitumor responses in HER2/neu tolerance transgenic mice.

Abstract Breast cancer is the most common cancer among women. Of all breast cancers cases, approximately 30% have amplifications of the self-antigen HER2/neu. Later studies have also found antibody response and T cell response specific for HER2/neu in cancer patients, indicating HER2/neu may be a good target for active immunotherapy. Following studies on HER2/neu targeted immunotherapeutic strategies all have been proven to be incapable of breaking tolerance towards HER2/neu, and couldn't elicit adequate antitumor immunity in curing HER2/neu positive breast cancer in spontaneously developed tumor transgenic mouse model, although both humeral and cellular immune responses could be detected. CD4+ T helper cells play an essential role in antitumor immunity. Reports have demonstrated that CD8+ T cells at resting or non-responsive state can be activated by providing cytokines and co-stimulatory signals, suggesting T cell help activation is crucial in breaking CD8+ CTL tolerance. The tetanus toxoid Th P30 has been found to be a universal and potent epitope in sensitizing and proliferating CD4+ T cells ex vivo. In this study, to evaluate the P30 effect on vaccine efficacy, we constructed two recombinant AdVs (AdVOVA-P30 and AdVHER2/neu-P30) expressing ovalbumin (OVA)-P30 and HER2/neu-P30, and two AdV transduced DC vaccines (DCOVA-P30 and DCHER2/neu-P30), and then compared the the immunization efficacy between vaccines with and without Th epitope P30 in the same vaccine modality in wild-type C57BL6 mouse model, a mouse model for OVA/OVA-P30 system study, and FVNneuN mouse model, a in vivo murine tumor model expressing the rat neu Ag. We demonstrate that both AdVOVA and AdVOVA-P30 vaccines could stimulate super high level of OVA-specific CD8+ T lymphocytes (CTLs) in wild-type C56BL/6 mice, but with no significant defference. However, when assess the immune responses induced by DCOVA and DCOVA-P30, we found both of CD4+ T cell and OVA-sepcific CD8+ T cell responses induced by DCOVA-P30 are significantly higher than that of DCOVA. Futher study on in vivo cytotoxic assay also confirmed this finding. Althogh both of DCOVA and DCOVA-P30 vaccines could lead to preventive long-term immunity against OVA-expressing BL6-10OVA melanoma in metastasizing in wild-type C56BL/6 mice, therapeutic antitumor immunity induced by DCOVA-P30 vaccine could kill significantly higher amount of pre-existing BL6-10OVA melanoma, suggesting that with the help of the universal potent Th epitope P30, vaccine efficacy could be enhanced. When apply such vaccine modality to HER2/neu-tolerance mouse model FVNneuN so as to enhace the anti-HER2/neu immunity, we also found DCneu-P30 could stimulate stronger CD8+ T cell response that of DCHER2/neu, leading to signigicantly stronger preventive antitumor immunity against HER2/neu expressing tumor cell line from FVNneuN, prolonging the life span of FVNneuN mice. Citation Format: Yuxiu Chen, Yufeng Xie, Jim Xiang. Potent Th cell epitope modified HER2/neu dendritc cell vaccine magnify antitumor responses in HER2/neu tolerance transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A26.

When yield gaps are poverty traps: The paradigm of ecological intensification in African smallholder agriculture

Yield gaps are pervasive in African smallholder agriculture, and are large for almost all crops in all regions. There is consensus that poor soil fertility and nutrient availability are the major biophysical limitations to agricultural production in the continent. We identify two major yield gaps: (1) the gap between actual yields (YA) and the water-limited yield potential (Yw), which is the maximum yield achievable under rainfed conditions without irrigation if soil water capture and storage is optimal and nutrient constraints are released, and (2) The gap between YA, and a locally attainable yield (YL) which corresponds to the water and nutrient-limited yields that can be measured in the most productive fields of resource endowed farmers in a community. Estimates of these two yield gaps are given for major crops, together with a framework for how yield gaps can be estimated in a pragmatic way for different farming systems. The paradigm of ecological intensification which focuses on yield potential, soil quality and precision agriculture is explored for the African context. Our analysis suggests that smallholder farmers are unable to benefit from the current yield gains offered by plant genetic improvement. In particular, continued cropping without sufficient inputs of nutrients and organic matter leads to localised but extensive soil degradation and renders many soils in a non-responsive state. The lack of immediate response to increased inputs of fertiliser and labour in such soils constitutes a chronic poverty trap for many smallholder farmers in Africa. This necessitates a rethink for development policy aimed to improve productivity and address problems of food insecurity.

