Immunology and Cell Biology (2015) 93, 326–327 & 2015 Australasian Society for Immunology Inc. All rights reserved 0818-9641/15

NEWS AND COMMENTARY MHCI and CD8 lineage commitment

Prolonged access to thymic epithelial MHCI seals CD8+ lineage commitment Stephen R Daley Immunology and Cell Biology (2015) 93, 326–327; doi:10.1038/icb.2015.28; published online 10 March 2015


he stage of thymocyte maturation when commitment to the CD8+ lineage is consolidated by recognition of major histocompatibility complex class I (MHCI) on thymic epithelial cells has been elucidated in a study published in Immunology and Cell Biology.1 Development of αβ T cells is blocked at the CD4+CD8+ double-positive (DP) stage in mice lacking Zap70.2 By complementing the defect in Zap70–/– mice via an inducible Zap70 transgene (Tet-Zap70), the authors previously examined the kinetics of phenotypic progressions during thymocyte development.3 This confirmed that CD4+ single-positive (SP) cells form earlier than CD8SP cells4 and enabled delineation of DP thymocytes into three substages (DP1–DP3) based on CD5 and TCRβ expression. DP1 (CD5−TCRβ−) encompasses the major population of unselected cells, DP2 (CD5hiTCRβint) is the population from which CD4+ T cells differentiate directly and DP3 (CD5intTCRβhi) cells represent direct precursors of CD8+ T cells.3 The inducible Tet-Zap70 system also focused attention on the developmental regulation of Zap70 expression during thymocyte maturation. A ‘Zap70-dependent positive feedback circuit’ was revealed when T-cell receptor (TCR) signaling was found to induce Zap70 upregulation and Zap70 upregulation correlated with responsiveness to TCR stimulation. In Tet-Zap70 mice, transgenic Zap70 expression is uncoupled from developmental regulation. Interestingly, this uncoupling partially blocks T-cell development at a later stage than the partial block caused by the hypomorphic Zap70SKG point-mutant allele.3 SR Daley is at Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia E-mail: [email protected]

Tet-Zap70 expression causes a partial block in T-cell development around the time of CD4SP or CD8SP cell formation. There is also evidence that Tet-Zap70 expression ‘misdirects’ a subset of MHCII-reactive cells into the DP3 pool, which is normally occupied only by MHCI-reactive precursors of CD8+ cells. Although framed somewhat differently in the paper,1 Sinclair et al.’s experiments predominantly compare ‘misdirected’ MHCIdeprived DP3 thymocytes with MHCI-replete DP3 thymocytes. The key question is stated nicely in the final sentence of a previous paper from the same laboratory: does ‘…instigation of CD8+ lineage specification…represent a default path of differentiation that occurs in the absence of mutually exclusive CD4 commitment or…(is it) dependent on continued and successful selection signaling at the DP3 stage (?)’.3 To disrupt ‘selection signaling’ within the DP3 precursors of CD8+ T cells, mice lacking functional MHCI expression due to absence of β2-microglobulin were used. These MHCIdeficient mice were irradiated and reconstituted with donor bone marrow so that the lack of MHCI expression was confined to radioresistant thymic epithelial cells (TECs). Most experiments used Tet-Zap70 donor bone marrow, presumably because this facilitated formation of DP3 thymocytes, which are rare in MHCI-deficient Zap70+/+ mice. This approach provided access to DP3 thymocytes, either at a differentiation ‘dead end’ in MHCI-deficient hosts or progressing toward the CD8+ lineage in wild-type or MHCII-deficient hosts. To assess the importance of ‘selection signaling’, Sinclair et al. compared DP3 thymocytes from these different hosts in two ways: (1) phenotypically and (2) for their competence to differentiate in vivo.

Phenotypically, absence of MHCI from TECs resulted in decreased expression within DP3 thymocytes of several proteins induced by TCR activation, such as Zap70, Nur77, Egr1 and Egr2. The effect was stage specific, as expression levels of Zap70 and Nur77 within DP2 thymocytes were similar in MHCI-deficient versus MHCII-deficient hosts. Bcl-2 was expressed at similar levels in DP3 thymocytes under all conditions examined.1 Thus, the thymocytes at the DP3 stage still require access to MHCI-dependent ligands on TECs for efficient CD8+ lineage specification. On the basis of time course experiments using the inducible Tet-Zap70 system, thymocytes enter the DP3 stage ~ 3 days after initiating selection.3 Competence to differentiate in vivo was tested by intrathymic transfer experiments. DP3 thymocytes from wild-type donors were known to differentiate into CD8SP but not CD4SP cells after intrathymic transfer.3 By contrast, after Tet-Zap70 DP3 thymocytes from MHCI-deficient donors were injected into the thymus of a wild-type host, a quarter of the surviving cells had differentiated into CD4SP cells. The absence of MHCI from TECs was important for this result as TetZap70 DP3 thymocytes from MHCIIdeficient donors differentiated into CD8SP but not CD4SP cells after intrathymic transfer, as expected.1 Thus, the thymocytes require interactions with MHCI-dependent ligands on TECs at or before the DP3 stage in order to repress CD4+ lineage differentiation. CD4/CD8 lineage choice is mediated by the mutually repressive transcription factors, ThPOK and Runx3, which act in CD4SP and CD8SP thymocytes, respectively.5,6 Using a Th-POK reporter mouse strain, Sinclair et al.1 found that a subset of DP3 thymocytes expresses Th-POK. Although Th-POK

