Targeting CD4 coreceptor expression to postselection thymocytes reveals that CD4/CD8 lineage choice is neither error-prone nor stochastic

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4(+)CD8(+) thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by "coreceptor rescue" experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8(I)-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8(I)-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8(I)-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8(I)-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.     

Thymocyte-intrinsic genetic factors influence CD8 T cell lineage commitment and affect selection of a tumor-reactive TCR

Selection of immature CD4CD8 double-positive (DP) thymocytes for CD4 or CD8-lineage commitment is controlled by the interaction of the TCR with stromal cell-expressed peptide/MHC. We show that thymocyte-intrinsic genes influence the pattern of expression of a MHC class I-restricted transgenic (tg) TCR so that in DBA/2 mice, DP thymocytes with a characteristically high expression of tg TCR, infrequently transit to CD8 single-positive thymocytes. In contrast, in B10.D2 mice, the same tg TCR is expressed at lower levels on a subpopulation of DP thymocytes that more frequently transit to CD8 single-positive thymocytes. These characteristics were not influenced by thymic stromal components that control positive selection. Radiation chimeras reconstituted with a mixture of BM from tg TCR mice of the two genetic backgrounds revealed that the relative frequency of transit to the CD8 lineage remained thymocyte-intrinsic. Identifying the gene products whose polymorphism controls CD8 T cell development may shed new light on the mechanisms controlling T cell commitment/selection in mice other than the most studied "C57BL/6"-based strains.     

Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice

Following successful gene rearrangement at alphabeta T-cell receptor (TCR) loci, developing thymocytes express both CD4 and CD8 co-receptors and undergo a life-or-death selection event, which is known as positive selection, to identify cells that express TCRs with potentially useful ligand specificities. Positively selected thymocytes must then differentiate into either CD4(+) helper T cells or CD8(+) cytotoxic T cells, a crucial decision known as CD4/CD8-lineage choice. In this Review, we summarize recent advances in our understanding of the cellular and molecular events involved in lineage-fate decision and discuss them in the context of the major models of CD4/CD8-lineage choice.     

Peptide specificity of thymic selection of CD4+CD25+ T cells.

The CD4(+)CD25(+) regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4(+)CD25(+) cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4(+)CD25(+) cells in the thymus. By comparing the development of CD4(+)CD25(-) and CD4(+)CD25(+) cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4(+)CD25(+) cells appear during natural selection of CD4(+) T cells. The proportion of CD4(+)CD25(+) cells in the population of CD4(+) thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4(+)CD25(+) cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4(+) T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4(+)CD25(+) thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.     

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.     

Analyzing expression of perforin, Runx3, and Thpok genes during positive selection reveals activation of CD8-differentiation programs by MHC II-signaled thymocytes

Intrathymic positive selection matches CD4-CD8 lineage differentiation to MHC specificity. However, it is unclear whether MHC signals induce lineage choice or simply select thymocytes of the appropriate lineage. To investigate this issue, we assessed thymocytes undergoing positive selection for expression of the CD8 lineage markers perforin and Runx3. Using both population-based and single-cell RT-PCR analyses, we found large subsets of MHC class II (MHC-II)-signaled thymocytes expressing these genes within the CD4+ 8+ and CD4+ 8(int), but not the CD4+ 8- populations of signaling competent mice. This indicates that MHC-II signals normally fail to impose CD4 differentiation and further implies that the number of mature CD8 single-positive (SP) thymocytes greatly underestimates CD8 lineage choice. We next examined whether MHC-II-restricted CD4+ 8- thymocytes remain competent to initiate CD8 lineage gene expression. In mice in which expression of the tyrosine kinase Zap70 and thereby TCR signaling were impaired selectively in SP thymocytes, MHC-II-signaled CD4+ 8- thymocytes expressed perforin and Runx3 and failed to up-regulate the CD4 marker Thpok. This indicated that impairing TCR signals at the CD4 SP stage switched gene expression patterns from CD4- to CD8-lineage specific. We conclude from these findings that MHC-II-signaled thymocytes remain competent to initiate CD8-specific gene expression even after CD8 down-regulation and that CD4 lineage differentiation is not fixed before the CD4 SP stage.     

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.     

The role of CD8 alpha' in the CD4 versus CD8 lineage choice.

During thymic development the recognition of MHC proteins by developing thymocytes influences their lineage commitment, such that recognition of class I MHC leads to CD8 T cell development, whereas recognition of class II MHC leads to CD4 T cell development. The coreceptors CD8 and CD4 may contribute to these different outcomes through interactions with class I and class II MHC, respectively, and through interactions with the tyrosine kinase p56lck (Lck) via their cytoplasmic domains. In this paper we provide evidence that an alternatively spliced form of CD8 that cannot interact with Lck (CD8 alpha') can influence the CD4 vs CD8 lineage decision. Constitutive expression of a CD8 minigene transgene that encodes both CD8 alpha and CD8 alpha' restores CD8 T cell development in CD8 alpha mutant mice, but fails to permit the development of mismatched CD4 T cells bearing class I-specific TCRs. These results indicate that CD8 alpha' favors the development of CD8-lineage T cells, perhaps by reducing Lck activity upon class I MHC recognition in the thymus.     

The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4+ single-positive T cells

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a ss-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4(-) CD8(-) double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4(+) CD8(+) double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4(+) cell differentiation proceeded, reaching a maximum at the CD4(+) CD8(-) single-positive stage. These CD4(+) cells expressing CCR8 were also CD69(high) CD62L(low) thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4(-) CD8(+) thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4(+) lineage.     