BMP9 regulates endoglin-dependent chemokine responses in endothelial cells

AbstractBMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.

Brain–computer interfaces for communication with nonresponsive patients

A substantial number of patients who survive severe brain injury progress to a nonresponsive state of wakeful unawareness, referred to as a vegetative state (VS). They appear to be awake, but show no signs of awareness of themselves, or of their environment in repeated clinical examinations. However, recent neuroimaging research demonstrates that some VS patients can respond to commands by willfully modulating their brain activity according to instruction. Brain-computer interfaces (BCIs) may allow such patients to circumvent the barriers imposed by their behavioral limitations and communicate with the outside world. However, although such devices would undoubtedly improve the quality of life for some patients and their families, developing BCI systems for behaviorally nonresponsive patients presents substantial technical and clinical challenges. Here we review the state of the art of BCI research across noninvasive neuroimaging technologies, and propose how such systems should be developed further to provide fully fledged communication systems for behaviorally nonresponsive populations.

Persistence of a biocontrol Pseudomonas inoculant as high populations of culturable and non-culturable cells in 200-cm-deep soil profiles

Little is known about the ecology of soil inoculants used for pathogen biocontrol, biofertilization and bioremediation under field conditions. We investigated the persistence and the physiological states of soil-inoculated Pseudomonas protegens (previously Pseudomonas fluorescens) CHA0 (108 CFU g−1 surface soil) in different soil microbial habitats in a planted ley (Medicago sativa L.) and an uncovered field plot. At 72 days, colony counts of the inoculant were low in surface soil (uncovered plot) and earthworm guts (ley plot), whereas soil above the plow pan (uncovered plot), and the rhizosphere and worm burrows present until 1.2 m depth (ley plot) were survival hot spots (105–106 CFU g−1 soil). Interestingly, strain CHA0 was also detected in the subsoil of both plots, at 102–105 CFU g−1 soil between 1.8 and 2 m depth. However, non-cultured CHA0 cells were also evidenced based on immunofluorescence microscopy. Kogure's direct viable counts of nutrient-responsive cells showed that many more CHA0 cells were in a viable but non-culturable (VBNC) or a non-responsive (dormant) state than in a culturable state, and the proportion of cells in those non-cultured states depended on soil microbial habitat. At the most, cells in a VBNC state amounted to 34% (above the plow pan) and those in a dormant state to 89% (in bulk soil between 0.6 and 2 m) of all CHA0 cells. The results indicate that field-released Pseudomonas inoculants may persist at high cell numbers, even in deeper soil layers, and display a combination of different physiological states whose prevalence fluctuates according to soil microbial habitats.

Transcriptional analysis of steroid hormone receptors in smooth muscle uterine leiomyoma tumors of postmenopausal patients

Smooth muscle tumors are histologically separated into benign leiomyomas and malignant leiomyosarcomas. Uterine leiomyomas represent benign clonal tumors often arising within the smooth muscle tissue of the human uterus. Uterine leiomyomas develop after the start of the menstrual cycle, become symptomatic during middle age, and in most postmenopausal patients tumor regression occurs. Rarely, leiomyomas progress to leiomyosarcomas, where many sarcomas have markedly reduced or no steroid hormone receptors, thus, evolve to a hormone non-responsive state. Premenopausal leiomyomas are known to express higher levels of estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ) and progesterone receptor (PGR) than control myometrium, whereas postmenopausal leiomyomas have not been so well characterized molecularly. In this present investigation, ERβ, ERα and PGR gene expression were assessed in leiomyomas and in matched adjacent myometrium from a cohort of 14 postmenopausal patients using semi-quantitative Realtime PCR and RT-PCR. The mean average results showed that ERβ was 2.5-fold statistically significantly over expressed in postmenopausal leiomyomas compared to patient matched myometrium ( p = 0.038), whereas ERα and PGR were not significantly differently expressed. These results showed that up-regulation of ERβ occurred at the transcriptional level in postmenopausal leiomyomas. Quantitation of steroid hormone receptors from benign uterine tumors may be important for a more tailored therapy. In addition, a role for steroid hormones, specifically ERβ, is discussed in terms of benign tumor regression or tumor maintenance in postmenopausal leiomyomas.

