The Role of CD8 Downregulation during Thymocyte Differentiation
Authors: Aneela Nomura, Ichiro Taniuchi
Journal: Trends in Immunology (2020)

Positive-selection signals by TCR engagement specifically result in CD8 downregulation in CD4+CD8+ thymocytes. Because CD4 and CD8 co-receptors aid in TCR–MHC engagement, differences in their kinetic expression have been proposed to promote differences in TCR signaling in MHC-I- and MHC-II-specific cells.CD8 downregulation serves as an important determinant segregating the developmental fates of MHC-II- and MHC-I-specific cells into helper and cytotoxic lineages. However, it remains unclear whether there any other functional roles of CD8 downregulation in T cell development.The effects of constitutive CD8 expression on enhancing TCR signaling in MHC-I-specific cells can promote the development of both innate-like CD4-CD8+ and iNKT2 cells in mice.Innate T cells can differentiate in the absence of CD8 expression; therefore, the function of CD8 in response to nonclassical MHC-I and in modulating TCR signaling remains uncharacterized. During mammalian T cell development, CD4+CD8+ double-positive (DP) thymocytes must make a lineage choice to become either conventional CD4+ or CD8+ T cells, dependent on their specificity for MHC-II or MHC-I, respectively. Alternatively, DP thymocytes can decide to become innate-like T cells in response to nonclassical MHC-I molecules. A key feature is the downregulation of CD8, which causes transient T cell receptor (TCR) signaling in MHC-I-selected DP thymocytes. Hence, this kinetic signaling model postulates that short or long duration of TCR signals during positive selection can direct the development of cytotoxic or helper T cell lineages. In this opinion article, we discuss the effects of constitutive expression of transgenic CD8 and prolonged TCR signaling on T cell lineage choice in MHC-I selected mouse thymocytes. During mammalian T cell development, CD4+CD8+ double-positive (DP) thymocytes must make a lineage choice to become either conventional CD4+ or CD8+ T cells, dependent on their specificity for MHC-II or MHC-I, respectively. Alternatively, DP thymocytes can decide to become innate-like T cells in response to nonclassical MHC-I molecules. A key feature is the downregulation of CD8, which causes transient T cell receptor (TCR) signaling in MHC-I-selected DP thymocytes. Hence, this kinetic signaling model postulates that short or long duration of TCR signals during positive selection can direct the development of cytotoxic or helper T cell lineages. In this opinion article, we discuss the effects of constitutive expression of transgenic CD8 and prolonged TCR signaling on T cell lineage choice in MHC-I selected mouse thymocytes. T lymphocytes are fundamental cells of the adaptive immune response. They express the TCR that recognizes antigens presented on MHC-class I and MHC-II molecules (Box 1). Each individual lymphocyte expresses a single TCR specific for a single antigen. Hence, there is selectivity of antigen receptors that underpins immune specificity and this selectivity is thoroughly shaped by a process known as positive selection (see Glossary) in the thymus [1.Klein L. et al.Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat. Rev. Immunol. 2014; 14: 377-391Crossref PubMed Scopus (729) Google Scholar,2.Carpenter A.C. Bosselut R. Decision checkpoints in the thymus.Nat. Immunol. 2010; 11: 666-673Crossref PubMed Scopus (194) Google Scholar]. After assembly of Tcra and Tcrb genes via V(D)J recombination, αβTCRs are expressed on thymocyte precursors known as CD4+CD8+ DP thymocytes. In order to form a T cell pool that expresses a variety of TCRs, DP thymocytes undergo selection processes that test the reactivity of the αβTCR to self-antigens presented by MHC molecules on each cell [1.Klein L. et al.Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat. Rev. Immunol. 2014; 14: 377-391Crossref PubMed Scopus (729) Google Scholar,2.Carpenter A.C. Bosselut R. Decision checkpoints in the thymus.Nat. Immunol. 2010; 11: 666-673Crossref PubMed Scopus (194) Google Scholar]. CD4 and CD8 are glycoproteins that serve as co-receptors to assist the recognition of antigen presented on MHC-II and MHC-I, respectively [3.Zamoyska R. CD4 and CD8: modulators of T-cell receptor recognition of antigen and of immune responses?.Curr. Opin. Immunol. 1998; 10: 82-87Crossref PubMed Scopus (87) Google Scholar]. These co-receptors function to couple SRC family kinase LCK to the TCR signaling complex, which, in turn, leads to the phosphorylation of TCR-associated chains and the initiation of the downstream signaling pathways (Box 1) [4.Bubeck Wardenburg J. et al.Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,5.Zhang W. et al.LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar]. The presentation of self-antigen-MHC complexes to DP thymocytes is mediated by thymic epithelial cells (TECs) [1.Klein L. et al.Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat. Rev. Immunol. 2014; 14: 377-391Crossref PubMed Scopus (729) Google Scholar]. DP thymocytes that have successfully undergone positive selection differentiate to either CD4+CD8− single positive (SP) and CD4−CD8+ SP thymocytes, thus committed to become helperT cells or cytotoxic T cells, respectively [6.Ellmeier W. et al.The regulation of CD4 and CD8 coreceptor gene expression during T cell development.Annu. Rev. Immunol. 1999; 17: 523-554Crossref PubMed Scopus (214) Google Scholar,7.Germain R.N. T-cell development and the CD4-CD8 lineage decision.Nat. Rev. Immunol. 2002; 2: 309-322Crossref PubMed Scopus (480) Google Scholar]. The outcome of this helper/cytotoxic lineage choice is perfectly matched with co-receptor expression and the MHC class that gives positive selection signals. Hence, MHC-II and MHC-I selected thymocytes develop into CD4+CD8− and CD4−CD8+ SP thymocytes [6.Ellmeier W. et al.The regulation of CD4 and CD8 coreceptor gene expression during T cell development.Annu. Rev. Immunol. 1999; 17: 523-554Crossref PubMed Scopus (214) Google Scholar,7.Germain R.N. T-cell development and the CD4-CD8 lineage decision.Nat. Rev. Immunol. 2002; 2: 309-322Crossref PubMed Scopus (480) Google Scholar].Box 1Structure of the TCR–CD3 Complex and Signaling Pathways in MiceThe TCRαβ heterodimer functions to provide antigen specificity. These dimers interact with pairs of CD3ζ chains, CD3εγ and CD3εδ to form the TCR-CD3 complex (Figure I). Upon TCR engagement with the antigen-MHCI/II, the CD3γ, δ, ε, and ζ chains propagate signaling via the immunoreceptor tyrosine-based activation motifs (ITAMs shown in red) located in the intracellular tails of CD3ζ chains. The colocalization of CD4 or CD8 co-receptor-associated LCK with TCR allows Lck to phosphorylate the tyrosine residues located in ITAMs (Step 1). This results in the recruitment of SRC homology 2 (SH2) domain-containing proteins such as ZAP-70 kinase, which becomes phosphorylated by LCK (Steps 2 and 3). Active ZAP-70 phosphorylates two scaffold proteins, LAT (linker for the activation of T cells) and SLP-76 (SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) (Step 5). Phosphorylation of SLP-76 allows its interaction with adapter proteins Gads, LAT, and PLC-γ, to form the LAT–SLP76 signalosome [4.Bubeck Wardenburg J. et al.Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,5.Zhang W. et al.LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar]. This signaling complex recruits various signaling proteins that ultimately result in the activation of the following pathways: IP3 and DAG; RAS/RAF/MEK/ERK; PI3K/PDK1/AKT; and mTORC1 (Step 5). The TCRαβ heterodimer functions to provide antigen specificity. These dimers interact with pairs of CD3ζ chains, CD3εγ and CD3εδ to form the TCR-CD3 complex (Figure I). Upon TCR engagement with the antigen-MHCI/II, the CD3γ, δ, ε, and ζ chains propagate signaling via the immunoreceptor tyrosine-based activation motifs (ITAMs shown in red) located in the intracellular tails of CD3ζ chains. The colocalization of CD4 or CD8 co-receptor-associated LCK with TCR allows Lck to phosphorylate the tyrosine residues located in ITAMs (Step 1). This results in the recruitment of SRC homology 2 (SH2) domain-containing proteins such as ZAP-70 kinase, which becomes phosphorylated by LCK (Steps 2 and 3). Active ZAP-70 phosphorylates two scaffold proteins, LAT (linker for the activation of T cells) and SLP-76 (SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) (Step 5). Phosphorylation of SLP-76 allows its interaction with adapter proteins Gads, LAT, and PLC-γ, to form the LAT–SLP76 signalosome [4.Bubeck Wardenburg J. et al.Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,5.Zhang W. et al.LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar]. This signaling complex recruits various signaling proteins that ultimately result in the activation of the following pathways: IP3 and DAG; RAS/RAF/MEK/ERK; PI3K/PDK1/AKT; and mTORC1 (Step 5). Understanding the mechanisms that control the CD4/CD8 lineage choice has been a central question in developmental immunology and immunologists have proposed various models of T cell lineage commitment [7.Germain R.N. T-cell development and the CD4-CD8 lineage decision.Nat. Rev. Immunol. 2002; 2: 309-322Crossref PubMed Scopus (480) Google Scholar, 8.Robey E.A. et al.Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage.Cell. 1991; 64: 99-107Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 9.Chan S.H. et al.Another view of the selective model of thymocyte selection.Cell. 1993; 73: 225-236Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 10.Davis C.B. et al.Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes.Cell. 1993; 73: 237-247Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 11.Janeway Jr., C.A. T-cell development. Accessories or coreceptors?.Nature. 