Suicide induced by cytolytic activity controls the differentiation of memory CD8(+) T lymphocytes.

Cytotoxic T lymphocytes (CTL) confer protection against intracellular pathogens, yet the mechanism by which some escape activation induced cell death (AICD) and give rise to long-lived memory cells is unclear. We studied the differentiation of transgenic TCR CD8(+) cells into CTL and memory cells using a novel system that allowed us to control cytolytic activity. The perforin/granzyme granules used to lyse targets induced the apoptosis of CTL in a fratricide-independent manner. After adoptive transfer to antigen-free mice, the ability of CTL to give generate memory cells was determined. We found that the extent of cytolysis by a common pool of CTL controlled the differentiation into memory cells, which were only generated under conditions of minimal cytolytic activity. Thus, the differentiation of naive CD8(+) cells into memory cells may not depend on the presence on a subset of committed CTL precursors, but rather is controlled by the extent of granule-mediated cytolysis.

Affinity of thymic self-peptides for the TCR determines the selection of CD8(+) T lymphocytes in the thymus.

Experiments with synthetic antigen peptides have suggested that a critical parameter that determines the developmental fate of an immature thymocyte is the affinity of interaction between TCR and self-peptide/MHC expressed on thymic stromal cells. To test the physiological relevance of this model for thymocyte development, we determined the affinity of the anti-HY TCR (B6.2.16) expressed on CD8(+) cells for thymic self-peptide/H-2D(b) tetramers, then examined the ability of these self-peptides to determine the outcome of B6.2.16 CD8 cell selection in the thymus. The B6.2.16 TCR bound the male HY self-antigen with high affinity. Thymic self-peptides, which are highly abundant on the surface of thymic epithelial cells, bound the B6.2.16 TCR with low affinity. The ability of self-peptides to trigger positive or negative selection of B6.2.16 CD8 cells in cultured fetal thymi was determined by the relative affinity of self-peptide/H-2D(b) for the B6.2.16 TCR. High-affinity binding of the HY self-peptide resulted in B6.2.16 TCR complex zeta chain phosphorylation and the negative selection of B6.2.16 CD8 cells. Low-affinity binding of thymic self-peptides to B6.2.16 TCR resulted in the positive selection of B6.2.16 CD8 cells. Differences between the binding affinities of self-peptides to B6.2.16 TCR accounted for the self-peptide specificity of B6.2.16 CD8 cell positive selection. We conclude that the relative affinity of TCR for thymic self-peptide/class I MHC is a critical parameter in determining fate of CD8(+) cells during thymic selection.

Linear differentiation of cytotoxic effectors into memory T lymphocytes.

A central question in immunology is the origin of long-lived T cell memory that confers protection against recurrent infection. The differentiation of naïve T cell receptor transgenic CD8+ cells into effector cytotoxic T lymphocytes (CTLs) and memory CD8+ cells was studied. Memory CD8+ cells that were generated after strong antigenic stimulation were the progeny of cytotoxic effectors and retained antigen-specific cytolytic activity 10 weeks after adoptive transfer to antigen-free recipient mice. Thus, potential vaccines based on CTL memory will require the differentiation of naïve cells into post-effector memory T cells.

Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules.

How memory T cells are maintained in vivo is poorly understood. To address this problem, a male-specific peptide (H-Y) was identified and used to activate female anti-H-Y T cells in vitro. Anti-H-Y T cells survived in vivo for at least 70 days in the absence of antigen. This persistence was not because of the intrinsic ability of memory T cells to survive in vivo. Instead, the survival and function of adoptively transferred memory cells was found to require transporter of antigen protein 1-dependent expression of self-peptide/major histocompatibility complex class I molecules in recipient animals. Therefore, it appears that the level of T cell receptor engagement provided by transporter of antigen protein 1-dependent, self-peptide/major histocompatibility complexes is sufficient to maintain the long-term survival and functional phenotype of memory cells in the absence of persistent antigen. These data suggest that positive selection plays a role not only in T cell development but also in the maintenance of T cell memory.

Limits to the differential avidity model of T cell selection in the thymus.

It has been postulated that the critical feature that determines the developmental fate of an immature thymocyte is the avidity of interaction between thymocyte TCR and peptide/MHC molecules on thymic stromal cells. However, it is possible that certain innate properties of peptides predispose them to triggering only positive or negative selection irrespective of their density on thymic stromal cells. To distinguish between these hypotheses, we examined the ability of several different peptides to induce the positive and negative selection of TCR transgenic (P14) antilymphocytic choriomeningitis virus (LCMV) CTLs in fetal thymus organ cultures (FTOC) from TAP1+ and TAP1- mice. We found that only relatively weak agonist peptides could induce the positive selection of anti-LCMV CTLs. A nonagonist peptide could induce positive selection but not negative selection; however, a weak agonist peptide could induce the positive selection of anti-LCMV CTLs in P14 TAP1- FTOC and negative selection in P14 TAP1+ FTOC. These data imply that there are upper and lower limits for the affinity of a peptide in triggering positive or negative selection, but that for peptides of intermediate affinity the overall avidity of interaction with the P14 TCR is the critical parameter in determining the developmental fate of thymocytes. Our observations also suggest a prominent role for low affinity self peptides in selecting a function repertoire of CD8+ T cells.

Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes.

To understand how thymic selection gives rise to T cells that are capable of major histocompatibility complex (MHC)-restricted recognition of antigen but are tolerant of self, we directly examined how peptide/MHC ligands expressed on thymic epithelial cells trigger the positive selection of immature thymocytes. We demonstrate that abundant self-peptides, purified from the H-2D(b) molecules of thymic epithelial cells, are specifically recognized during the positive selection of CD8+ T cells, implying that positive selection generates a repertoire of T cells that is weakly self-reactive. We also found that this recognition is somewhat cross-reactive, thereby providing an explanation for how the specific recognition of a limited repertoire of thymic self-peptides can select a diverse repertoire of T cells.

Presentation of endogenous viral proteins in association with major histocompatibility complex class II: on the role of intracellular compartmentalization, invariant chain and the TAP transporter system.

Major histocompatibility complex (MHC) class II-associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously synthesized antigens. We have studied the MHC class II-associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis, it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II-associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non-professional APC. We found that class II-associated presentation of endogenously synthesized membrane associated LCMV-GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II-associated presentation of endogenously synthesized cytosolic LCMV-NP was not observed even in Ii-deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV-GP apparently does not require processing in acidic endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading.

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and beta 2-microglobulin mutant mice.

TAP1 -/- and beta 2-microglobulin (beta 2m) -/- mice (H-2b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8+ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8+ T cells than beta 2m -/- mice. Alloreactive CD8+ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in beta 2m -/- mice. Alloreactive CTL generated in TAP1 -/- and beta 2m -/- mice cross-react with H-2b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from beta 2m -/- mice than with similar cells from TAP1 -/- mice. The beta 2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2b MHC class I molecules. Such H-2b-reactive CD8+ CTL from beta 2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2b-reactive CD8+ CTL from neither beta 2m -/- mice nor TAP1 -/- mice killed beta 2m -/- cells. In line with these results, beta 2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8+ T cells differs between TAP1 -/- and beta 2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and beta 2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8+ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.

Altered peptidase and viral-specific T cell response in LMP2 mutant mice.

MHC class I molecules present peptides generated by processing of endogenously synthesized proteins to CD8+ T lymphocytes. Recently, large proteolytic complexes, termed proteasomes, were implicated in antigen processing. Two proteasomal subunits, LMP2 and LMP7, are encoded within the MHC class II region, but their precise role in antigen processing is unknown. We have generated mice that harbor a disruption in their LMP2 gene. Proteasomes purified from spleen and liver of these mutant mice exhibit altered peptidase activities, and antigen-presenting cells showed reduced capacity to stimulate a T cell hybridoma specific for H-2Db plus a nucleoprotein epitope of an influenza A virus. The mutant mice have reduced (60%-70% of wild type) levels of CD8+ T lymphocytes and generate 5- to 6-fold fewer influenza nucleoprotein-specific cytotoxic T lymphocyte precursors. These findings indicate that LMP2 influences antigen processing.

A differential-avidity model for T-cell selection.

The processes of positive and negative selection during thymic development shape the repertoires of antigen specificities displayed by T cells. This rids the animal of potentially autoreactive T cells and, at the same time, ensures that they are capable of major histocompatibility complex (MHC)-restricted recognition of antigen. Paradoxically, both processes involve the engagement of the T-cell recepetor (TCR) on immature thymocytes with peptide/MHC complexes expressed on thymic stromal cells. Here, Philip Ashton-Rickardt and Susumu Tonegawa suggest that the critical parameter determining the outcome of this interaction is the number of TCRs occupied by peptide/MHC complexes and that this, in turn, is determined by the avidity of the TCR-MHC interaction: low avidity resulting in positive selection and high avidity resulting in negative selection.

Positive selection of self- and alloreactive CD8+ T cells in Tap-1 mutant mice.

Mice with a homozygous deletion in their Tap-1 gene (-/- mice) express very low levels of cell membrane major histocompatibility complex class I molecules and have < 1% peripheral CD8+ T cells. We show that these -/- mice but not their +/- littermates display strong primary syngeneic anti-H-2Kb and -Db-specific responses mediated by CD8+ T cells. These responses are augmented by in vivo priming. Further, -/- mice primed in vivo with H-2d alloantigens generate an anti-H-2d response which appears nearly as strong as that found in +/- littermates. Both -/- anti-H-2b and anti-H-2d T cells do not recognize target cells from Tap-1 -/- animals or Tap-2-deficient RMA-S cells. Thus, some CD8+ anti-self and alloreactive T cells can be selected in the absence of Tap proteins.