Extracellular Matrix as a Strategy for Treating Chronic Wounds

The dermis normally directs all phases of skin wound healing following tissue trauma or disease. However, in chronic wounds, the dermal matrix is insufficient to stimulate healing and assistance by external factors is needed for wound closure. Although the concept of the extracellular matrix directing wound healing is not new, ideas about how best to provide the extracellular matrix components required to 'jump-start' the healing process are still evolving. Historically, these strategies have included use of enzyme-inhibiting dressing materials, which bind matrix metalloproteinases and remove them from the chronic wound environment, or direct application of purified growth factors to stimulate fibroblast activity and deposition of neo-matrix. More recently, the application of a structurally intact, biochemically complex extracellular matrix, designed to provide the critical extracellular components of the dermis in a single application, has allowed for the reconstruction of new, healthy tissue and restoration of tissue integrity in the previously chronic wound. This review focuses on this third mechanism as an emerging tactic in effective wound repair. Intact extracellular matrix can quickly, easily, and effectively provide key extracellular components of the dermis necessary to direct the healing response and allow for the proliferation of new, healthy tissue. Its application may promote the healing of wounds that have been refractory to other, more conventional treatment strategies, and may eventually show utility when used earlier in wound healing treatment with the goal of preventing wounds from reaching a truly chronic, nonresponsive state.

State-Dependent Mechanisms of LTP Expression Revealed by Optical Quantal Analysis

The expression mechanism of long-term potentiation (LTP) remains controversial. Here we combine electrophysiology and Ca(2+) imaging to examine the role of silent synapses in LTP expression. Induction of LTP fails to change p(r) at these synapses but instead mediates an unmasking process that is sensitive to the inhibition of postsynaptic membrane fusion. Once unmasked, however, further potentiation of formerly silent synapses leads to an increase in p(r). The state of the synapse thus determines how LTP is expressed.

Membrane-Associated TGF-β1 Inhibits Human Memory T Cell Signaling in Malignant and Nonmalignant Inflammatory Microenvironments

TGF-beta1 is present on cells derived from the microenvironment of human lung tumors and nonmalignant inflammatory tissues. We establish that this cell-associated cytokine mediates hyporesponsiveness of the memory T cells in these microenvironments in situ by blocking TCR signaling. T cells derived from these tissues failed to translocate NF-kappaB to the nucleus in response to CD3 + CD28 cross-linking. This nonresponsiveness was reversed by an anti-TGF-beta1-neutralizing Ab. Refractoriness of the memory T cells to TCR activation was also reversed by the removal of TGF-beta1 by briefly pulsing the cells in a low pH buffer. Addition of exogenous TGF-beta1 to eluted T cells re-established their nonresponsive state. Neither TGF-beta1, anti-TGF-beta1 Ab, nor low pH affected TCR signaling potential of peripheral blood T cells. We conclude that TGF-beta1 mediates a physiologically relevant regulatory mechanism, selective for memory T cells present in the tumor microenvironment and nonmalignant chronic inflammatory tissues.