1988; 335: 208-210Crossref PubMed Scopus (90) Google Scholar]. One model proposed by Dr. Singer's group focuses on the unique kinetic expression of the CD8 co-receptor (Figure 1): positively selected DP thymocytes terminate Cd8 gene transcription and become CD4+CD8lo intermediate thymocytes [12.Cibotti R. et al.CD8 coreceptor extinction in signaled CD4(+)CD8(+) thymocytes: coordinate roles for both transcriptional and posttranscriptional regulatory mechanisms in developing thymocytes.Mol. Cell. Biol. 2000; 20: 3852-3859Crossref PubMed Scopus (19) Google Scholar,13.Brugnera E. et al.Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.Immunity. 2000; 13: 59-71Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar]. This process occurs regardless of the class of MHC molecule that engages with the TCR during positive selection. On the one hand, because the CD8 co-receptor functions specifically for MHC-I-restricted TCR ligands, the reduced amounts of CD8 result in the cessation of TCR signaling in MHC-I-selected CD4+CD8lo thymocytes [13.Brugnera E. et al.Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.Immunity. 2000; 13: 59-71Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar] (Figure 1). On the other hand, TCR signaling in MHC-II-selected cells remain persistent. Therefore, this model – known as the kinetic signaling model [13.Brugnera E. et al.Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.Immunity. 2000; 13: 59-71Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar,14.Singer A. et al.Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.Nat. Rev. Immunol. 2008; 8: 788-801Crossref PubMed Scopus (326) Google Scholar] – argues that short or long duration of TCR signals during positive selection directs the development towards the cytotoxic or helper lineages, respectively. Moreover, loss of TCR signals in MHC-I-restricted thymocytes renders them responsive to the cytokine IL-7 [15.Park J.H. et al.Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells.Nat. Immunol. 2010; 11: 257-264Crossref PubMed Scopus (176) Google Scholar,16.McCaughtry T.M. et al.Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus.J. Exp. Med. 2012; 209: 2263-2276Crossref PubMed Scopus (69) Google Scholar]. IL-7 receptor signaling in MHC-I-selected thymocytes is thought to promote the transcriptional induction of Runx3, a central transcription factor (TF) that drives a developmental program for cytotoxic T cell function [17.Taniuchi I. et al.Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.Cell. 2002; 111: 621-633Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 18.Setoguchi R. et al.Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.Science. 2008; 319: 822-825Crossref PubMed Scopus (190) Google Scholar, 19.Sato T. et al.Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes.Immunity. 2005; 22: 317-328Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar]. While the Cd8 gene is transcriptionally repressed in MHC-II-selected thymocytes, it is reactivated in MHC-I-selected thymocytes. Thus, the expression of CD8 is temporally downregulated and reactivated specifically during differentiation into cytotoxic T cells [13.Brugnera E. et al.Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.Immunity. 2000; 13: 59-71Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar]. Hence, the downregulation of CD8 has been proposed to play a central role in segregating helper and cytotoxic lineages via modulating the duration of TCR-mediated positive selection signals [14.Singer A. et al.Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.Nat. Rev. Immunol. 2008; 8: 788-801Crossref PubMed Scopus (326) Google Scholar]. However, is this the only functional significance of CD8 downregulation? Recent work from our laboratory has shed new light on the novel role of CD8 downregulation in balancing invariant natural killer T (iNKT) cell subset differentiation in mice [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar]. In this Opinion, we discuss the significance of kinetic CD8 expression in controlling conventional helper and cytotoxic T cell lineage fates, and in balancing iNKT cell subset differentiation. We hypothesize that downregulation of the CD8 co-receptor in MHC-I-restricted cells does not primarily stop them from becoming CD4+ helper T cells, but rather, prevents them from becoming innate memory-like CD8+ SP thymocytes. In addition, we propose an unappreciated role for CD8 in controlling iNKT subset differentiation by modulating TCR signaling in response to the nonclassical MHC-I-like molecule CD1d. The significance of different kinetic expression patterns between CD4 and CD8 co-receptors was initially tested by a genetic approach. For instance, several mutant mouse lines in which CD4 expression was lost after positive selection were generated [21.Adoro S. et al.Coreceptor gene imprinting governs thymocyte lineage fate.EMBO J. 2012; 31: 366-377Crossref PubMed Scopus (23) Google Scholar,22.Sarafova S.D. et al.Modulation of coreceptor transcription during positive selection dictates lineage fate independently of TCR/coreceptor specificity.Immunity. 