Abstract
Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step.
Introduction
Initially, αβ T cells are selected in the thymic cortex through interactions with MHC/peptide ligands present in that environment. This event triggers multiple gene-expression and functional changes, including initial survival through upregulation of Bcl-2, migration to the medulla through upregulation of CCR7, and fixation of receptor specificity through termination of RAG gene expression (1).This is followed immediately by signals that fix the cytotoxic/helper lineage to MHC class recognition in emerging CD4 or CD8 single-positive (SP) thymocytes (2). If we consider the process of positive selection as the “birth” of a T cell, we should also consider that the full expression of T cell functional properties requires critical developmental events that occur later in thymic development or during “adolescence.” Some of these even occur after progenitors egress from the thymus or “leave home.” These events, which we collectively refer to as maturation, include the development of the competence to proliferate when stimulated through the Ag receptor (AgR), resistance to death from external stimuli (e.g., TNF), egress, licensing, and survival in the periphery (Fig. 1).
Functional changes that occur throughout development and their anatomic locations. TCRαβ rearrangement is completed at the preselection DP (Pre-DP) stage as the progenitor resides in the thymic cortex. MHC-dependent positive (and negative) selection events initiate here. Positive selection results in the upregulation of genes that facilitate survival, migration, and receptor fixation, leading to a readily identifiable postselection DP (Post-DP) cell. As the cell migrates toward the medulla, coreceptor gene expression changes as part of the lineage-commitment process. Initially, these medullary SP cells remain susceptible to apoptosis induction and are so-called “SM” cells. SM cells that acquire the competence to proliferate are considered mature (M1). Protection from death receptor signaling occurs concurrently with this. Over time, M1 cells acquire the competence to emigrate and are considered fully mature (M2). M2 cells that have egressed from the thymus into the periphery are considered RTEs and are functionally distinct from MNTs in that their cytokine production is further enhanced in secondary lymphoid organs (SLOs). T cell progenitors that enter the circulation also require protection from complement, a property that is acquired late in the maturation process.
Functional changes that occur throughout development and their anatomic locations. TCRαβ rearrangement is completed at the preselection DP (Pre-DP) stage as the progenitor resides in the thymic cortex. MHC-dependent positive (and negative) selection events initiate here. Positive selection results in the upregulation of genes that facilitate survival, migration, and receptor fixation, leading to a readily identifiable postselection DP (Post-DP) cell. As the cell migrates toward the medulla, coreceptor gene expression changes as part of the lineage-commitment process. Initially, these medullary SP cells remain susceptible to apoptosis induction and are so-called “SM” cells. SM cells that acquire the competence to proliferate are considered mature (M1). Protection from death receptor signaling occurs concurrently with this. Over time, M1 cells acquire the competence to emigrate and are considered fully mature (M2). M2 cells that have egressed from the thymus into the periphery are considered RTEs and are functionally distinct from MNTs in that their cytokine production is further enhanced in secondary lymphoid organs (SLOs). T cell progenitors that enter the circulation also require protection from complement, a property that is acquired late in the maturation process.
