Antagonist Peptide Selects Thymocytes Expressing a Class II Major Histocompatibility Complex–restricted T Cell Receptor into the CD8 Lineage

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4⁺ cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8⁺ cells in thymus organ culture from mice impaired in CD4⁺ cell development (invariant chain–deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.     

CD4+8- thymocytes bearing major histocompatibility complex class I-restricted T cell receptors: evidence for homeostatic control of early stages of CD4/CD8 lineage development.

During thymus development CD4+ CD8+ precursor cells differentiate into mature CD4+ and CD8+ T cells expressing T cell receptors (TCR) that recognize foreign antigens in association with major histocompatibility complex (MHC) class II or I molecules, respectively. Studies with TCR transgenic mice have shown that the accumulation of mature CD4+ and CD8+ thymocytes is strongly skewed by the MHC restriction specificity of the TCR, thus suggesting that commitment of CD4+ CD8+ precursors to the CD4 or CD8 lineage is a direct consequence of TCR/MHC interactions. However, we show here that CD4+ cells expressing an inappropriate (MHC class I-specific) TCR appear transiently in the neonatal thymus of TCR transgenic mice and can also be found in the periphery of adult TCR transgenic recombination-deficient SCID mice. These data argue that the early stages of CD4 and CD8 lineage development in the thymus are (at least in part) controlled by homeostatic mechanisms independent of appropriate TCR/MHC interactions.     

Lineage Commitment in the Thymus: Only the Most Differentiated (TCRhibcl-2hi) Subset of CD4+CD8+Thymocytes Has Selectively Terminated CD4 or CD8 Synthesis

Lineage commitment is a developmental process by which individual CD4⁺CD8⁺ (double positive, DP) thymocytes make a decision to differentiate into either CD4⁺ or CD8⁺ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5^hiCD69^hiTCR^hibcl-2^hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct subpopulations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCR^hibcl-2^hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.     

Commitment of Immature CD4+8+ Thymocytes to the CD4 Lineage Requires CD3 Signaling but Does Not Require Expression of Clonotypic T Cell Receptor (TCR) Chains

As a consequence of positive selection in the thymus, immature CD4⁺8⁺ double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4⁺ or CD8⁺ T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)–CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or ζ chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.     

CD3 Ligation on Immature Thymocytes Generates Antagonist-like Signals Appropriate for CD8 Lineage Commitment, Independently of T Cell Receptor Specificity

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4⁺) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8⁺ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8⁺ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab′)₂ reagent (CD3fos-F(ab′)₂). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab′)₂ have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8⁺ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56^lck activation is responsible for directing commitment to the CD8 or CD4 lineages.     

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34⁺ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8⁺ and CD4⁺ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8⁺ and CD4⁺ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34⁺ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.     

The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells

In experiments to study the impact of deficiency in CD4+ T cell help on the magnitude of CD8+ cytotoxic T cell response to pathogens, it was noted that in CD4 gene knockout mice, the CD8 population made significant responses to several nominally major histocompatibility complex (MHC) class II-restricted epitopes in addition to the expected responses to MHC class I-restricted epitopes. A similar response by CD8+ T cells to class II-restricted epitopes was not observed in wild-type mice, or in mice that had been acutely depleted of CD4+ T cells just before the immunization. Coincident with this unexpected response to class II-restricted epitopes, it was also observed that the CD8+ response to the class I-restricted epitopes was consistently lower in CD4-/- mice than in wild-type mice. Further experiments suggested that these two observations are linked and that the CD8 population in CD4-/- mice may contain a majority of T cells that were actually selected by recognition of MHC class II molecules in the thymus. These results have implications for understanding CD4 versus CD8 lineage commitment in the thymus, and for the practical use of CD4-/- mice as models of helper deficiency.     

An antagonist peptide mediates positive selection and CD4 lineage commitment of MHC class II-restricted T cells in the absence of CD4

The CD4 coreceptor works together with the T cell receptor (TCR) to deliver signals to the developing thymocyte, yet its specific contribution to positive selection and CD4 lineage commitment remains unclear. To resolve this, we used N3.L2 TCR transgenic, RAG-, and CD4-deficient mice, which are severely impaired in positive selection, and asked whether altered peptide ligands can replace CD4 function in vivo. Remarkably, in the presence of antagonist ligands that normally deleted CD4+ T cells in wild-type mice, we induced positive selection of functional CD4 lineage T cells in mice deficient in CD4. We show that the kinetic threshold for positive and negative selection was lowered in the absence of CD4, with no evident skewing toward the CD8 lineage with weaker ligands. These results suggest that CD4 is dispensable as long as the affinity threshold for positive selection is sustained, and strongly argue that CD4 does not deliver a unique instructional signal for lineage commitment.     