2005; 23: 75-87Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Results using these lines showed that the differentiation of MHC-II-selected cells was redirected towards CD8+ thymocytes with cytotoxic gene signatures [22.Sarafova S.D. et al.Modulation of coreceptor transcription during positive selection dictates lineage fate independently of TCR/coreceptor specificity.Immunity. 2005; 23: 75-87Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. These results indicate that continuous TCR signaling is essential for CD4+ helper T cell development in mouse cells. Another study showed that the upregulation of CD4 after positive selection was necessary to prevent errors in MHC-II/helper matching that occurred in a minor proportion of MHC-II-restricted cells [23.Sarafova S.D. et al.Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice.Immunity. 2009; 31: 480-490Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. The disruption of Cd4 upregulation interfered with TCR signaling and subsequently, misdirected a proportion of MHC-II-selected thymocytes towards the CD8 lineage pathway [23.Sarafova S.D. et al.Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice.Immunity. 2009; 31: 480-490Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. Thus, this finding suggests that continuous TCR signaling ensures that all MHC-II-selected cells choose a helper T cell fate. There are however some contradictory findings that do not fit with a requirement for CD4-dependent TCR signals. Specifically, helper-like MHC-II-restricted cells in CD8+ T cells were reported to persist in Cd4−/− mice, relative to wild-type (WT) mice [24.Tyznik A.J. et al.The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells.J. Exp. Med. 2004; 199: 559-565Crossref PubMed Scopus (108) Google Scholar]. Therefore, loss of Cd4 alone was not sufficient to completely abolish positive selection of MHC-II-restricted thymocytes and the generation of helper T cells. In addition, the expression of the Cd4 gene in murine thymocytes is controlled by two stage-specific enhancers, Cd4 proximal and maturation enhancers [25.Issuree P.D. et al.Heritable gene regulation in the CD4:CD8 T cell lineage choice.Front. Immunol. 2017; 8: 291Crossref PubMed Scopus (24) Google Scholar]. The Cd4 proximal enhancer (E4p) is responsible for activating the Cd4 gene during the transition of double-negative to the DP stage [26.Chong M.M. et al.Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells.Genes Dev. 2010; 24: 659-669Crossref PubMed Scopus (52) Google Scholar]. Consequently, DP thymocytes in mice lacking the E4p enhancer (Cd4ΔE4p/ΔE4P mice) express low amounts of CD4. However, it was surprising to see that a significant number of CD4 SP thymocytes could still develop in a MHC-II-dependent manner in Cd4ΔE4p/ΔE4P mice [26.Chong M.M. et al.Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells.Genes Dev. 2010; 24: 659-669Crossref PubMed Scopus (52) Google Scholar], which subsequently led to the discovery of the Cd4 maturation enhancer (E4m) [27.Kojo S. et al.Runx-dependent and silencer-independent repression of a maturation enhancer in the Cd4 gene.Nat. Commun. 2018; 9: 3593Crossref PubMed Scopus (7) Google Scholar,28.Issuree P.D. et al.Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development.Nat. Commun. 2018; 9: 3594Crossref PubMed Scopus (16) Google Scholar]. Nevertheless, it is still unclear how precursors expressing MHC-II-restricted TCRs receive positive selection signals with little expression of CD4 in Cd4ΔE4p/ΔE4P mice. Thus, we suspect that some MHC-II-restricted TCRs may respond to antigen and transmit TCR signaling in a CD4-independent manner, although this remains to be tested. From another angle, the effects of constitutive expression of the CD8 co-receptor have been tested with the expectation that persistent TCR signaling might guide MHC-I-restricted cells into CD4+ helper T cells (Figure 2) [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar,29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Initial approaches using transgenic mouse lines expressing CD8α and CD8β chains under the control of human CD2 promoter/enhancer sequences have resulted in a partial redirection in differentiation of MHC-I-selected cells. However, approximately two thirds of MHC-I selected cells remained able to differentiate into CD4− cells [29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. So, why did constitutive expression of the CD8 co-receptor in MHC-I-restricted cells guide a significant proportion of them to become CD4− cytotoxic cells? Given that this experiment was conducted in mouse lines expressing the endogenous Cd8b gene, it was argued that a transgenic CD8α chain paired with an endogenous CD8β chain was downregulated after positive selection [29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. So, if