Acquiring the competence to divide
To understand the mechanisms guiding T cell maturation in the thymus, it is important to delineate stages of SP thymocyte maturation by cell surface markers, how they change with time, and how such phenotypic changes are related to functional changes. SP thymocytes reside predominantly in the medulla; however, SP thymocytes are not a homogenous population. It was initially reported that SP thymocytes gradually proceed to downregulate heat-stable Ag (also called CD24) and upregulate Qa2 after positive selection (3). In 1997, Kishimoto and Sprent (4) first defined CD24hiQa2lo SP thymocytes as “semimature” (SM) and showed that they are still susceptible to apoptosis when triggered through the TCR. This is in contrast to fully mature SP thymocytes and thymic emigrants, which proliferate when triggered through the TCR. Subsequently, other groups (5–7) showed that a number of other cell surface proteins, including CD69 and various cytokine and chemokine receptors, change dramatically during maturation (Table I). Mice expressing GFP under the control of the RAG1 or RAG2 regulatory elements have been instrumental in defining the temporal order of phenotypic changes during maturation, because RAG gene transcription is high in double-positive (DP) thymocytes, but acutely terminated upon positive selection. Thus the degradation of GFP provides an approximate molecular timer for thymic development after positive selection (8), and GFP expression in peripheral T cells provides the best contemporary means to identify recent thymic emigrants (RTEs) (9). The temporal ordering of the molecules shown in Table I has been confirmed in RAGGFP models (K.A. Hogquist, Y. Xing, F.-C. Hsu, and V.S. Shapiro, unpublished observations). Examining these using comprehensive microarray and flow cytometric analysis, we recently discovered that MHC class I upregulation most precisely defines the boundary between SM and proliferation-competent cells (Table I). However, not all MHC class Ihi cells are competent to emigrate. Further upregulation of CD62L and S1P1 and downregulation of CD69 are associated with tissue egress (discussed below). Thus, we propose the designation of three stages of SP maturation as a useful paradigm for evaluating molecular mechanisms: SM cells that are CD69+MHC-I− and undergo apoptosis when stimulated through the AgR, mature 1 (M1) cells that are CD69+MHC-I+ and competent to proliferate when stimulated through the AgR, and mature 2 (M2) cells that are CD69−MHC-I+, competent to proliferate, and express molecules associated with egress. M2 cells are the “oldest” SP thymocytes present in the thymus (among naive CD4 or CD8 SP thymocytes) and have a phenotype largely identical to RTEs in the periphery.
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MalII, mSiglecE, and SNBL are lectins that preferentially detect the products of distinct sialyltransferase enzymes, indicated in parentheses. Peanut agglutinin (PNA) binding is inhibited by sialic acids (SA); thus, PNA binding inversely correlates with the extent of sialylation. Symbols indicate level of expression. Shading indicates minimal (white) to maximal (dark grey) for each molecule.
Pre-DP, preselection DP; Post-DP, postselection DP.
Protection from death receptor signaling at M1
Mice deficient in a number of genes in the NF-κB pathway exhibit defects in postselection maturation, with near-normal numbers of total thymocytes but few peripheral T cells. For example, mice with CD4-Cre–directed deficiency in TGF-β–activated kinase 1 (TAK1), a key upstream kinase of the IKK complex have normal development to the SM SP stage but very few mature SP thymocytes (10–12). Deficiency in molecules downstream of TAK1 in the NF-κB pathway results in similar defects, including IKK2 (13), RelB (14), and NEMO (15). Also, deficiency in Ubc13, an E2 ubiquitin-conjugating enzyme that provides a scaffold for TAK1 activation and regulates NF-κB activation, leads to peripheral T cell lymphopenia, with near normal thymocyte development (16). The TAK1/NF-κB pathway is activated downstream of multiple cell surface receptors, including TCR, IL-1R, TLR, and TNF family receptors (17). Because NF-κB is well known to protect cells from TNF-induced death, the role of NF-κB signaling at the M1 stage may be to protect developing T cells from constitutive death receptor signals. Upon TNFR ligation, the serine/threonine kinase RIPK1 prevents a proapoptotic complex from forming and facilitates the activation of the canonical NF-κB pathway, promoting the expression of antiapoptotic molecules, such as c-FLIP (18). RIPK1 was shown to be required for T cell development (19), and thymocytes lacking c-FLIP display a block precisely at the SM-to-mature transition (20). c-FLIP inhibits death receptor–induced apoptosis by interfering with procaspase-8 recruitment to the adaptor FADD. In mice lacking c-FLIP, this unopposed death occurs at the transition from an SM to mature stage, preventing development of Qa2+ CD62L+ SP thymocytes. c-FLIP protects mature T cells from apoptosis induced by the death receptors Fas and TNFR, as well as from TCR-mediated and spontaneous apoptosis (21, 22). Thymocytes with the above mutations showed increased apoptosis after stimulation with TNF, TCR, and/or Fas (Table II), demonstrating that a survival defect contributes to the peripheral T cell lymphopenia. In particular, the cell-intrinsic apoptosis that occurred in mature T cells in the absence of c-FLIP could be rescued by Bim deficiency (22). There was also a partial rescue of thymocyte cellularity when TNFR1 and IKK2 were concurrently deleted in T cells (13). Thus, the ability to activate the NF-κB pathway during T cell maturation is critical for survival in response to death receptor or TCR signaling. Because NF-κB signals are critical for M1 survival, their role in differentiation remains unclear at this point.
