New approaches to eliciting protective immunity through T cell repertoire manipulation: the concept of thymic vaccination

Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp33-41, a Db-restricted peptide derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein, and of VSV8, a Kb-restricted peptide from the vesicular stomatitis virus (VSV) nucleoprotein, have been designed and their influence on thymic maturation of specific TCR-bearing transgenic thymocytes examined in vivo using irradiation chimeras. Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists.     

A critical role for CD2 in both thymic selection events and mature T cell function

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.     

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.     

Quantitative and qualitative adjustment of thymic T cell production by clonal expansion of premigrant thymocytes

In normal mice, single-positive thymocytes proliferate before being exported into the peripheral T cell pool. We measured the in vivo proliferation rates of mature thymocytes in several TCR transgenic mice. Different monoclonal TCR transgenic single-positive thymocytes proliferated at different rates in a given MHC context. Conversely, mature thymocytes expressing a given TCR, generated in mice of different MHC haplotypes, also showed different rates of proliferation. In p59(fyn)-deficient mice, the proliferation rate of mature thymocytes was diminished. Thus, premigrant thymocyte expansion is TCR mediated and depends on TCR affinity for self peptide/MHC ligands. In addition, we show that mature thymocyte expansion is clonotypic, increases the daily thymic T cell output, and modifies the TCR repertoire of newly produced T cells.     

Fine tuning of the threshold of T cell selection by the Nck adapters

Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire.     

Antigen challenge inhibits thymic emigration

T cell development in the thymus involves a series of TCR-mediated control points including TCR-beta selection and positive and negative selection. Approximately half of the thymic sojourn is spent in the medulla, where thymocytes undergo final maturation before emigrating to the periphery. Although it is acknowledged that thymic emigration is an active process, relatively little is known about how this is regulated, why it takes so long, and whether TCR-mediated signaling can influence this step. Using wild-type and TCR transgenic mice, we found that Ag injected i.v. or intrathymically led to a striking reduction in the number of recent thymic emigrants (RTE) in the periphery. This was caused by inhibition of T cell export rather than peripheral deletion, because a cohort of RTE that was already released before in vivo Ag challenge was not depleted, and similar results were observed in Bim-deficient mice, which have impaired T cell deletion. Within the thymus, the loss of RTE was associated with retention of medullary thymocytes rather than increased negative selection. In addition to Ag-specific inhibition of export, some TCR-independent suppression of emigration was also observed that appeared to be partly the result of the inflammatory cytokine TNF. Thus, in addition to its accepted role in intrathymic selection events, TCR signaling can also play an important role in the regulation of thymic emigration.     

A NK1.1+ thymocyte-derived TCR beta-chain transgene promotes positive selection of thymic NK1.1+ alpha beta T cells

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR beta-chain derived from conventional alphabeta T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion alpha-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a beta-chain derived from nonclassical, NK1.1+ alphabeta T cells, the thymus-derived, CD1. 1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 beta Tg thymocytes revealed that the frequency of the parental alpha-chain sequence, in this instance the Valpha14-Jalpha281 canonical alpha-chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1+ alphabeta T cells in DN32H6 beta Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR beta-chain from a thymus-derived NK1.1+ Valpha14+ T cell hybridoma promotes positive selection of thymic NK1.1+ alphabeta T cells. These observations indicate that the strong influence of productive beta-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical alphabeta T cells and therefore represents a general phenomenon in intrathymic alphabeta T lymphocyte development.     

Inefficient cell spreading and cytoskeletal polarization by CD4+CD8+ thymocytes: regulation by the thymic environment

CD4(+)CD8(+) double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4(+)) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.     

Fetal exposure to high-avidity TCR ligand enhances expansion of peripheral T regulatory cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

Nuclear export of histone deacetylase 7 during thymic selection is required for immune self‐tolerance

Histone deacetylase 7 (HDAC7) is a T‐cell receptor (TCR) signal‐dependent regulator of differentiation that is highly expressed in CD4/CD8 double‐positive (DP) thymocytes. Here, we examine the effect of blocking TCR‐dependent nuclear export of HDAC7 during thymic selection, through expression of a signal‐resistant mutant of HDAC7 (HDAC7‐ΔP) in thymocytes. We find that HDAC7‐ΔP transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection‐associated gene expression programme in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self‐tolerance.     

Effects of a constitutively active form of calcineurin on T cell activation and thymic selection

Calcineurin is a calcium/calmodulin-dependent phosphatase whose activity is required for the induction of T cell lymphokine production and proliferation. Although its specific role in T cell development is less well defined, studies with the immunosuppressive drugs cyclosporin A and FK-506 suggest that it is involved in both positive and negative selection of immature thymocytes. To more completely characterize a role for calcineurin in T cell development in vivo, we have generated transgenic mice that express an activated form of this enzyme in thymocytes and peripheral T cells. We find that the transgene causes a block in early thymic development, resulting in a reduction in the steady-state number of CD4 and CD8 double positives, but not on the number of mature T cells. We also find that thymocytes and mature T cells expressing this transgene are more sensitive to signals through their TCR. In thymocytes this sensitivity difference is manifested as an increase in positive selection, although negative selection seems to remain unaffected. Therefore, these studies confirm and extend past reports that suggested a role for calcineurin in thymic development and selection.     

Transgenic expression of Ly-49A in thymocytes alters repertoire selection

A T cell-specific Ly-49A transgene inhibits TCR-mediated activation in the presence of H-2Dd. Expression of this transgene by developing thymocytes impairs negative selection evidenced by a failure to delete potentially autoreactive T cells and development of a graft-vs-host-disease-like syndrome. In mice carrying both the Ly-49A and a class II-restricted TCR transgene, positive selection was lost, but only when H-2Dd was present on thymic epithelium. These results are consistent with models suggesting that thymic selection is dependent on the perceived intensity of TCR signaling. More interestingly, these results show that Ly-49A does not simply provide a strict on/off switch for T cell responses. Since Ly-49A may shift the signaling threshold of TCR-induced triggering, inducible expression of Ly-49A may regulate peripheral memory/activated T cells by raising the threshold for T cell reactivation.     

The quantity of TCR signal determines positive selection and lineage commitment of T cells

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.     

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Normal T Cell Selection Occurs in CD205-Deficient Thymic Microenvironments

The thymus imparts a developmental imprint upon T cells, screening beneficial and self-tolerant T cell receptor (TCR) specificities. Cortical thymic epithelial cells (CTEC) present self-peptide self-MHC complexes to thymocytes, positively selecting those with functional TCRs. Importantly, CTEC generate diverse self-peptides through highly specific peptide processing. The array of peptides utilized for positive selection appears to play a key role in shaping TCR repertoire and influencing T cell functionality. Whilst self-peptide diversity influences T cell development, the precise source of proteins generating such self-peptide arrays remains unknown, the abundance of apoptotic thymocytes failing thymic selection may provide such a pool of self-proteins. In relation to this notion, whilst it has been previously demonstrated that CTEC expression of the endocytic receptor CD205 facilitates binding and uptake of apoptotic thymocytes, the possible role of CD205 during intrathymic T cell development has not been studied. Here, we directly address the role of CD205 in normal thymocyte development and selection. Through analysis of both polyclonal and monoclonal transgenic TCR T-cell development in the context of CD205 deficiency, we demonstrate that CD205 does not play an overt role in T cell development or selection.