Thymic Deletion Research Articles

Regulated Expression of a Tumor-Associated Antigen Reveals Multiple Levels of T-Cell Tolerance in a Mouse Model of Lung Cancer

Maximizing the potential of cancer immunotherapy requires model systems that closely recapitulate human disease to study T-cell responses to tumor antigens and to test immunotherapeutic strategies. We have created a new system that is compatible with Cre-LoxP-regulatable mouse cancer models in which the SIY antigen is specifically overexpressed in tumors, mimicking clinically relevant TAAs. To show the utility of this system, we have characterized SIY-reactive T cells in the context of lung adenocarcinoma, revealing multiple levels of antigen-specific T-cell tolerance that serve to limit an effective antitumor response. Thymic deletion reduced the number of SIY-reactive T cells present in the animals. When potentially self-reactive T cells in the periphery were activated, they were efficiently eliminated. Inhibition of apoptosis resulted in more persistent self-reactive T cells, but these cells became anergic to antigen stimulation. Finally, in the presence of tumors overexpressing SIY, SIY-specific T cells required a higher level of costimulation to achieve functional activation. This system represents a valuable tool in which to explore sources contributing to T-cell tolerance of cancer and to test therapies aimed at overcoming this tolerance.

Cyclin D1–Specific Cytotoxic T Lymphocytes Are Present in the Repertoire of Cancer Patients: Implications for Cancer Immunotherapy

Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.

Intrahepatic Murine CD8 T-Cell Activation Associates With a Distinct Phenotype Leading to Bim-Dependent Death

Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo. Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen. Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

Through Regulation of TCR Expression Levels, anIdd7Region Gene(s) Interactively Contributes to the Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in Nonobese Diabetic Mice

When expressed in NOD, but not C57BL/6 (B6) genetic background mice, the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose the ability to mediate the thymic deletion of autoreactive CD8+ T cells contributing to type 1 diabetes (T1D). This indicated some subset of the T1D susceptibility (Idd) genes located outside the MHC of NOD mice interactively impair the negative selection of diabetogenic CD8+ T cells. In this study, using both linkage and congenic strain analyses, we demonstrate contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chromosome 7 predominantly, but not exclusively, determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells as illustrated using the AI4 TCR transgenic system. The polymorphic Idd7 region gene(s) appears to control events that respectively result in high vs low expression of the AI4 clonotypic TCR alpha-chain on developing thymocytes in B6.H2g7 and NOD background mice. This expression difference likely lowers levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. These findings provide further insight to how susceptibility genes, both within and outside the MHC, may interact to elicit autoreactive T cell responses mediating T1D development in both NOD mice and human patients.

Generation and characterization of transgenic mice expressing a T‐cell receptor specific for the tumour‐associated antigen MDM2

T-cell-based antigen-specific immunotherapy targeting tumour-associated antigens offers the potential for cancer immunotherapy. However, the majority of identified tumour-associated antigens are also expressed at low levels in normal tissues and mechanisms of tolerance induction are likely to affect the quality of T-cell responses to such antigens. In this study a T-cell receptor transgenic model was developed to determine the magnitude of T-cell tolerance to the tumour-associated antigen murine double minute-2 (MDM2), a widely expressed protein that is found at elevated levels in many tumours. The analysis of transgenic mice showed that thymic deletion was responsible for purging large numbers of MDM2-specific T cells from the repertoire. However, some T cells with specificity for MDM2 were able to escape thymic deletion and persisted in the peripheral T-cell pool. Functional analysis revealed that these T cells displayed defects in antigen-driven expansion. This functional impairment of the MDM2-specific T cells was maintained following adoptive transfer of the T cells into hosts that were unable to present the T-cell-receptor-recognized antigen. This study demonstrates that thymic deletion and the functional impairment of T cells present in the periphery both operate to establish T-cell tolerance to the tumour-associated antigen MDM2. Furthermore, the tolerant phenotype was stable and did not require continuous MDM2 peptide presentation in normal tissues.