Gene . | Notes . | Refs. . |
---|---|---|
Cell Division Competence in Response to CD3/CD28 (M1) | ||
TAK1 | Decreased CD4 SP thymocyte proliferation (CD4-Cre cKO) | (12) |
Decreased thymocyte proliferation (Lck-Cre cKO) | (10, 52) | |
c-FLIP | Decreased splenic T cell proliferation (KO/Rag KO chimeras) | (54) |
Decreased thymocyte proliferation (Lck-Cre cKO) | (21) | |
Ubc13 | Decreased splenic T cell proliferation (Lck-Cre KO) | (16) |
RelB | Decreased thymocyte proliferation in response to anti-CD3 (KO) | (14) |
IKK2 | Decreased thymocyte proliferation to anti-CD3 in IKK2/TNFR1 KO | (13) |
ZFAT | Decreased splenic CD4 T cell proliferation (CD4-Cre cKO) | (48) |
Casp8 | Decreased splenic CD4 T cell proliferation (Lck-Cre cKO) | (55) |
Bclaf1 | Decreased splenic T cell proliferation (KO/Rag2 KO chimeras) | (56) |
Protection from TCR and Death Receptor Signals (M1) | ||
c-FLIP | Increased thymocyte and peripheral T cell apoptosis in response to TCR, TNF and Fas signals (Lck-Cre cKO, ER-Cre cKO) | (20, 21) |
TAK1 | Increased thymocyte apoptosis in response to TNF, CD3 (Lck-Cre cKO) | (10, 53) |
NEMO | Increased thymocyte apoptosis at steady-state (CD4-Cre) | (15) |
IKK2 | Increased thymocyte apoptosis to TNF, Fas (KO fetal liver stem cell transplant), partial rescue of thymocyte numbers in IKK2/TNFR1 KO | (13) |
RIP1 | Increased thymocyte apoptosis in response to Fas, TNF (KO) | (19) |
RelB | Increased thymocyte apoptosis in response to anti-CD3 (KO) | (14) |
Ubc13 | Regulates Tak1 and NF-κB signaling in thymocytes (Lck-Cre cKO) | (16) |
Gpx4 | Increased ferroptosis in splenic T cells to superantigen or anti-CD3 (CD4-Cre) | (51) |
Egress (M2) | ||
S1P1 | Impaired egress in deficient thymocytes (KO fetal liver chimeras) | (25) |
KLF2 | Impaired egress in deficient thymocytes with reduced S1P1 (KO fetal liver chimeras, CD4-Cre) | (24) |
PAK2 | Impaired egress associated with reduced KLF2/S1P1 (CD4-Cre) | (29) |
Mst1/2 | Impaired egress with normal KLF2/S1P1 expression (Lck-Cre) | (28) |
mDia-1 | Impaired egress in deficient thymocytes (KO) | (27) |
Coro1 | Impaired egress with normal S1P1 expression (point mutant) | (26) |
Foxo1 | Decreased expression of KLF2 (CD4-Cre cKO), regulates thymic egress with Foxo3 (unpublished observations) | (30) |
Paxip1 | Decreased S1P1, but normal KLF2, Foxo1 expression (KO) | (32) |
IL-2 Production to CD3/CD28 Stimulation (Begins at M1 and Is Optimal at MNT) | ||
NKAP | Decreased IL-2 production by splenic CD4+ RTE (CD4-Cre cKO) | (38) |
ZFAT | Decreased IL-2 production by splenic CD4 T cells (CD4-Cre cKO) | (48) |
Casp8 | Decreased IL-2 production by splenic T cells (Lck-Cre cKO) | (55) |
TAK1 | Decreased IL-2 production in CD4 SP thymocytes (CD4-Cre cKO) | (12) |
c-FLIP | Decreased IL-2 production in thymocytes (Lck-Cre cKO) | (20) |
TNF Licensing to CD3/CD28 Stimulation (Begins at M2/RTE and Is Optimal at MNT) | ||
HDAC3 | Defect in TNF production in CD4 and CD8 RTE and MNT (CD4-Cre cKO) | (42) |
NKAP | Defect in TNF production in CD4 and CD8 RTE and MNT (CD4-Cre cKO) | Hsu and Shapiro, unpublished observations |
MNT Survival or Maintenance (Peripheral T Cell Lymphopenia) | ||
NKAP | Peripheral T cells eliminated by complement (CD4-Cre cKO) | (50) |
Zfp335 | Few peripheral T cells, not rescued by a Bcl-2 transgene (point mutant) | (57) |