News and Commentary 327 DP3 DP3 Differentiation Phenotype competence TEC MHCI


Zap70 Nur77 Runx3 Egr1 Egr2


Zap70 Nur77 Runx3 Egr1 Egr2


stage, they also fall outside the classical definition of ‘death by neglect’. Whether and how many wild-type thymocytes die by this mechanism remains unclear, but some at-risk cells may survive by becoming more sensitive to TCR stimulation via the Zap70dependent positive feedback loop.1,3 Thus, collectively the study by Sinclair et al. provides some novel and unexpected insights into T-cell lineage determination.

Zap70 expression







Time since initiation of TCR signaling (d)

Figure 1 Access to MHCI on TECs promotes TCR activation and CD8+ specification in DP3 thymocytes. Lower section shows thymocyte populations with distinct competencies to enter CD4+ or CD8+ T-cell lineages positioned to indicate kinetics of formation on x axis versus Zap70 expression on y axis.3 Upper section shows the effect of depriving TECs of MHCI or MHCII on DP3 thymocyte phenotype and competence to enter CD4 or CD8 lineages.1

expression in DP3 cells was considerably lower than that in CD4SP cells, this observation, and the intrathymic transfer results discussed above, indicates that the DP3 population can include cells with the potential for CD4+ development. It was known, however, that many DP3 thymocytes express Runx3, consistent with commitment to the CD8+ lineage.3,7 Sinclair et al. observed that DP3 thymocytes from MHCI-deficient mice have reduced Runx3 expression, relative to wild-type controls, indicating that Runx3 upregulation within DP3 thymocytes directly or indirectly requires MHCI expression on TECs.1 The findings regarding Th-POK and Runx3, which were all based on the analysis of Zap70+/+ mice, suggest that instigation of

CD8+ lineage specification is not an ‘automatic’ consequence of entering the DP3 stage. Rather, the firm commitment of wild-type DP3 cells to the CD8+ lineage that was observed in intrathymic transfer experiments3 relies on an intact Zap70dependent positive feedback circuit and/or intact MHCI expression by TECs (Figure 1). An interesting hypothesis discussed by Sinclair et al.1 is the existence of a novel mode of thymocyte death. In Tet-Zap70 mice many ‘misdirected’ thymocytes die at the DP3 stage.8 On the basis of their low expression of TCR activation markers,1 these cells are dissimilar to cells undergoing clonal deletion.9,10 As these thymocytes did receive a productive TCR signal to reach the DP3

1 Sinclair C, Ono M, Seddon B. A Zap70-dependent feedback circuit is essential for efficient selection of CD4 lineage thymocytes. Immunol Cell Biol 2015; 93: 406–416. 2 Negishi I, Motoyama N, Nakayama K, Nakayama K, Senju S, Hatakeyama S et al. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 1995; 376: 435–438. 3 Saini M, Sinclair C, Marshall D, Tolaini M, Sakaguchi S, Seddon B. Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds. Sci Signal 2010; 3: ra23. 4 Lucas B, Vasseur F, Penit C. Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression. J Immunol 1993; 151: 4574–4582. 5 Egawa T, Littman DR. ThPOK acts late in specification of the helper T cell lineage and suppresses Runxmediated commitment to the cytotoxic T cell lineage. Nat Immunol 2008; 9: 1131–1139. 6 Setoguchi R, Tachibana M, Naoe Y, Muroi S, Akiyama K, Tezuka C et al. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development. Science 2008; 319: 822–825. 7 Sinclair C, Seddon B. Overlapping and asymmetric functions of TCR signaling during thymic selection of CD4 and CD8 lineages. J Immunol 2014; 192: 5151–5159. 8 Sinclair C, Bains I, Yates AJ, Seddon B. Asymmetric thymocyte death underlies the CD4:CD8 T-cell ratio in the adaptive immune system. Proc Natl Acad Sci USA 2013; 110: E2905–E2914. 9 Daley SR, Hu DY, Goodnow CC. Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NFkappaB. J Exp Med 2013; 210: 269–285. 10 Stritesky GL, Xing Y, Erickson JR, Kalekar LA, Wang X, Mueller DL et al. Murine thymic selection quantified using a unique method to capture deleted T cells. Proc Natl Acad Sci USA 2013; 110: 4679–4684.

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MHCI and CD8 lineage commitment. Prolonged access to thymic epithelial MHCI seals CD8⁺ lineage commitment.

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