Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by self-epitopes.

The invariant chain (Ii) binds to newly synthesized MHC class II molecules with the CLIP region of Ii occupying the peptide-binding groove. Here we demonstrate that recombinant Ii proteins with the CLIP region replaced by antigenic self-epitopes are highly efficient in activating and silencing specific T cells in vitro and in vivo. The Ii proteins require endogenous processing by antigen-presenting cells for efficient T cell activation. An Ii protein encompassing the epitope myelin basic protein amino acids 84-96 (Ii-MBP84-96) induced the model autoimmune disease experimental allergic encephalomyelitis (EAE) with a higher severity and earlier onset than the peptide. When applied in a tolerogenic manner, Ii-MBP84-96 abolished antigen-specific T cell proliferation and suppressed peptide-induced EAE more effectively than peptide alone. Importantly, i.v. administration of Ii proteins after EAE induction completely abrogated the disease, whereas peptides only marginally suppressed disease symptoms. Ii fusion proteins are thus more efficient than peptide in modulating CD4(+) T cell-mediated autoimmunity, documenting their superior qualities for therapeutic antigen delivery in vivo.

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands.

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function.

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.

Commonly used mouse strains.

This appendix lists commonly used mouse strains and selected cell-surface molecules expressed by these strains. H-2 is the gene locus residing on chromosome 17 of the mouse which encodes major histocompatibility proteins. Only those H-2 loci involved in immunologic interactions frequently cited in this manual are listed here--i.e., Aa-, Ab-, Ea-, and Eb--region-encoded MHC class II molecules, and K-and D-region-encoded MHC class I molecules. In addition to H-2 loci, others are listed that encode immunologically important proteins. Congenic strains listed in this appendix represent inbred strains that were derived from their origin (BALB/c, C57Bl/10, and C3H) by selective matings, such that they differ from that originating strain at only one independently segregating genetic locus. The locus of choice in these cases was H-2; thus, these strains differ from their strain of origin only in their H-2 complex, but not in any other loci. During the production of the congenic lines, several strains were derived that had undergone intra-H-2 recombination events. These strains are listed here and have been invaluable in defining the major roles of various MHC glycoproteins.

Isolation of mouse mononuclear cells.

This unit covers techniques for preparing lymphoid cell suspensions and removing red blood cells and dead cells. These basic manipulations are required for most of the subsequent protocols in this chapter, i.e., measuring functional responses of mouse lymphocytes.

In vivo depletion of CD4- and CD8-specific T cells.

In vivo depletion of CD4- and CD8-specific T cells is a means of studying the role of these subpopulations in the initiation and effector phases of particular in vivo immune responses. In this unit, a protocol is provided for harvesting anti-CD4 or anti-CD8 monoclonal antibody- producing ascites fluid or tissue culture supernatant from rat or mouse T cell hybridomas. The antibody (preferably IgG) is then purified and injected intraperitoneally into adult mice. Depletion of the appropriate subset of T cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on in vivo immune responses. The depleted condition is maintained by repeated injections of the monoclonal antibody.

Circumventing T-cell tolerance to tumour antigens.

During past decades, many attempts have been made to induce or enhance tumour-specific T-cell immunity in cancer patients by vaccination. However, it has become apparent that in a large number of cases the naturally occurring tumour-specific T-cell repertoire is of low affinity and therefore inefficient in mediating tumour rejection. Because of the potential therapeutic value of high affinity TCRs with tumour/lineage specificities, we set out to develop a number of new technologies that can be used to create improved tumour-specific T-cell immunity. These strategies entail: (i) the efficient expansion of low affinity T cells specific for self antigens through the use of variant peptides with improved TCR-binding characteristics; (ii) a retroviral library-based technology to improve the affinity of (self-specific) T-cell receptors in vitro, and (iii) proof of principle for the feasibility of TCR gene transfer as a means to generate T-cell populations with a desired antigen-specificity in vivo. Collectively this toolbox should allow us to create improved T-cell receptors for human tumour antigens, which can subsequently be used to impose tumour-reactivity on to peripheral T cells.

Branching out to gain control: how the pre-TCR is linked to multiple functions.

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Tumor size at the time of adoptive transfer determines whether tumor rejection occurs.

Here we investigate the minimal requirements for induction of an anti-tumor response in CD8 T cells in vivo. We compare the efficacy of adoptive transfer of CD8 T cells with a transgenic TCR specific for the main cytotoxic T lymphocyte epitope of the influenza virus nucleoprotein (NP) on the growth of NP-expressing EL4 tumors under different conditions. In a setting in which tumor rejection is solely dependent on tumor-specific CD8 T cells, small immunogenic tumors fail to induce a rejection response, despite the fact that they are not ignored: tumor-specific CD8 T cells are activated, differentiate into effector cells and infiltrate the tumor bed. Nevertheless, tumor rejection does not occur. In sharp contrast, the same immunogenic tumor, when growing as a large tumor mass, is rejected by transferred tumor-specific CD8 T cells. The main features which distinguish the rejection response to a large tumor mass from the response to a small tumor is that, in the latter case, activated CD8 T cells appear much later, and in much smaller numbers. Efficacy of adoptive transfer is thus dictated by the size of the tumor mass at the time of transfer. These findings predict that treatment of minimal residual disease with adoptive transfer will fail, unless vaccination is also provided at the time of transfer.