HDAC3 | Peripheral T cells eliminated by complement (CD4-Cre cKO) | (42) |
TAK1 | Decreased response to IL-7 but normal IL-7Rα expression (CD4-Cre cKO) | (12) |
ZFAT | Decreased survival, IL-7Rα and Bcl-2 expression (CD4-Cre cKO) | (48) |
Foxo1 | Decreased survival, IL-7Rα and Bcl-2 expression (CD4-Cre cKO) | (30) |
IKK2 | Decreased survival, IL-7Rα but not Bcl-2 expression (CD4-Cre cKO) | (46, 47) |
Other: Cause for Defects Largely Unknown | ||
FLVCR | Normal thymic populations but few peripheral T cells (CD4-Cre cKO) | (58) |
Casp8 | Normal thymic populations but few peripheral T cells (Lck-Cre cKO) | (55) |
Bptf | Loss of mature SP thymocytes, decreased IL-7Rα expression but not rescued by Bcl-2 transgene (Lck-Cre cKO) | (49) |
Gene . | Notes . | Refs. . |
---|---|---|
Cell Division Competence in Response to CD3/CD28 (M1) | ||
TAK1 | Decreased CD4 SP thymocyte proliferation (CD4-Cre cKO) | (12) |
Decreased thymocyte proliferation (Lck-Cre cKO) | (10, 52) | |
c-FLIP | Decreased splenic T cell proliferation (KO/Rag KO chimeras) | (54) |
Decreased thymocyte proliferation (Lck-Cre cKO) | (21) | |
Ubc13 | Decreased splenic T cell proliferation (Lck-Cre KO) | (16) |
RelB | Decreased thymocyte proliferation in response to anti-CD3 (KO) | (14) |
IKK2 | Decreased thymocyte proliferation to anti-CD3 in IKK2/TNFR1 KO | (13) |
ZFAT | Decreased splenic CD4 T cell proliferation (CD4-Cre cKO) | (48) |
Casp8 | Decreased splenic CD4 T cell proliferation (Lck-Cre cKO) | (55) |
Bclaf1 | Decreased splenic T cell proliferation (KO/Rag2 KO chimeras) | (56) |
Protection from TCR and Death Receptor Signals (M1) | ||
c-FLIP | Increased thymocyte and peripheral T cell apoptosis in response to TCR, TNF and Fas signals (Lck-Cre cKO, ER-Cre cKO) | (20, 21) |
TAK1 | Increased thymocyte apoptosis in response to TNF, CD3 (Lck-Cre cKO) | (10, 53) |
NEMO | Increased thymocyte apoptosis at steady-state (CD4-Cre) | (15) |
IKK2 | Increased thymocyte apoptosis to TNF, Fas (KO fetal liver stem cell transplant), partial rescue of thymocyte numbers in IKK2/TNFR1 KO | (13) |
RIP1 | Increased thymocyte apoptosis in response to Fas, TNF (KO) | (19) |
RelB | Increased thymocyte apoptosis in response to anti-CD3 (KO) | (14) |
Ubc13 | Regulates Tak1 and NF-κB signaling in thymocytes (Lck-Cre cKO) | (16) |
Gpx4 | Increased ferroptosis in splenic T cells to superantigen or anti-CD3 (CD4-Cre) | (51) |
Egress (M2) | ||
S1P1 | Impaired egress in deficient thymocytes (KO fetal liver chimeras) | (25) |
KLF2 | Impaired egress in deficient thymocytes with reduced S1P1 (KO fetal liver chimeras, CD4-Cre) | (24) |
PAK2 | Impaired egress associated with reduced KLF2/S1P1 (CD4-Cre) | (29) |
Mst1/2 | Impaired egress with normal KLF2/S1P1 expression (Lck-Cre) | (28) |
mDia-1 | Impaired egress in deficient thymocytes (KO) | (27) |
Coro1 | Impaired egress with normal S1P1 expression (point mutant) | (26) |
Foxo1 | Decreased expression of KLF2 (CD4-Cre cKO), regulates thymic egress with Foxo3 (unpublished observations) | (30) |
Paxip1 | Decreased S1P1, but normal KLF2, Foxo1 expression (KO) | (32) |
IL-2 Production to CD3/CD28 Stimulation (Begins at M1 and Is Optimal at MNT) | ||
NKAP | Decreased IL-2 production by splenic CD4+ RTE (CD4-Cre cKO) | (38) |
ZFAT | Decreased IL-2 production by splenic CD4 T cells (CD4-Cre cKO) | (48) |