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4⁺CD25⁺ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4⁺ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4⁺CD25⁺ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4⁺CD25⁻ cells, the CD4⁺CD25⁺ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4⁺CD25⁺ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25⁻ T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4⁺CD25⁺ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.     

Downregulation of CD1 Marks Acquisition of Functional Maturation of Human Thymocytes and Defines a Control Point in Late Stages of Human T Cell Development

We have investigated whether in the human thymus transition of CD4⁺CD8⁺ double positive (DP) to CD4⁺ or CD8⁺ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a⁻ cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II– positive, but not MHC class II–negative, mouse thymic microenvironments support differentiation of human progenitors into TCRαβ⁺CD4⁺ SP cells, indicating that mouse MHC class II can positively select TCRαβ⁺CD4⁺ SP human cells. Strikingly, these SP are arrested in the CD1a⁺ stage and could not be expanded in vitro with PHA and IL-2. CD1a⁺CD4⁺ SP thymocytes do not represent an end stage population because purified CD1a⁺CD4⁺ SP thymocytes differentiate to expandable CD1a⁻ cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a⁺ DP TCRαβ^low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCRαβ⁺ cells.Tcell progenitors that develop in the thymus to mature T cells are submitted to a series of selective events (reviewed in reference 1), the first of which takes place when immature CD4⁻CD8⁻CD3⁻ cells differentiate into CD4⁺ CD8⁺ double positive (DP)¹ cells. A second selection occurs when DP thymocytes differentiate into CD4⁺ or CD8⁺ mature T cells, and is generally referred to as positive selection. It is well established that positive selection involves sustained interactions of the TCR αβ heterodimer with complexes of peptides and MHC antigens on thymic stromal cells (reviewed in references 2–4). During this selection process, either CD4 or CD8 is downregulated. There is current debate over whether downregulation of CD4 or CD8, and thus commitment to CD4⁺ or CD8⁺ T cells, is dictated by the MHC specificity of the TCR (instructive model) (5, 6) or whether it occurs in a stochastic fashion independent of TCR/MHC interactions (selective model) (7–9). In the majority of the studies addressing the issue of positive selection, all CD3^high thymocytes with a CD4 or CD8 single positive (SP) phenotype were considered to have completed the process of positive selection and to be functionally mature. However, recent studies in the mouse indicate that not all SP thymocytes that have been submitted to positive selection signals are functionally mature. It is known that SP cells are phenotypically heterogeneous with respect to CD24 (heat stable antigen) and CD69 (10, 11). In addition, CD4⁺ SP thymocytes with intermediate levels of CD24 express very low levels of CD8 when analyzed with a sensitive panning method (11). More recently, it has been demonstrated that although the CD4⁺CD8^low cells had hallmarks of positive selection such as CD69 and high levels of TCR, they were not able to induce a lethal Graft versus host disease upon transfer into irradiated allogeneic recipients and to survive in the periphery (12). The immature CD3^highCD4⁺CD8^low cells require the thymic environment to reach the end stage of positive selection (12). These data suggest that when functional immunocompetence of T cells is taken as the end stage of positive selection, this process is not necessarily completed when CD4 or CD8 are downregulated.Here we report on the identification of downregulation of CD1a as a hallmark for functional maturation, not only of SP human thymocytes, but also of DP cells. To arrive at this model, we made use of the observations that DP cells contain in vitro clonogenic cells both in human (13, 14) and mouse (15). These observations were intriguing because if one accepts that maturity of T cells is appropriately reflected by their capacity to expand in vitro, some DP cells should have been submitted to a maturation signal. The presence of both mature clonogenic DP cells and immature CD4⁺ SP cells (12) is difficult to reconcile with a linear model of thymocyte differentiation from immature CD3⁺CD4⁺CD8⁺ DP via immature to mature SP cells. A possible explanation for the existence of both in vitro clonogenic mature DP thymocytes and presumably immature SP cells could be that there are bifurcations in the pathway of later stages of T cell development. The data presented here are consistent with this notion, since it was found that acquisition of functional maturity correlates perfectly with downregulation of CD1a and, most importantly, not with downregulation of CD4 or CD8. Moreover, we show here that MHC class II–positive, but not MHC class II–negative, mouse thymic microenvironments can support differentiation of human progenitors into CD3⁺CD4⁺ SP cells. However, human TCRαβ⁺ CD4⁺ SP cells selected on mouse MHC class II continue to express CD1a and exhibit poor clonogenic potential in vitro, suggesting that a species-specific signal is required for downregulation of CD1a and induction of functional maturity in the CD4 TCRαβ lineage.     