Danger-free autoimmune disease in Aire-deficient mice

The danger theory of immune tolerance asserts that environmental factors hold primacy over lymphocyte autoreactivity in initiating autoimmune disease. We sought to test this contention using the Aire-deficient mouse model of the human disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a multiorgan autoimmune disorder rooted in a lesion in thymic tolerance. Compound screens stimulating a broad range of innate immune system pathways failed to show any modulation of disease characteristics in Aire(-/-) mice on either the C57BL/6 or NOD genetic backgrounds. Furthermore, deficiency in the Toll-like receptor adaptor Myd88 increased the lifespan of NOD.aire(-/-) mice but did not prevent the initiation of autoimmunity. Finally, germ-free NOD.aire(-/-) mice exhibited autoimmunity in all organs normally targeted in this model, indicating that microbial conditioning is not required for activation of autoreactive T cells relevant to this disease. Together, these data suggest that the stochastic genesis of dangerous T cell clones can initiate autoimmune disease without the need for environmental stimulation, underlining the importance of Aire-dependent thymic deletion.

Ecoimmunity: immune tolerance by symmetric co‐evolution

It is widely accepted that immune tolerance toward "self" is established by central and peripheral adaptations of the immune system. Mechanisms that have been demonstrated to play a role in the induction and maintenance of tolerance include thymic deletion of self-reactive T cells, peripheral T cell anergy and apoptosis, as well as thymic and peripheral induction of regulatory T cells. However, a large body of experimental findings cannot be rationalized solely based on adaptations of the immune system to its environment. Here we propose a new model termed Ecoimmunity, where the immune system and the tissue are viewed as two sides of a continuously active and co-evolving predator-prey system. Ecoimmunity views self-tolerance, not as an equilibrium in which autoimmunity is chronically suppressed, but as a symmetrical balanced conflict between the ability of immune cells to destroy tissue cells by numerous mechanisms, and the capacity of adapted tissue cells to avoid predation. This balance evolves during ontogeny, in parallel to immune adaptations, embryonic tissue cells adapt their phenotype to the corresponding immune activity by developing the ability to escape or modulate damaging local immune responses. This phenotypic plasticity of tissue cells is directed by epigenetic selection of gene expression pattern and cellular phenotype amidst an ongoing immune pressure. Thus, whereas some immune cells prey predominantly on pathogens and infected cells, self-reactive cells continuously prey on incompetent tissue cells that fail to express the adapted phenotype and resist predation. This model uses ecological generalization to reconcile current contradictory observations as well as classical enigmas related to both autoimmunity and to tolerance toward foreign tissues. Finally, it provides empirical predictions and alternative strategies toward clinical challenges.

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Participation of FLIP, RIP and Bcl‐xL in Fas‐mediated T‐cell Death

Apart from the conventional Fas signalling pathway, alternative pathways including the mitochondrial caspase-dependent and RIP-mediated cell death routes have been proposed to operate during Fas-mediated cell death. To evaluate the contribution of different Fas signalling pathways, mice overexpressing FLIP(L), Bcl-x(L), a kinase-deficient form of RIP (RIPDeltakin) or combinations thereof were generated by retroviral gene transfer of haematopoietic stem cells. Such mice did not show overt abnormalities in haematopoietic development, defects in thymic deletion, accumulation of double-negative T cells or signs of autoimmunity. Fas-mediated death of mitogen-activated T cells was caspase dependent and could be blocked by FLIP(L) overexpression only with the minor involvement of Bcl-x(L) or RIPDeltakin inhibitable pathways. Fas-mediated death of resting CD4(+) and CD8(+) T cells was mainly caspase dependent but could only partly be blocked by FLIP(L) overexpression. Both Bcl-x(L) or RIPDeltakin expression resulted in partial protection of CD8(+) T cells against Fas-mediated cell death. These results indicate that yet uncharacterized signalling pathways from the Fas receptor are critically involved in lymphoproliferative and autoimmune disease observed in lpr mice and autoimmune lymphoproliferative syndrome patients.

