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.     

Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4⁺ and CD8⁺ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8⁺ T cells is qualitatively different from that in CD4⁺ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8⁺ T cells, whereas those in CD4⁺ T cells were intact. PLCβ4-deficient CD8⁺ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP₃ generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8⁺ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8⁺ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4⁺ and CD8⁺ T cells and selectively promotes CD8⁺ T cell–dependent adaptive immunity.     

Aplastic Anemia Rescued by Exhaustion of Cytokine-secreting CD8+ T Cells in Persistent Infection with Lymphocytic Choriomeningitis Virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P^0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8⁺, but not of CD4⁺, T cells and accelerated by increasing the frequency of LCMV-specific CD8⁺ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8⁺ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8⁺ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P^0/0 and P^0/0 × TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-α and interferon (IFN)-γ produced by CD8⁺ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P^0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P^0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-γ–producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-γ–producing CD8⁺ T cells. Thus, in the absence of IFN-γ and/or TNF/LT-α, exhaustion of virus-specific T cells was not hampered.     

T cell repertoire following autologous stem cell transplantation for multiple sclerosis

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies. In clinical trials, HSCT has been evaluated for severe autoimmunity as a method to “reset” the immune system and produce a new, non-autoimmune repertoire. While the feasibility of eliminating the vast majority of mature T cells is well established, accurate and quantitative determination of the relationship of regenerated T cells to the baseline repertoire has been difficult to assess. Here, in a phase II study of HSCT for poor-prognosis multiple sclerosis, we used high-throughput deep TCRβ chain sequencing to assess millions of individual TCRs per patient sample. We found that HSCT has distinctive effects on CD4⁺ and CD8⁺ T cell repertoires. In CD4⁺ T cells, dominant TCR clones present before treatment were undetectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8⁺ clones were not effectively removed, and the reconstituted CD8⁺ T cell repertoire was created by clonal expansion of cells present before treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. These results demonstrate that TCR characterization during immunomodulatory treatment is both feasible and informative, and may enable monitoring of pathogenic or protective T cell clones following HSCT and cellular therapies.     

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.     

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.     

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.     

An Opposite Pattern of Selection of a Single T Cell Antigen Receptor in the Thymus and among Intraepithelial Lymphocytes

The differentiation of intestinal intraepithelial lymphocytes (IEL) remains controversial, which may be due in part to the phenotypic complexity of these T cells. We have investigated here the development of IEL in mice on the recombination activating gene (RAG)-2^−/− background which express a T cell antigen receptor (TCR) transgene specific for an H-Y peptide presented by D^b (H-Y/D^b × RAG-2⁻ mice). In contrast to the thymus, the small intestine in female H-Y/D^b × RAG-2⁻ mice is severely deficient in the number of IEL; TCR transgene⁺ CD8αα and CD8αβ are virtually absent. This is similar to the number and phenotype of IEL in transgenic mice that do not express the D^b class I molecule, and which therefore fail positive selection. Paradoxically, in male mice, the small intestine contains large numbers of TCR⁺ IEL that express high levels of CD8αα homodimers. The IEL isolated from male mice are functional, as they respond upon TCR cross-linking, although they are not autoreactive to stimulator cells from male mice. We hypothesize that the H-Y/D^b TCR fails to undergo selection in IEL of female mice due to the reduced avidity of the TCR for major histocompatibility complex peptide in conjunction with the CD8αα homodimers expressed by many cells in this lineage. By contrast, this reduced TCR/CD8αα avidity may permit positive rather than negative selection of this TCR in male mice. Therefore, the data presented provide conclusive evidence that a TCR which is positively selected in the thymus will not necessarily be selected in IEL, and furthermore, that the expression of a distinct CD8 isoform by IEL may be a critical determinant of the differential pattern of selection of these T cells.     

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8⁺ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8⁺ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8⁺ lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8⁺ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8⁺ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8⁺PD-1⁺ compared with CD8⁺PD-1^– TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8⁺ and the CD8⁺PD-1⁺ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8⁺ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.     

Active surveillance characterizes human intratumoral T cell exhaustion

Intratumoral T cells that might otherwise control tumors are often identified in an “exhausted” state, defined by specific epigenetic modifications and upregulation of genes such as CD38, cytotoxic T-lymphocyte–associated protein 4 (CTLA4), and programmed cell death 1 (PD1). Although the term might imply inactivity, there has been little study of this state at the phenotypic level in tumors to understand the extent of their incapacitation. Starting with the observation that T cells move more quickly through mouse tumors the longer they reside there and progress toward exhaustion, we developed a nonstimulatory, live-biopsy method for the real-time study of T cell behavior within individual patient tumors. Using 2-photon microscopy, we studied native CD8⁺ T cell interaction with antigen-presenting cells (APCs) and cancer cells in different microniches of human tumors and found that T cell speed was variable by region and by patient and was inversely correlated with local tumor density. Across a range of tumor types, we found a strong relationship between CD8⁺ T cell motility and the exhausted T cell state that corresponded with our observations made in mouse models in which exhausted T cells moved faster. Our study demonstrates T cell dynamic states in individual human tumors and supports the existence of an active program in “exhausted” T cells that extends beyond incapacitating them.     

STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8⁺ cells, CD4⁺ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/β sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti–PD-1 treatment in MC38 and B16F10 tumor models.     