Survival of Mature CD4 T Lymphocytes Is Dependent on Major Histocompatibility Complex Class II–expressing Dendritic Cells

Thymic T cell development is controlled by T cell receptor (TCR)–major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR–MHC contact has not been described so far. To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells. Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II–positive embryonic thymic tissue, which had been depleted of bone marrow derived cells. Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4⁺ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient. As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II⁺ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II⁺ dendritic cells might play an important role for the longevity of CD4 T cells.Thymic positive selection is a process that generates mature CD4⁺ and CD8⁺ single-positive T lymphocytes from CD4⁺CD8⁺ double-positive thymocytes. The mechanistic control of positive selection is the interaction between TCR on thymocytes and MHC-encoded molecules on thymic epithelial cells. Mature CD4⁺ and CD8⁺ single-positive thymocytes, selected on MHC class II and I, respectively, subsequently leave the thymus and seed the peripheral lymphoid organs (1–3). Consequently, CD4⁺ single-positive thymocytes and CD4⁺ peripheral T cells are nearly absent in class II–deficient mice (4, 5).The further survival of peripheral T cells seems not to be dependent on antigen-specific TCR–MHC interactions. Transfer experiments performed with T cells from TCR-transgenic mice in the presence or absence of antigen (6, 7) showed that specific Ag is not necessary for T cell survival. In another experimental model, Sprent et al. (8) demonstrated that when unseparated lymph node cell suspensions were injected into H-2 identical SCID hosts, they formed a self-sufficient pool of lymphocytes. T cells survived in this system without reduction in numbers in the absence of antigen. However, conflicting results have been reported on the survival of T cells in the absence of MHC molecules expressed on hematopoietic cells. When irradiated normal mice received bone marrow from class II–deficient mice, normal CD4 T cell repopulation was observed in one study (9). Others doing the same experiment could not detect reconstitution of the CD4 compartment in the MHC class II–negative environment of such mice (10). Huss et al. speculated that this discrepancy could have been caused by the different time spans of bone marrow inoculum in the host mice used by the two groups or different bone marrow treatments (e.g., T cell depletion) before injection (10). Therefore, these experiments could not definitely clarify the question of whether peripheral CD4 T cell survival is dependent of peripheral MHC class II expression.In a recent report, Takeda et al. (11) transplanted untreated fetal thymi from MHC class II⁺ mice under the kidney capsules of class II⁺, as well as class II–deficient, hosts. The authors observed an identical initial donor type CD4⁺ T cell accumulation in the periphery of both hosts. In comparison to the MHC class II⁺ mice, the class II–deficient hosts showed faster declining numbers of peripheral CD4⁺ T cells. These results suggested that interactions between CD4⁺ T cells and MHC class II⁺ peripheral cells are not necessary for short-term survival, but might be important for longevity of T cells. However, a potential contamination of the MHC class II–deficient peripheral organs of the hosts with MHC class II⁺ donor type cells originating from the transplanted thymi (thymic dendritic cells, B cells, macrophages) cannot be excluded when the thymus grafts (TGs),¹ are not depleted of hemopoietic cells before transplantation. Furthermore, the initial export of large numbers of donor-type thymocytes from untreated grafts (12) might not reflect the actual kinetics of thymocyte export from a developing thymus.To avoid the presence of donor-type thymocytes and to exclude the possibility of contamination of the hosts with thymus-derived MHC class II–positive cells, in this report MHC class II⁺ fetal TGs were depleted of hematopoietic cells before transplantation. Then survival of host-type CD4 T cells in a host lacking MHC class II expression completely (4) was compared to CD4 T cell survival in an environment where only dendritic cells (DCs) express MHC class II (13). Although TGs in both types of hosts were repopulated with similar numbers of phenotypically identical host-type thymocytes, only the mice expressing MHC class II on DCs, but not the conventional MHC class II–deficient hosts, showed accumulation of CD4 T cells in blood and peripheral lymphoid organs. The conclusion from these experiments is that CD4 T cells, after having left the thymus, do need interaction with MHC class II–positive peripheral cells for survival. MHC class II–positive DCs are sufficient to ensure this survival.     

Liver Damage Preferentially Results from CD8+ T Cells Triggered by High Affinity Peptide Antigens

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)–restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4⁻CD8⁻B220⁺ T cells occurs more frequently from a CD8⁺ precursor than from CD4⁺ T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8⁺ cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC–restricted TCR. The findings show that CD4⁻CD8⁻B220⁺ T cells preferentially derive from a CD8⁺ precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.