The role of B cells in the induction of peripheral T cell tolerance

The immune system of vertebrates is designed to protect against invading pathogens. To be able to deal with different antigenic threats, the T cell repertoire is very diverse. This diversity, though useful, can cause autoimmune diseases if any imbalances in the function of the immune system occur. However, the immune system has its own mechanisms for suppressing or regulating the potentially dangerous, autoreactive T cells. The first mechanism is central tolerance, where potentially self-reactive thymocytes, which encounter APC expressing self-encoded molecules, are triggered to undergo apoptosis so that the thymocytes are clonally deleted in the thymus. However, thymic deletion of self-reactive T cells seems only to affect cells of the highest affinity, and some autoreactive T cells (with low affinity to self-molecules) still manage to gain access to the periphery [1, 2]. Furthermore, only T cells, which recognize antigens expressed within the thymus, are clonally deleted. This means that T cells, which are capable of recognizing tissue-restricted antigens, can still manage to make it to the periphery [2]. To deal with the threat posed by these self-reactive T cells, there are mechanisms for induction of peripheral T cell tolerance. Tolerance can be defined as the state of unresponsiveness to a specific antigen as a result of the presence of one or more mechanisms, which suppress the immune reaction. Thus, tolerance is an active process and is not just the absence of an immune response. B cells are professional APC, which activate or tolerize T cells to help induce or suppress immune responses. There are several mechanisms by which B cells induce tolerization of the T cell compartment. Identifying how peripheral T cell tolerance can be induced will serve to design new therapeutic strategies for regulating the balance between tolerance and active immunity in cases such as autoimmune diseases, tumor immunity, and transplant tolerance.

Association between PTPN22 and endometriosis

PTPN22 is currently one of the few known shared-autoimmunity genes and is therefore a candidate marker for endometriosis. Our data show that female carriers of the PTPN22( *)T variant are significantly more susceptible to endometriosis than controls.

256. Ultrasound Guided Gene Transfer to the Fetal Thymus Results in Thymic Epithelial Cell Transduction and Deletion of Transgene Specific T Cells

256. Ultrasound Guided Gene Transfer to the Fetal Thymus Results in Thymic Epithelial Cell Transduction and Deletion of Transgene Specific T Cells

A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

BackgroundMost tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells.Methodology/Principal FindingsWe used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-γ, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-γ production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR.Conclusions/SignificanceThese data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2.

CD24 inhibits thymic deletion of myelin antigen-specific T cells (129.34)

Abstract CD24 is a glycosylphosphatidylinositol (GPI) anchored cell surface glycoprotein that is expressed in hematopoietic cells and cells of the central nervous system (CNS). We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model of human disease multiple sclerosis. In CD24−/− mice, normal levels of myelin oligodendrocyte glycoprotein (MOG) specific T cell were primed, however these T cells were non-pathogenic. To understand this issue, we bred CD24−/− mice with 2D2 TCR transgenic mice, which bear TCR specific to MOG, and generated 2D2 TCR transgenic mice with or without CD24. Here we show that 2D2 TCR transgenic mice with CD24-deficiency (2D2/CD24−/−) have remarkably withered thymus. In peripheral lymphoid organs, transgenic T cells from 2D2/CD24−/− mice have an immature phenotype (CD4−CD8−), do not respond to MOG peptide stimulation, and fail to cause autoimmune inflammation in the CNS and optical nerves. In contrast, OT-2 TCR transgenic mice with CD24 deficiency (OT-2/CD24−/−), which bear TCR specific to chicken ovalbumin (OVA), have normal thymus and their peripheral T cells have a normal response to OVA peptide. These data suggest that CD24 inhibits thymic deletion of myelin antigen, but not foreign antigen-reactive T cells.

Tracing the action of IL‐2 in tolerance to islet‐specific antigen

Genetic variants of interleukin 2 (IL-2) and its receptor are associated with murine and human susceptibility to Type 1 diabetes, yet the role of IL-2 in controlling pancreatic islet-reactive T cells is unknown. Here, we develop a model where IL-2 deficiency precipitates a breakdown of self-tolerance and progression to diabetes, and its action upon diabetogenic islet-specific CD4 T cells can be tracked. We find that IL-2 is not required for Aire-dependent thymic clonal deletion of high-avidity diabetogenic clones, but is essential for thymic formation of islet-specific Foxp3-expressing CD4 T cells. The absence of IL-2 results in the expansion of low-avidity Foxp3(-) islet-reactive CD4 T cells. The mechanism by which IL-2 prevents diabetes is therefore through the establishment of a repertoire of islet-reactive Foxp3(+) T cells within the thymus, and limitation of the peripheral activation of low-avidity islet-reactive T cells that normally escape thymic negative selection.