Casp8 | Decreased IL-2 production by splenic T cells (Lck-Cre cKO) | (55) |
TAK1 | Decreased IL-2 production in CD4 SP thymocytes (CD4-Cre cKO) | (12) |
c-FLIP | Decreased IL-2 production in thymocytes (Lck-Cre cKO) | (20) |
TNF Licensing to CD3/CD28 Stimulation (Begins at M2/RTE and Is Optimal at MNT) | ||
HDAC3 | Defect in TNF production in CD4 and CD8 RTE and MNT (CD4-Cre cKO) | (42) |
NKAP | Defect in TNF production in CD4 and CD8 RTE and MNT (CD4-Cre cKO) | Hsu and Shapiro, unpublished observations |
MNT Survival or Maintenance (Peripheral T Cell Lymphopenia) | ||
NKAP | Peripheral T cells eliminated by complement (CD4-Cre cKO) | (50) |
Zfp335 | Few peripheral T cells, not rescued by a Bcl-2 transgene (point mutant) | (57) |
HDAC3 | Peripheral T cells eliminated by complement (CD4-Cre cKO) | (42) |
TAK1 | Decreased response to IL-7 but normal IL-7Rα expression (CD4-Cre cKO) | (12) |
ZFAT | Decreased survival, IL-7Rα and Bcl-2 expression (CD4-Cre cKO) | (48) |
Foxo1 | Decreased survival, IL-7Rα and Bcl-2 expression (CD4-Cre cKO) | (30) |
IKK2 | Decreased survival, IL-7Rα but not Bcl-2 expression (CD4-Cre cKO) | (46, 47) |
Other: Cause for Defects Largely Unknown | ||
FLVCR | Normal thymic populations but few peripheral T cells (CD4-Cre cKO) | (58) |
Casp8 | Normal thymic populations but few peripheral T cells (Lck-Cre cKO) | (55) |
Bptf | Loss of mature SP thymocytes, decreased IL-7Rα expression but not rescued by Bcl-2 transgene (Lck-Cre cKO) | (49) |
Egress
After ∼4 d of medullary residency, SP thymocytes egress from the organ (8). The transcription factor Kruppel-like factor 2 (KLF2) plays a key role in egress and is expressed at the late M1 stage (23). KLF2 directly promotes both CD62L and S1P1 transcription (24). Deficiency in either KLF2 (24) or S1P1 (25) dramatically impairs thymic egress. Molecules involved in actin polymerization are also required for egress. Coronins are one type of actin regulator, and mice with a mutation in Coronin-1A showed impaired egress and peripheral T cell lymphopenia, despite normal expression of chemokine and S1P receptors (26). This is consistent with the phenotype of mice lacking another actin-regulating protein, mDia1 (27). The mammalian sterile twenty-like (Mst) 1/2 kinases control Rho GTPase activation and the migratory response of T cells. Again, although KLF2 and S1P1 were expressed normally in Mst1/2 double-deficient thymocytes, egress was strongly impaired (28). P21-activated kinase 2 (PAK2) is an effector for the Rac and CDC42 GTPases that regulates actin cytoskeletal remodeling. PAK2 deficiency in thymocytes led to peripheral lymphopenia and accumulation of mature phenotype CD4 and CD8 SP thymocytes. PAK2-deficient thymocytes showed reduced KLF2, S1P1, and CD62L expression (29), suggesting that PAK2 is required for signals that turn on KLF2. Foxo1 signaling is required for the optimal upregulation of KLF2 (30, 31), although it is not known whether Foxo1 and PAK2 act in the same signaling pathway. Another molecule, Paxip1, which is involved in histone methylation and double-strand break repair, also was shown to be required for S1P1 expression, but this was independent of Foxo1 and KLF2, leading the investigators to speculate that genomic integrity is a prerequisite of thymic egress (32).