transgenic CD8α and transgenic CD8β were to be expressed together, this transgenic co-receptor should in theory, be continuously expressed in this setting. To address this argument accurately, a recent study generated a mouse model that constitutively expressed the CD8 co-receptor in a genetic background lacking both endogenous Cd8a and Cd8b genes(Cd8a−/− : Cd8b1−/−: Rosa26Cd8a/Cd8b mice) [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar]. This approach almost recapitulated the results from the preliminary study discussed above [29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar], resulting in two thirds of MHC-I-restricted thymocyte precursors acquiring the CD4−CD8+ phenotype. It should be acknowledged, however, that the expression of the transgenic CD8αβ co-receptor was lower than that of the endogenous one, and was nearly equivalent to that on CD4+CD8lo intermediate thymocytes from a WT control [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar]. Hence, it is possible that weakened TCR signaling – as a result of reduced CD8 expression amounts – was not sufficient to direct all MHC-I-restricted cells into CD4+ cells, but theoretically, weakened TCR signaling must have also been continuous due to constitutive CD8 co-receptor expression [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar]. Of note, although transgenic CD8αβ expression in the previous study was equivalent to the endogenous one [29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar], the results of these two studies were similar. Therefore, constitutive expression of CD8αβ co-receptor was not sufficient to redirect MHC-I-selected cells towards the CD4+ helper fate. To further understand the functional characteristics of CD4−CD8+ SP thymocytes being generated under constitutive CD8 co-receptor expression, these cells were subjected to RNA-sequencing analysis; a next-generation sequencing technique that was not available in the preliminary study [29.Bosselut R. et al.Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.Immunity. 2001; 14: 483-494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. A significant proportion of CD4−CD8+ SP thymocytes expressed high amounts of the promyelocytic zinc finger transcription factor (PLZF) and Eomes transcripts [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar], which were also abundantly present in innate memory-like CD8+ T cells. Thus, CD4−CD8+ SP thymocytes from a CD8αβ transgenic mouse model in our laboratory, acquired transcriptional signatures similar to those of innate memory-like CD8+ SP thymocytes (Box 2) [30.Weinreich M.A. et al.T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells.Nat. Immunol. 2010; 11: 709-716Crossref PubMed Scopus (196) Google Scholar]. Coincidentally, constitutive CD8 co-receptor expression specifically skewed the balance of iNKT subset differentiation towards the iNKT2 subset and, because of this, promoted the survival and expansion of these innate memory-like CD8+ SP thymocytes. Thus, analyses of the underlying mechanisms for the emergence of innate memory-like CD8+ SP thymocytes uncovered novel significance for the downregulation of CD8 in regulating iNKT cell differentiation [20.Kojo S. et al.Constitutive CD8 expression drives innate CD8(+) T-cell differentiation via induction of iNKT2 cells.Life Sci. Alliance. 2020; 3e202000642Crossref PubMed Scopus (3) Google Scholar] (discussed later).Box 2Characteristics of Conventional and Unconventional T Cells in MiceT cells expressing αβ TCR (αβ T cells) originate from CD4+CD8+ thymocytes and can be functionally classified as either conventional or unconventional. Conventional CD4+ and CD8+ T cells express a diverse repertoire of αβ TCR but their activation is restricted by antigen–MHC complexes. On the one hand, during primary immune responses, the generation of effector cells from conventional naïve T cells requires antigen priming and this can take weeks to occur. On the other hand, unconventional T cells have both innate and adaptive characteristics [52.Godfrey D.I. et al.The burgeoning family of unconventional T cells.Nat. Immunol. 2015; 16: 1114-1123Crossref PubMed Scopus (445) Google Scholar]. They are generally responsive to MHC-I-like molecules encoded by genes outside MHC loci. They also immediately respond to antigens and release large amounts of cytokines. Thus, they are also known as innate-like T cells. The main groups of unconventional αβ T cells include CD8αα+ intraepithelial T lymphocytes (IELs), innate memory-like CD8+ T cells, iNKT cells, and MAIT cells [52.Godfrey D.I. et al.The burgeoning family of unconventional T cells.Nat. Immunol. 2015; 16: 1114-1123Crossref PubMed Scopus (445) Google Scholar,53.Legoux F. et al.Unconventional or preset αβ T cells: evolutionarily conserved tissue-resident T cells recognizing nonpeptidic ligands.Annu. Rev. Cell Dev. 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