Regulatory T Cells Are Resistant to Apoptosis via TCR but Not P2X7

Regulatory T cells (Tregs) are relatively autoreactive yet, paradoxically, have been found to display normal sensitivity to thymic deletion. The relationship between self-avidity, apoptosis, and the selection of Tregs therefore remains unclear. We show that thymic Tregs develop efficiently, even at low self-avidity, and are moderately resistant to apoptosis in comparison to conventional thymocytes. Consistent with this, although conventional self-reactive T cell populations undergo chronic peripheral deletion, self-reactive Tregs are largely spared removal. Similarly, the distribution of Tregs among peripheral CD4(+) cells exhibits a linear inverse relationship with CD45RB expression, indicating relative apoptosis resistance of Tregs in chronic responses to environmental Ags. We also show that appropriate controls for CD45RB levels are important for comparisons of Treg and conventional T cell activity. When thus controlled, and contrary to previous reports, Tregs exhibit normal sensitivity to cell death through TCR-independent stimuli, such as the purinergic receptor, P2X(7). Finally, although absence of CD45 in gene-targeted mice results in profound T cell hyporesponsiveness, there is little or no effect on thymic Treg frequency. In summary, the data support a model in which signal strength plays little part in Treg lineage specification, though moderate resistance of self-reactive Tregs to apoptosis may result in progressive biasing of peripheral Treg TCRs toward autoreactivity in comparison to those of conventional T cells.

Double Negative T Cells Influence TCR VB Variablity to Induce Allotolerance.

Immune alloresponsiveness following allogeneic HSCT is influenced by the dynamics of immune reconstitution and development of allotolerance. In general, tolerance is induced by thymic clonal deletion (central) and apoptosis or suppression of alloresponsive lymphocytes by regulatory T cells in the periphery. We have recently demonstrated that the size of the TCR repertoire within the CD4 and CD8 compartments can be assessed using VB spectrum by flow cytometry, and expansions/losses of individual TCR VB families can be used as a surrogate marker of TCR variability. (Exp. Hem. 32: 1010–1022; Br. J. Haematol. 129:411–419). Additionally, regulatory T cells can also impact the clonal contractions and expansions within the TCR VB repertoire. Various types of regulatory T cells have been described including CD4+CD25+, CD8+, NK T−cells, and CD3+CD4/CD8− double negative T cells (DN Tregs). In our current study we investigated the role of DN Tregs on the restoration of immune repertoire diversity. We hypothesized that alloresponsiveness clinically detected as a manifestation of GvHD may be associated with oligoclonal T−cell expansions, and in this context decreased numbers of regulatory T cells suggest deficient tolerizing function by regulatory T cells including DN Tregs. Here we studied a cohort of 60 HSCT recipients (AML, CML, CLL, NHL, AA, and PV), of which 25 patients received matched unrelated donor grafts and 35 received matched sibling donor grafts. Blood was sampled between 2003–2006 at monthly intervals after HSCT, and flow cytometry for TCR repertoire in CD4 and CD8 cells as well as the numbers of DN cells were recorded. Additionally, separate samples were collected for measurement of chimerism and were included in analysis when donor lymphoid chimerism was > 60%. A subset analysis was performed based on the presence/absence of GvHD. For the 27/60 (45%) patients with episodes of GvHD, results were obtained at the time of diagnosis of GvHD (grade > 2), while for patients in whom notable GvHD was not captured, the steady−state values at corresponding times were used for analysis. For all patients serial evaluations were available. For the purpose of this study, significant VB expansions/contractions were defined as +/− 2 standard deviation over the average VB family size. Using Cox proportional hazards analysis to identify univariate risk factors for GVHD, CD8 VB TCR contractions > 14 VB families (58.3% contraction of entire CD4 VB repertoire) constituted a strong indicator for increased risk (HR=7.61, p=0.011). This observation is consistent with the fact that oligoclonality of alloreactive T cell clones is frequently accompanied by a significant contraction/loss of remaining VB families and may herald heightened alloresponsiveness as a manifestation of GvHD. Estimation for correlation by Pearson's correlation coefficient also demonstrated that percentage of DN cells strongly correlated with a normalization of CD4 VB TCR repertoire (lower number of expansions; N=57, R= −0.51, p=0.027), supporting our hypothesis that DN cells participate in peripheral tolerance and suppress proliferative, alloresponsive CD4 clones. In summary, our results further characterize TCR variability post HSCT and define the role of DN cells in the induction of allotolerance.

Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura

Immunoregulatory CD4+CD25+ T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. Therefore, CD4+CD25+ T cells are believed to possibly play an important role in pathogenic autoimmune diseases. We measured the count of CD4+CD25+ T cells in 44 patients with idiopathic thrombocytopenic purpura (ITP), and the number of CD4+CD25+ T cells and clinical features were then analyzed. By using a flow cytometric analysis, the number of CD4+CD25+ T cells in the patients with ITP showed a very wide distribution in comparison to healthy volunteers. The number of CD4+CD25+ T cells was significantly lower in the ITP patients in the severe phase, and in patients positive for anti-glycoprotein IIb­IIIa antibody. However, the number of those cells increased in the patients at the complete remission phase, especially after a splenectomy. The Foxp3 mRNA levels of peripheral blood mononuclear cells (PBMC) of ITP patients were higher with an improved platelet count than in those with a low platelet count. In addition, the Foxp3 mRNA levels closely correlated with the number of CD4+CD25+ cells. These mechanisms remain to be fully elucidated, however, the count of CD4+CD25+ T cells is considered to possibly be related to the severity of ITP.

Accumulation of CD8+ T Cells in Advanced-Stage Tumors and Delay of Disease Progression following Secondary Immunization against an Immunorecessive Epitope

Self-reactive T cells that survive the process of positive and negative selection during thymocyte development represent potential effector cells against tumors that express these same self-Ags. We have previously shown that CD8+ T lymphocytes (T(CD8)) specific for an immunorecessive epitope, designated epitope V, from the SV40 large T Ag (Tag) escape thymic deletion in line SV11 Tag-transgenic mice. In contrast, these mice are tolerant to the three most dominant Tag epitopes. The majority of the residual epitope V-specific T(CD8) have a low avidity for the target epitope, but a prime/boost regimen can expand higher avidity clones in vivo. Whether higher avidity T(CD8) targeting this epitope are affected by Tag-expressing tumors in the periphery or can be recruited for control of tumor progression remains unknown. In the current study, we determined the fate of naive TCR-transgenic T(CD8) specific for Tag epitope V (TCR-V cells) following transfer into SV11 mice bearing advanced-stage choroid plexus tumors. The results indicate that TCR-V cells are rapidly triggered by the endogenous Tag and acquire effector function, but fail to accumulate within the tumors. Primary immunization enhanced TCR-V cell frequency in the periphery and promoted entry into the brain, but a subsequent booster immunization caused a dramatic accumulation of TCR-V T cells within the tumors and inhibited tumor progression. These results indicate that epitope V provides a target for CD8+ T cells against spontaneous tumors in vivo, and suggests that epitopes with similar properties can be harnessed for tumor immunotherapy.

Functional and Molecular Comparison of Anergic and Regulatory T Lymphocytes

Tolerance in vivo is maintained by multiple mechanisms that function to prevent autoimmunity. An encounter of CD4+ T cells with a circulating self-Ag leads to partial thymic deletion, the development of CD25+ regulatory T cells (Tregs), and functional anergy in the surviving CD25- population. We have compared anergic and regulatory T cells of the same Ag specificity generated in vivo by the systemic self-Ag. Anergic cells are unresponsive to the self-Ag that induces tolerance, but upon transfer into a new host and immunization, anergic cells can induce a pathologic autoimmune reaction against tissue expressing the same Ag. Tregs, in contrast, are incapable of mediating harmful reactions. To define the basis of this functional difference, we have compared gene expression profiles of anergic and regulatory T cells. These analyses show that Tregs express a distinct molecular signature, but anergic cells largely lack such a profile. Anergic cells express transcripts that are associated with effector differentiation, e.g., the effector cytokines IL-4 and IFN-gamma. Anergic cells do not produce these cytokines in response to self-Ag, because the cells exhibit a proximal signaling block in response to TCR engagement. Thus, anergy reflects an aborted activation pathway that can readily be reversed, resulting in pathologic effector cell responses, whereas Treg development follows a distinct developmental pathway that extinguishes effector functions.