Thymocytes egress via blood vessels and not lymphatics (33), although it is unclear precisely how transmigration is achieved. CD4 and CD8 SP thymocytes are presumed to emigrate via the same mechanism, because the gene deficiencies discussed above influence both lineages similarly. However, once in circulation, CD8 and CD4 T cells may take different routes to tissues. Notably, CD8 RTEs were shown to traffic more efficiently to the gut (34).
Licensing
As adolescent SP thymocytes mature and prepare to leave home, they become licensed to produce cytokines, including TNF-α, IL-2, IL-4, and IFN-γ. More than 25 y ago, Boyer and Rothenberg (35) demonstrated that DP thymocytes are unable to express IL-2Rα after activation, in contrast to mature T cells. Mature SP thymocytes produce IL-2 and upregulate IL-2Rα after TCR activation, but SM cells neither proliferate nor upregulate IL-2Rα (4), similar to DP thymocytes. However, after thymic egress as an RTE, these adolescent T cells still drive on a “junior license,” with restricted ability to produce cytokines compared with their “senior” long-lived mature naive counterparts. RTEs produce less IL-2, IFN-γ, and IL-4 under neutral/nonpolarizing conditions compared with mature naive T cells (MNTs) (9, 36). Similarly, there is minimal production of TNF-α by SP thymocytes, whereas MNTs produce substantial quantities of TNF-α, and RTEs have an intermediate ability (37). RTEs that have a postthymic maturation block, such as NKAP-deficient RTEs, produce even less IL-2 (38) and TNF-α (F.-C. Hsu and V.S. Shapiro, unpublished observations) than do wild-type (WT) RTEs. The relatively poor ability to produce cytokines upon stimulation is not due to decreased TCR signaling, because ZAP-70 and Erk are phosphorylated to greater extents in CD8+ RTEs than in MNTs in response to low-affinity TCR ligands, although they produce less cytokines (39). However, changes in transcription of cytokine loci during maturation correlate with epigenetic changes (36). The competence to express IL-2 is linked to epigenetic marking of the IL2 gene at a distal cis-regulatory region (40). Dimethylation of histone H3 Lysine 4 was fully elevated across the distal cis-regulatory region of the IL2 gene in mature T cells before stimulation but was not detected in DP thymocytes (40). The IL-2 and IL-4 promoters in CD4+ RTEs are hypermethylated compared with CD4+ MNTs. Reversing the hypermethylation by 5-azacytidine increased the ability of RTEs to make IL-2 and IL-4 to a level closer to that of MNTs, demonstrating the critical role of DNA methylation in regulating cytokine production in RTEs (36). Hendricks and Fink (41) demonstrated that RTEs express higher levels of the DNA methyltransferases DMNT1, DMNT3, and TET1 compared with MNTs, which indicates that this may be the mechanism responsible for increased DNA methylation and decreased cytokine production in RTEs. Histone acetylation also has a role in cytokine production during maturation, because RTEs and MNTs from CD4-Cre HDAC3 conditional knockout (cKO) mice fail to produce TNF-α upon CD3/CD28 stimulation (42). Thus, epigenetic changes at cytokine promoters first occur during thymocyte maturation and continue to improve after emigration to the periphery but are not completed until entry into the long-lived MNT pool.