Tracing and characterization of the low-avidity self-specific T cell repertoire.

It is well established that expression of self antigens results in the deletion of the functional high-avidity self-specific T cell repertoire. Due to the low frequency of naturally occurring low-avidity self-specific T cells, a detailed evaluation of their ability to survive and differentiate into effector and memory populations in vivo has yet to be obtained. We here employ tetramer technology to characterize and determine the in vivo fate of a self-specific CD8(+) T cell population specific for a ubiquitously expressed T cell epitope. We find that in influenza nucleoprotein (NP)-transgenic mice (B10NP mice) an oligoclonal population of NP(366 - 374)-specific T cells can be triggered by live influenza virus exposure. The main hallmark of this self-specific T cell population is its diminished avidity for the tetrameric MHC / NP peptide complex. These low-avidity T cells are not deleted and do not down-regulate their antigen or CD8 receptors, and exhibit cytolytic activity towards tumor cells expressing NP endogenously. Strikingly, a secondary influenza infection generates a typical memory response in the low-avidity repertoire. The observation that low-avidity T cells persist in vivo and can differentiate into memory T cells underscores their potential role in anti-tumor immunity.

Selective expansion of cross-reactive CD8(+) memory T cells by viral variants.

The role of memory T cells during the immune response against random antigenic variants has not been resolved. Here, we show by simultaneous staining with two tetrameric major histocompatibility complex (MHC)-peptide molecules, that the polyclonal CD8(+) T cell response against a series of natural variants of the influenza A nucleoprotein epitope is completely dominated by infrequent cross-reactive T cells that expand from an original memory population. Based on both biochemical and functional criteria, these cross-reactive cytotoxic T cells productively recognize both the parental and the mutant epitope in vitro and in vivo. These results provide direct evidence that the repertoire of antigen-specific T cells used during an infection critically depends on prior antigen encounters, and indicate that polyclonal memory T cell populations can provide protection against a range of antigenic variants.

Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells.

Signaling through the pre-TCR is essential for early T cell development and is severely impaired in mice lacking the CD3 gamma chain of the pre-TCR. We here address the molecular mechanisms underlying this defect. Impaired pre-TCR signaling is shown to be associated with a profound increase in the number of apoptotic CD4- CD8- (DN) thymocytes. Introduction of p53 deficiency into CD3 gamma-deficient mice completely reverses the cell survival defect in CD3 gamma-deficient DN thymocytes and rescues the block in pre-T cell differentiation. In addition, the CD4+ CD8+ (DP) compartment is expanded to its normal size. These findings suggest that the pre-TCR regulates progression through the DNA-damage checkpoint of the DN to DP transition by inactivating p53.

Systemic T cell expansion during localized viral infection.

In a local immune response, the priming and expansion of the antigen-specific T cell population has been thought to largely take place in the draining lymphoid tissue. This model was primarily based on indirect enumeration of antigen-specific T cells by limiting dilution analyses. Here, tetrameric MHC class I complexes were used to evaluate the contribution of different secondary lymphoid organs in a local immune response by following the CD8+ T cell responses against the immunodominant epitopes of influenza A virus and herpes simplex virus-1. Mice were either intranasally infected with influenza A virus and developed pneumonia or were intradermally injected with herpes simplex virus-1. Remarkably, even though these viruses cause a local infection, the spleen of infected animals contains approximately 50-fold more antigen-specific cytotoxic T cells than the draining lymph nodes. Although antigen-specific T cells in spleen appear not to have experienced any recent encounter with antigen, this population is actively dividing, and over time, the formation of a memory T cell population is observed. These data reveal that there is a remarkably large and distinct population of antigen-specific T cells in spleen in the course of a local antigenic challenge. This T cell compartment may not only form the foundation of a memory T cell pool but could also provide a safeguard against systemic spreading of an infection.

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR.

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.

Costimulatory signals are required for induction of transcription factor Nur77 during negative selection of CD4(+)CD8(+) thymocytes.