Human regulatory T cells and their role in autoimmune disease

As self-recognition is fundamental to the efficient operation of the immune system, a number of mechanisms have evolved to keep this potential pathologic self-reactivity in check. Thus, even though the majority of strongly self-reactive T cells are deleted in the thymus during T-cell maturation, a number of mature T cells that recognize self-antigens can be found in the peripheral circulation in healthy individuals as well as in patients with autoimmune disease. These self-reactive cells are kept in a non-responsive state in healthy individuals while they appear to be involved in the etiology of a number of autoimmune diseases in patients. The primary role of a relatively recently identified T-cell population, referred to as natural CD4+ CD25+ regulatory T cells, is to modulate the activity of these self-reactive cells. Although it is still unclear how these regulatory cells function, they can inhibit the activation of other potentially pathologic T cells in in vitro assays. Using such assays, regulatory T cells isolated from patients with a number of autoimmune diseases have been shown to exhibit reduced inhibitory function as compared with those isolated from healthy individuals. In this review, we discuss human natural regulatory T cells, what is known about their function, and their associations with specific autoimmune diseases.

Proliferation Response of Leukemic Cells to CD70 Ligation Oscillates with Recurrent Remission and Relapse in a Low-Grade Lymphoma

Interactions of the TNF-related cell surface ligand CD70 with its receptor CD27 provide a costimulatory signal in B and T cell activation. Functional CD70-CD27 interactions could contribute to lymphoma and leukemia progression. This possibility was studied using DNA microarrays on a unique case of low-grade lymphoma/leukemia characterized by recurrent cycles of acute leukemic phase alternating with spontaneous remission. Upon induction of the acute phase expression of CD70 and CD27 in the leukemic cells increased 38- and 25-fold, respectively. Coexpression of membrane CD70 and CD27 on the leukemic (CD5+CD19+) cells was maximal 2-3 days following initiation of the attack. Soluble CD27 in the patient's serum was elevated during remission and further increased in the attack. Functional tests showed that neither anti-CD70 nor anti-CD27 Abs affect the rate of apoptosis. However, the anti-CD70 Ab specifically enhanced proliferation of the remission phase leukemic cells, whereas proliferation of the acute-phase counterparts that express higher level of membrane CD70 was unaffected. Hence, in this lymphoma/leukemia, membrane CD70 is presented on the leukemic cells in a responsive state during the remission and a nonresponsive state during the attack. Presumably, CD70 in its responsive state provides a costimulatory receptor for initiating the next acute phase while its nonresponsive state enables the remission.

Anergy and suppression as coexistent mechanisms for the maintenance of peripheral T cell tolerance.

Using T cell receptor (TCR) V(beta)8.1 transgenic mice, we have developed an in vivo system for the study of peripheral T cell tolerance, in which two distinct mechanisms of peripheral tolerance are observed to act simultaneously during the maintenance phase of the nonresponsive state. These two mechanisms, anergy and suppression, have been studied using the CD4+ T cell lineage markers 6C10 and CD25, which can be employed to purify the cells involved in each form of tolerance. Findings and perspectives gained through the study of peripheral tolerance in our model, as well as relevant observations from the literature, will be reviewed.

Photosensitivity disorders: cause, effect and management.

Abnormal photosensitivity syndromes form a significant and common group of skin diseases. They include primary (idiopathic) photodermatoses such as polymorphic light eruption (PLE), chronic actinic dermatitis (CAD), actinic prurigo, hydroa vacciniforme and solar urticaria, in addition to drug- and chemical-induced photosensitivity and photo-exacerbated dermatoses. They can be extremely disabling and difficult to diagnose. PLE, characterized by a recurrent pruritic papulo-vesicular eruption of affected skin within hours of sun exposure, is best managed by restriction of ultraviolet radiation (UVR) exposure and the use of high sun protection factor (SPF) sunscreens. If these measures are insufficient, prophylactic phototherapy with PUVA, broadband UVB or narrowband UVB (TL-01) for several weeks during spring may be necessary. CAD manifests as a dermatitis of chronically sun-exposed skin. Again, UVR exposure needs to be restricted; cyclosporine, azathioprine or PUVA may also be necessary. Actinic prurigo is characterized by the presence of excoriated papules and nodules on the face and limbs, most prominent and numerous distally. Actinic prurigo is managed again by restriction of UVR and the use of high SPF sunscreens; PUVA or broadband UVB therapy, or low doses of thalidomide may be necessary. Hydroa vacciniforme causes crops of discrete erythematous macules, 2 to 3mm in size, that evolve into blisters within a couple of days of sun exposure. Treatment for this rare disease is difficult; absorbent sunscreens and restricted UVR exposure may help. Solar urticaria is characterized by acute erythema and urticarial wealing after exposure to UVR. Treatment options for solar urticaria include non-sedating antihistamines such as fexofenadine and cetirizine; other options include absorbent sunscreens, restriction of UVR at the relevant wavelength, maintenance of a non-responsive state with natural or artificial light exposure and plasmapheresis. Industrial, cosmetic and therapeutic agents can induce exogenous drug- or chemical-induced photosensitivity. The clinical pattern is highly varied, depending on the agent; treatment is based on removal of the photosensitizer along with restriction of UVR exposure. Predominantly non-photosensitive dermatoses may also be exacerbated or precipitated by UVR; exposure to UVR should be reduced and sunscreens should be advocated, along with appropriate treatment of the underlying disease.