Interestingly, the inability of RTEs to produce Th1 cytokines, in particular IFN-γ, upon Ag stimulation may promote peripheral tolerance (43). CD4+ RTEs convert to the induced regulatory T cell (iTreg) lineage in the periphery more rapidly than do CD4+ MNTs, especially in GALT, after cells are adoptively transferred into recipient mice or in an oral tolerance model (43, 44). In vitro, (Foxp3−) CD4+ SP thymocytes and RTEs preferentially convert to Foxp3+ iTregs compared with CD4+ MNTs in both mouse and human (44, 45). The increased sensitivity to all-trans retinoic acid is due to the increased expression of RARα and RARβ in RTEs. In addition, the relatively lower IFN-γ production by RTEs contributes to enhanced iTreg generation. Exogenous IFN-γ inhibited the ability of CD4+ RTEs to become iTregs, and inhibiting IFN-γ with blocking Abs enhanced the ability of CD4+ MNTs to convert to iTregs (43). Thus, differences in sensitivity to all-trans retinoic acid and in the level of inflammatory cytokine production by RTEs compared with MNTs have a role in peripheral tolerance by influencing the size of the iTreg pool.
Survival in the periphery
In the periphery, T cells depend on IL-7 stimulation for Bcl-2 expression and continued survival. IL-7Rα is not expressed in DP thymocytes but is turned on after positive selection at the SP stage, and its expression continues to increase with maturation as RTEs transit into MNTs. Decreased expression or function of IL-7 or IL-7Rα leads to a defect in peripheral T cell homeostasis. T cells deficient in Foxo1, IKK2, Bptf, or ZFAT have decreased expression of IL-7Rα on the cell surface, which contributes to a defect in T cell maintenance in the periphery (30, 46–49). There are consensus Foxo1 and NF-κB binding sites in the IL-7Rα promoter, but the mechanisms behind IL-7Rα regulation by Bptf and ZFAT are not known. T cells from CD4-Cre TAK1 cKO mice have normal levels of IL-7Rα on the cell surface but decreased IL-7 signaling (12). T cells that lack ZFAT have normal levels of Bcl-2 expression; however, its expression does not increase in response to IL-7Rα stimulation (48). As IL-7Rα levels continue to rise during peripheral T cell maturation, decreased expression of IL-7Rα may indicate a block in maturation in general and not simply a specific effect on IL-7Rα expression. The decreases in peripheral T cell cellularity in Zfp335 mutant mice or mice with conditional deletion of Bptf or NKAP could not be rescued by a Bcl-2 transgene, demonstrating that additional mechanisms must regulate T cell persistence and survival in the periphery during maturation.