A major question in end-stage T cell development is how T cell receptor(TCR) ligation on immature CD4(+)CD8(+) double positive thymocytes is translated into either survival (positive selection) or apoptotic (negative selection) signals. Because different types of antigen-presenting cells (APCs) induce positive or negative selection in the thymus and express different costimulatory molecules, involvement of such costimulatory molecules in determining cell fate of DP thymocytes is considered here. If TCR-generated signals are modulated by APCs, this should be reflected in the activation of distinct biochemical pathways. We here demonstrate that costimulatory signals involved in negative selection also are required for induction of protein expression of Nur77 and its family members. These transcription factors are critically involved in negative but not positive selection. In contrast, the signals that costimulate negative selection are not required for induction of several molecular events associated with positive selection. These include activation of the immediate early gene Egr-1, the mitogen-activated protein kinase ERK2, and surface expression of the CD69 marker. Thus, costimulation for negative selection selectively provides signals for activation of apoptotic mediators. These data provide molecular insights into how TCR-engagement by ligands on different thymic APCs can determine cell fate.

Thymocyte selection: not by TCR alone.

During development of T cells in the thymus, T-cell receptor (TCR)-mediated recognition of self-MHC/self-peptide complexes on thymic stroma dictates the developmental fate of immature CD4+CD8+ (double positive) thymocytes. Intriguingly, TCR-generated intracellular signals can elicit two entirely different cellular responses in such thymocytes: apoptosis or further differentiation. The critical issue in understanding end-stage T-cell development is how TCR occupancy can be perceived in such markedly different ways by the TCR. Here, we review the cytoplasmic and nuclear events that result from TCR signaling during thymocyte selection. Studies aimed at distinguishing molecular components involved in positive selection (resulting in signals for further differentiation) and negative selection (resulting in apoptosis) will help solve this fascinating feature of T-lymphocyte biology. We also discuss how non-TCR-derived signaling might serve to fine tune the TCR-driven selection events in thymocytes. Central to this aspect of the conceptual framework needed to explain thymocyte selection is the observation that thymic antigen-presenting cells appear to be specialized in the induction of either positive or negative selection. Finally, we suggest a hypothesis that integrates the facts currently available on developing thymocytes, and which may serve to refine our exploration of unresolved issues in thymocyte selection. This hypothesis expands our focus to include signals from receptors other than TCRs as modulating and amplifying factors in thymocyte signaling.

Functional analysis of Ran/TC4 as a protein regulating T-cell costimulation.

Antigen (Ag)-triggered activation of T cells requires engagement of both the T-cell Ag receptor and a costimulatory receptor, for which CD28 can function as a prototypical example. CD80 and CD86 represent ligands for this receptor, and although they are present on professional Ag-presenting cells, these molecules are absent from most tumors. Yet some tumors are still able to costimulate a T-cell response, while others cannot. Therefore, a key question concerns the molecular basis for the costimulation of T cells by those tumor cells not expressing the CD28 ligands CD80 and CD86. Upon screening a cDNA library of such a tumor cell line in a transient COS cell transfection assay for costimulatory activity, we identified Ran/TC4 as a protein whose overexpression results in costimulatory activity. Ran/TC4 is a ubiquitously expressed member of the Ras gene superfamily of small guanosine triphosphate-binding proteins and is involved in nuclear transport; Ran/TC4 cDNA-transfected COS cells specifically costimulate CD8 T cells and not CD4 T cells. Transfection of Ran/TC4 into the costimulation-deficient murine RMA lymphoma cell line introduced costimulatory capacity for CD8 T cells and resulted in markedly elevated levels of nuclear Ran/TC4 protein expression. In addition, in vivo priming of mice with Ran/TC4-transfected RMA cells induced protection against wild-type (wt) RMA tumor cells. Ran/TC4-transfected RMA cells and wt RMA tumor cells exhibit comparable in vivo growth rates in mice lacking T and B cells, and Ran/TC4-mediated tumor rejection thus involves B and/or T cells. This possibility is substantiated by the observation that T cells from normal mice challenged with Ran/TC4-transfected RMA cells can mount a cytotoxic T-cell response not only against the Ran/TC4-transfected tumor cells but also against wt RMA tumor cells. Based on these results, we conclude that gene transfer-mediated elevations in Ran/TC4 can confer costimulatory function for CD8 T cells to tumor cells. This finding suggests a novel application of Ran/TC4 as a protein capable of regulating costimulation in tumor cells.

The TNF receptor family member CD27 signals to Jun N-terminal kinase via Traf-2.

CD27 is a lymphocyte-specific member of the TNF receptor (TNFR) family. It is a costimulatory molecule for peripheral T cells, as defined by its ability to enhance the TCR-induced proliferative response. We show here that CD27 augments TCR-induced Jun N-terminal kinase (JNK) activity in primary murine lymph node T cells. To investigate how CD27 couples to JNK, we performed a yeast two hybrid screen with the CD27 cytoplasmic tail. This revealed that CD27 directly associates with Traf-2. Transfection experiments using dominant negative Traf-2 indicated that CD27 communicates with JNK via Traf-2. These findings group CD27 together with other members of the TNFR family, TNFR-1, -2, CD30 and CD40, which have all been shown to couple to Traf proteins. Since Traf proteins have been reported to initiate an anti-apoptotic signaling pathway, our data suggest that CD27 not only regulates proliferation, but also survival of T lymphocytes.