CD64 surface expression on neutrophils is transiently upregulated in patients with septic shock.

To clarify the changes in total leukocyte counts, CD64 neutrophil receptor expression and serum granulocyte colony-stimulating factor (G-CSF) concentrations in critically ill patients without infection and sepsis and in patients with septic shock. Prospective study. Intensive care unit (ICU) and research laboratory of a university hospital. Eleven critically ill patients without infections and 22 patients with proven infections in septic shock for the first time and of at least 3 days' duration. Over a 6month period, a longitudinal analysis of expression of the monomeric Fc receptor type I (CD64, FcgammaRI) on neutrophils was performed by flow cytometric analysis on a daily basis in all postoperative/post-traumatic patients admitted to the ICU until discharge from the ICU or death. Out of 273 patients, 11 patients without sepsis had organ failure and 22 patients with proven infections had septic shock for the first time and of at least 3 days' duration. Ten out of the 22 patients survived, 12 died. CD64 expression was greater in patients with septic shock than in patients without sepsis. Moreover, CD64 expression was only initially and transiently elevated in most survivors (9/10) and non-survivors (8/12) of septic shock. In survivors, G-CSF serum concentrations were markedly decreased in the 2nd week. Decreased neutrophil CD64 expression in an acutely ill population with septic shock may reflect the development of a non-responsive state as well as the early downregulation of neutrophil activation prior to the resolution of an ongoing infection.

Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8(+) T cells with a defect in effector function that is reversible on extended interleukin-2 incubation.

A vigorous expansion of antigen-specific CD8(+) T cells lacking apparent effector function was observed in a rhesus macaque acutely infected with the simian immunodeficiency virus (SIV) strain SIVmac239. Antigen-specific CD8(+) T cells were identified using antigenic-peptide class I major histocompatibility complex tetramers. As many as 8.3% of CD8(+) cells recognized the Mamu-A*01-associated SIV epitope Gag(181-189) (CTPYDINQM); however, these cells demonstrated no effector function when presented with peptide-incubated targets, as measured by intracellular cytokine staining for gamma interferon (IFN-gamma), interleukin-2 (IL-2) production, or direct cellular lysis. Similar results were observed with three other SIV peptide antigens. Nonresponsiveness did not correlate with apoptosis of the CD8(+) cells, nor were cells from this macaque impaired in their ability to present peptide antigens. Associated with the nonresponsive state was a lack of IL-2 production and decreased IL-2 receptor expression. Exogenous IL-2 treatment for 1 week in the absence of antigenic stimulation restored antigen-specific responses and the quantitative correlation between tetramer recognition and antigen-responsive IFN-gamma secretion. This case report suggests a regulatory mechanism that may impede the effector function of antigen-specific T cells during acute infection with SIV or human immunodeficiency virus in some cases. This mechanism may participate in the failure of the immune system to limit infection.