Evidence for a differential avidity model of T cell selection in the thymus.

Positive and negative selection of a lymphocytic choriomeningitis virus (LCMV) peptide-specific, H-2Db-restricted T cell clone (P14) was studied using TAP1- and TAP1+ mice transgenic for P14 T cell receptor (TCR) alpha and beta genes. Positive selection of transgenic CD8+ P14 cells was impaired in TAP1- mice. Addition of the LCMV peptide to TAP1- fetal thymic organ cultures (FTOCs) at low and high concentrations induced positive and negative selection of CD8+ P14 cells, respectively, while addition of the same peptide to TAP1+ FTOCs induced negative selection even at low concentrations. Both types of selection were peptide specific. Thus, a critical parameter that controls the fate of a thymocyte seems to be the number of TCRs engaged with complexes of peptide and major histocompatibility complex. When this number is low, positive selection occurs, and when it is high, negative selection takes place. These findings support a differential avidity model of T cell selection.

Peptide length and sequence specificity of the mouse TAP1/TAP2 translocator.

The transporter associated with antigen processing (TAP) delivers peptides to the lumen of the endoplasmic reticulum in an adenosine triphosphate (ATP) dependent fashion for presentation by major histocompatibility complex class I molecules. We show that the mouse TAP translocator (H-2b haplotype) selects peptides based on a minimal size of nine residues, and on the presence of a hydrophobic COOH-terminal amino acid. The preponderance of COOH-terminal hydrophobic amino acids in peptides capable of binding to mouse class I molecules thus fits remarkably well with the specificity of the TAP translocator. In addition to transport in the lumenal direction, efflux of peptide in the cytosolic direction is observed in an ATP- and temperature-dependent manner. By maintaining a low peptide concentration at the site of class I assembly, this efflux mechanism may ensure that class I molecules are loaded preferentially with high affinity peptides.

The role of peptide in the positive selection of CD8+ T cells in the thymus.

Recent experiments using fetal thymus organ culture (FTOC) systems derived from class I deficient mice have provided evidence supporting the view that specific recognition of class I MHC plus peptide complexes is required for the positive selection of CD8+ T cells. The addition of class I binding peptides to FTOC systems derived from beta 2-microglobulin and TAP 1 deficient mice induced the positive selection of CD8+ T cells in a peptide specific manner. The specific recognition of peptide during positive selection implies that the repertoire of specificity's exhibited by CD8+ T cells is at least in part determined by the repertoire of self-peptides presented by class I MHC in the thymus.

TAP1-dependent peptide translocation in vitro is ATP dependent and peptide selective.

T cells detect infection of cells by recognizing peptide fragments of foreign proteins bound to class I molecules of the major histocompatibility complex (MHC) on the surface of the infected cell. MHC class I molecules bind peptide in the endoplasmic reticulum, and analysis of mutant cells has demonstrated that an adequate supply of peptides requires the presence of two genes in the MHC class II locus that encode proteins called transporters associated with antigen processing (TAP) 1 and 2. TAP1 and TAP2 are members of the ATP-binding cassette family of membrane translocators. In this study, we demonstrate in a cell-free system that TAP1 is part of an ATP-dependent, sequence-specific, peptide translocator.

Peptide contributes to the specificity of positive selection of CD8+ T cells in the thymus.

Mice deficient in the gene encoding the peptide transporter associated with antigen processing (TAP1) have drastically reduced levels of surface expression of MHC class I molecules and few CD8+ T cells. Addition of class I binding peptides to cultured fetal thymi from TAP1 mutant mice invariably allowed the rescue of class I expression, while only some of these peptides promoted the positive selection of CD8+ T cells, which were polyclonal and specific for the peptide-MHC complex. A nonselecting peptide was converted to a mixture of selecting peptides when the residues involved in the interaction with TCRs were altered. A mixture of self-peptides derived from C57BL/6 thymi induced positive selection of CD8+ T cells at concentrations that gave relatively low class I surface expression. The implication of these observations is that self-peptides determine, in part, the repertoire of specificities exhibited by CD8+ T cells.

TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells.

The transporter associated with the antigen processing 1 (TAP1) gene encodes a subunit for a transporter, presumed to be involved in the delivery of peptides across the endoplasmic reticulum membrane to class I molecules. We have generated mice with a disrupted TAP1 gene using embryonic stem cell technology. TAP1-deficient mice are defective in the stable assembly and intracellular transport of class I molecules and consequently show severely reduced levels of surface class I molecules. These properties are strikingly similar to those described for the TAP2 mutant cell line RMA-S. Cells from the TAP1-deficient mice are unable to present cytosolic antigens to class I-restricted cytotoxic T cells. As predicted from the near absence of class I surface expression, TAP1-deficient mice lack CD4-8+ T cells.