CD4+ T Cells Prevent Spontaneous Experimental Autoimmune Encephalomyelitis in Anti–Myelin Basic Protein T Cell Receptor Transgenic Mice

Autoimmune diseases result from a failure of tolerance. Although many self-reactive T cells are present in animals and humans, their activation appears to be prevented normally by regulatory T cells. In this study, we show that regulatory CD4⁺ T cells do protect mice against the spontaneous occurrence of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Anti–myelin basic protein (MBP) TCR transgenic mice (T/R⁺) do not spontaneously develop EAE although many self-reactive T cells are present in their thymi and peripheral lymphoid organs. However, the disease develops in all crosses of T/R⁺ mice with recombination-activating gene (RAG)-1 knockout mice in which transgenic TCR-expressing cells are the only lymphocytes present (T/R⁻ mice). In this study, crosses of T/R⁺ mice with mice deficient for B cells, CD8⁺ T cells, NK1.1 CD4⁺ T (NKT) cells, γ/δ T cells, or α/β T cells indicated that α/β CD4⁺ T cells were the only cell population capable of controlling the self-reactive T cells. To confirm the protective role of CD4⁺ T cells, we performed adoptive transfer experiments. CD4⁺ T cells purified from thymi or lymph nodes of normal mice prevented the occurrence of spontaneous EAE in T/R⁻ mice. To achieve full protection, the cells had to be transferred before the recipient mice manifested any symptoms of the disease. Transfer of CD4⁺ T cells after the appearance of symptoms of EAE had no protective effect. These results indicate that at least some CD4⁺ T cells have a regulatory function that prevent the activation of self-reactive T cells.     

CD4+ and CD8+ T cell–dependent antiviral immunity requires STIM1 and STIM2

Calcium signaling is critical for lymphocyte function, and intracellular Ca²⁺ concentrations are regulated by store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release–activated Ca²⁺ (CRAC) channels. In patients, loss-of-function mutations in CRAC channel components ORAI1 and STIM1 abolish SOCE and are associated with recurrent and chronic viral infections. Here, using mice with conditional deletion of Stim1 and its homolog Stim2 in T cells, we determined that both components are required for the maintenance of virus-specific memory CD8⁺ T cells and recall responses following secondary infection. In the absence of STIM1 and STIM2, acute viral infections became chronic. Early during infection, STIM1 and STIM2 were required for the differentiation of naive CD8⁺ T cells into fully functional cytolytic effector cells and mediated the production of cytokines and prevented cellular exhaustion in viral-specific CD8⁺ effector T cells. Importantly, memory and recall responses by CD8⁺ T cells required expression of STIM1 and STIM2 in CD4⁺ T cells. CD4⁺ T cells lacking STIM1 and STIM2 were unable to provide “help” to CD8⁺ T cells due to aberrant regulation of CD40L expression. Together, our data indicate that STIM1, STIM2, and CRAC channel function play distinct but synergistic roles in CD4⁺ and CD8⁺ T cells during antiviral immunity.     

CD1d-mediated Recognition of an α-Galactosylceramide by Natural Killer T Cells Is Highly Conserved through Mammalian Evolution

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid α-galactosylceramide (α-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Vα24 TCR expression, uniformly recognize α-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with α-GalCer led to the dramatic expansion of NK T cells with an invariant (Vα24⁺) TCR and the release of large amounts of cytokines. Because invariant Vα14⁺ and Vα24⁺ NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that α-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.     

A participant-derived xenograft model of HIV enables long-term evaluation of autologous immunotherapies

HIV-specific CD8⁺ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4⁺ T cells from HIV⁺ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8⁺ T cell response have a profound impact on the emergence and persistence of escape mutations. This “participant-derived xenograft” model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.     

CD5 Expression Is Developmentally Regulated By T Cell Receptor (TCR) Signals and TCR Avidity

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4⁻CD8⁻ (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56^lck. At the CD4⁺CD8⁺ (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR–major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4⁺ or CD8⁺ (single positive, SP) thymocytes is induced by engagement of the α/β-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR–MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.     

TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

Tumor cells frequently escape from CD8⁺ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/D^b complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-I^lo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8⁺ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.     

Positive Selection of Extrathymically Developed T Cells by Self-antigens

Most T cells develop through the thymus, where they undergo positive and negative selection. Some peripheral T cells are known to develop in the absence of thymus, but there is insufficient information about their selection. To analyze the selection of extrathymically developed T cells, we reconstituted thymectomized male or female recipient mice with bone marrow cells of mice transgenic for male H-Y antigen–specific T cell receptor (TCR). It was revealed that the T cells bearing self-antigen–specific TCR were not deleted in thymectomized male recipients. More importantly, the absence of H-Y antigen–specific T cells in thymectomized female recipients suggests positive selection of extrathymically developed T cells by the self-antigen. The extrathymically developed T cells in male mice expressed interleukin (IL)-2 receptor β chain (IL-2Rβ) and intermediate levels of CD3 (CD3^int) but were natural killer cell (NK)1.1⁻. They rapidly produced interferon γ but not IL-4 after TCR cross-linking. Furthermore, a similar pattern of cytokine production was observed in CD3^intIL-2Rβ⁺NK1.1⁻ cells in normal mice which have been shown to develop extrathymically. These results suggest that extrathymically developed CD3^intIL-2Rβ⁺NK1.1⁻ cells in normal mice are also positively selected by self-antigens.