Drive: neurobiological and molecular mechanisms of sexual motivation

What arouses an animal or human from an inactive, nonresponsive state to a condition of activity and responsiveness? What are the biological mechanisms for this change? In this book Donald W. Pfaff focuses on a reproductive behavior typical of many female animals. Sensory stimuli from the male trigger responses in a well-defined circuit of nerve cells. At the top of the circuit, certain nerve cells receive and retain sex hormones such as estrogens and progesterone. As a result, specific genes in these nerve cells are turned on at specific times, affecting in turn the rest of the neural circuit and causing a state of sexual responsiveness.According to Pfaff, the biological bases for the most primitive human drives are largely explained by mechanisms uncovered in animal brains that have not changed in their fundamental properties over millions of years of evolution. Focusing on a single instinctive behavior, in this case the sex drive, is an important step toward understanding the biological reasons for the change from unmotivated to motivated animal behavior.

Single cell analysis of cytokine expression kinetics by human CD4+ T-cell clones during activation or tolerance induction.

Exposure to optimal peptide antigen concentrations induces human CD4+ T-cell clones to proliferate and secrete various cytokines. Higher (> 10-fold optimal) antigen concentrations cause long-term proliferative unresponsiveness, which can be reversed by exogenous interleukin-2 (IL-2). We call this condition 'tolerance'. We used intracellular cytokine staining and flow cytometric analysis to investigate the kinetics of interferon-gamma, tumour necrosis factor-alpha, IL-4 and IL-5 production during the initial phase of tolerance induction. Single cell analysis of interferon-gamma and IL-4 or IL-5 coexpression showed functional heterogeneity of cloned human CD4+ T cells. Superstimulation with phorbol 12-myristate 13-acetate and ionomycin (PI) revealed enhanced responsiveness shortly after tolerizing treatment, followed by reduced responsiveness. Both tolerized and activated T cells had similarly reduced cytokine responses when further stimulated with antigen during the following 48 hr, with limited enhancement following additional stimulation with PI. We conclude that cytokine induction is normally followed by a refractory phase, but that the expression of cytokines is enhanced in the initial phase of tolerance induction.

Sustained Activation of the Raf-MEK-ERK Pathway Elicits Cytokine Unresponsiveness in T Cells

Activation of T cells via the TCR and other costimulatory receptors triggers a number of signaling cascades. Among them, the Ras-activated Raf-mitogen-activated protein/extracellular signal-related kinase (ERK) kinase (MEK)-ERK signaling cascade has been demonstrated to be crucial for both T cell development and activation. It has previously been demonstrated that high doses of Ag or anti-CD3 mAb are able to induce in T cells a nonresponsive state to subsequent treatment with cytokines such as IL-2. The precise biochemical mechanisms underlying this effect are not fully characterized. In this study, we demonstrate that cytokine nonresponsiveness is accompanied by the induction of the cyclin-dependent kinase inhibitor p21Cip1 that is mediated, at least in part, by the activation of the Raf-MEK-ERK pathway. Furthermore, we demonstrate that selective activation of the Raf-MEK-ERK signaling pathway in T cells is sufficient to induce cytokine nonresponsiveness in both a T cell clone and naive primary T cells. In this case, nonresponsiveness is accompanied by the induction of p21Cip1 and the prevention of p27Kip1 down-regulation, leading to inhibition of cyclin E/cyclin-dependent kinase 2 activity. These data suggest that anti-CD3 mAb-induced cytokine nonresponsiveness may be a consequence of hyperactivation of the Raf-MEK-ERK pathway, leading to alterations in the expression of key cell cycle regulators. These observations may provide a novel insight into the mechanisms of induction of peripheral tolerance.

CTLA-4 Blockade Reverses CD8 + T Cell Tolerance to Tumor by a CD4 + T Cell– and IL-2-Dependent Mechanism

A tumor-specific CD8 + T cell response was studied using adoptive transfer of OT-I TCR transgenic cells. Upon i.p. challenge with E.G7 tumor, OT-I cells undergo CD4 + T cell–independent expansion at the tumor site and develop lytic function. Before tumor elimination, however, they leave the peritoneal cavity (PC) and appear in the LN and spleen where they exhibit “split anergy” and cannot further proliferate to antigen. Administering anti-CTLA-4 mAb early caused sustained OT-1 expansion in the PC, and late administration caused the OT-I cells to return to the PC and further expand; in both cases, tumor was controlled. These effects required CD4 + T cells and IL-2 and appear to result from reversal of the nonresponsive state of the CD8 + T cells.