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.     

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.     

Cross-positive selection of thymocytes expressing a single TCR by multiple major histocompatibility complex molecules of both classes: implications for CD4+ versus CD8+ lineage commitment

This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8+ CTL clone. The Idhigh+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, Dq or Lq molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2q background, mature Idhigh+ T cells appeared among both CD4+ and CD8+ subsets in periphery, even in the absence of RAG-2 gene. When examined on beta2-microglobulin-/- background, CD4+, but not CD8+, Idhigh+ T cells developed, suggesting that maturation of CD8+ and CD4+ Idhigh+ cells was MHC class I (Dq/Lq) and class II (I-Aq) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4+/CD8+ lineage commitment are discussed.     

Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein,CpG DNA,and lipopolysaccharide

Pathogen-associated molecular patterns (PAMPs) signal through Toll-like receptors (TLRs) to activate immune responses, but prolonged exposure to PAMPs from Mycobacterium tuberculosis (MTB) and other pathogens inhibits class II MHC (MHC-II) expression and Ag processing, which may allow MTB to evade CD4(+) T cell immunity. Alternate class I MHC (MHC-I) processing allows macrophages to present Ags from MTB and other bacteria to CD8(+) T cells, but the effect of PAMPs on this processing pathway is unknown. In our studies, MTB and TLR-signaling PAMPs, MTB 19-kDa lipoprotein, CpG DNA, and LPS, inhibited alternate MHC-I processing of latex-conjugated Ag by IFN-gamma-activated macrophages. Inhibition was dependent on TLR-2 for MTB 19-kDa lipoprotein (but not whole MTB or the other PAMPs); inhibition was dependent on myeloid differentiation factor 88 for MTB and all of the individual PAMPs. Inhibition of MHC-II and alternate MHC-I processing was delayed, appearing after 16 h of PAMP exposure, as would occur in chronically infected macrophages. Despite inhibition of alternate MHC-I Ag processing, there was no inhibition of MHC-I expression, MHC-I-restricted presentation of exogenous peptide or conventional MHC-I processing of cytosolic Ag. MTB 19-kDa lipoprotein and other PAMPs inhibited phagosome maturation and phagosome Ag degradation in a myeloid differentiation factor 88-dependent manner; this may limit availability of peptides to bind MHC-I. By inhibiting both MHC-II and alternate MHC-I Ag processing, pathogens that establish prolonged infection of macrophages (>16 h), e.g., MTB, may immunologically silence macrophages and evade surveillance by both CD4(+) and CD8(+) T cells, promoting chronic infection.     

A role for accessibility to self-peptide-self-MHC complexes in intrathymic negative selection

Whether intrathymic-positive and -negative selection of conventional alpha beta T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (V alpha 1/V beta 6) TCR, both directed against the 52--68 fragment of the I-E alpha-chain (E alpha 52--68) bound to I-A(b), we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-E alpha transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-E alpha double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of V beta 6(+)CD4(+) cells and were unresponsive to E alpha 52--68 in vitro. The absolute number of CD4(+)CD8(+) thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4(+)CD8(+) thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.     

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.     

The level of CD8 expression can determine the outcome of thymic selection.

During thymic development, thymocytes that can recognize major histocompatability complex (MHC) molecules on thymic epithelial cells are selected to survive and mature (positive selection), whereas thymocytes that recognize MHC on hematopoietic cells are destroyed (negative selection). It is not known how MHC recognition can mediate both death and survival. One model to explain this paradox proposes that thymocytes whose T cell antigen receptors (TCRs) recognize MHC with high affinity are eliminated by negative selection, whereas low affinity TCR-MHC interactions are sufficient to mediate positive selection. Here we report that, while the expression of a 2C TCR transgene leads to positive selection of thymocytes in H-2b mice, expression of both a CD8 transgene and a 2C TCR transgene causes negative selection. This observation indicates that quantitative differences in TCR-MHC recognition are a critical determinant of T cell fate, a finding predicted by the affinity model for thymic selection.     

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.     

Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.     

Potential role of NKG2D/MHC class I-related chain A interaction in intrathymic maturation of single-positive CD8 T cells

The nonclassical MHC class I molecule MHC class I-related chain A (MICA) interacts with the NKG2D receptor expressed at the surface of most peripheral CD8 T cells, gammadelta T cells, and NK cells. We investigated the role of MICA-NKG2D interactions in the selection or maturation of the T cell repertoire within the thymus using MICA tetramers and anti-MICA mAbs. MICA tetramers identified a small population of late stage CD8 single-positive, CD45RA(+) CD62L(+) CCR7(+) CD69(-) thymocytes, a phenotype compatible with that of fully mature CD8(+) cells ready to emigrate to the periphery as naive cells. MICA molecules were expressed in the outer layer of Hassal's corpuscles within the medulla of normal thymus. In thymomas, an overexpression of MICA in cortical and medullar epithelial cells was observed. This was associated with a decreased percentage of NKG2D-positive thymocytes, which expressed a less mature phenotype than in normal thymus. These results indicate that CD8(+) thymocytes up-regulate NKG2D as they complete their developmental program before leaving the thymic medulla to seed the periphery, and identify NKG2D as a potential regulator of the developmental processes in T cells that are essential for immune homeostasis.