CD4-Cre NKAP cKO mice have a block in peripheral T cell maturation, and the naive T cell pool is composed almost entirely of functionally and phenotypically immature RTEs. The disappearance of naive NKAP-deficient T cells in the periphery is not due to defective IL-7 signaling, and expression of either Bcl-2 or Bcl-xl transgenes did not restore T cell cellularity in CD4-Cre NKAP cKO mice, indicating that apoptosis was unlikely to be responsible (38, 50). Instead, NKAP-deficient T cells are targeted by the classical arm of the complement pathway and, thus, are eliminated in the periphery (50). NKAP-deficient RTEs and MNTs exhibit deposition of C3, C4, C1q, and IgM on the cell surface. As SP thymocytes prepare for egress into the bloodstream, they upregulate the incorporation of sialic acid into cell surface glycans (Table I). Stripping peripheral lymphocytes of sialic acid by neuraminidase induces binding of natural IgM and complement deposition. NKAP-deficient T cells fail to incorporate α2,8-linked sialic acids into cell surface glycans, which may be responsible for the IgM binding and activation of the classical complement pathway. In addition, expression of the complement inhibitor CD55 (decay accelerating factor) is normally induced upon maturation but is not in the absence of NKAP. It is not known whether alterations in sialylation or complement activation are present in other mice, such as Bptf or Zfp335 knockouts (KOs), which have defects in maturation or peripheral T cell numbers that cannot be rescued by a Bcl-2 transgene. However, conditional deletion of HDAC3, which is a binding partner of NKAP, causes a similar block in postthymic maturation, leading to targeting by complement (42). Thus, during maturation, T cells must be able to respond to IL-7 for survival and avoid elimination by complement to persist in the periphery and enter into the long-lived T cell pool.
Recent studies showed that reactive oxygen species play a role as signaling molecules in regulating T cell activation and expansion. Glutathione peroxidase 4 (Gpx4) is one of the antioxidant enzymes that directly reduces phospholipid hydroperoxides and oxidized lipoproteins. Matsushita et al. (51) reported that mice in which Gpx4 is conditionally deleted by CD4-Cre have normal thymic development but a significantly reduced number of peripheral CD8+ T cells. Gpx4-deficient CD4+ and CD8+ T cells displayed a substantial disadvantage in homeostatic expansion in the presence of competing WT cells. Gpx4-deficient T cells also accumulate lipid peroxidates and die more rapidly upon TCR stimulation or under neutral condition compared with WT cells. The cell death of Gpx4-deficient cells could not be attributed to autophagy, necroptosis, or apoptosis. However, it could be rescued by inhibition of lipoxygenase and ferroptosis, suggesting that there is an entirely new mechanism that allows for survival of T cells in the periphery during maturation.
Conclusions
T cell maturation is as important as positive selection for the generation of a diverse peripheral naive T cell pool; however, it has been poorly understood, at least in part, because of the lack of genetic models with specific blocks at this stage of T cell development. Work in the last few years discovered the role that many proteins (Table II) play at specific points in the maturation process as particular competencies are gained: proliferation in response to CD3/CD28, protection from TCR and death receptor signals, thymic egress, cytokine production/TNF licensing, and peripheral survival. Although we are gaining insight into the process, what is not currently known are the signals that drive T cell maturation. For example, it remains unknown what receptor(s) activate TAK1 in medullary SP thymocytes. RTE entry into secondary lymphoid organs is required for later maturational events; however, it does not appear to be dependent on either TCR or IL-7 signals (although IL-7 is critical for peripheral T cell survival, it cannot induce other competencies during maturation) (23, 52). In addition, it is not clear whether maturation is the same in CD4 T cells as in CD8 T cells or whether it is the same in regulatory T cells as in conventional T cells. However, with recent advances into the pathways and molecules required, additional insight into the receptors that signal, as well as the cells that induce, T cell maturation should be gained. In doing so, the process by which these young T cell adolescents mature into adult participants in immune system society will be revealed.
Acknowledgements
We thank Michael Shapiro and Barsha Dash for critical reading of the manuscript.
Footnotes
This work was supported by National Institutes of Health Grants R37 AI039560 and R01 AI088209 (to K.A.H.) and R01 AI083279 (to V.S.S.).
Abbreviations used in this article:
- AgR
Ag receptor
- cKO
conditional knockout
- DP
double positive
- Gpx4
glutathione peroxidase 4
- iTreg
induced regulatory T cell
- KLF2
Kruppel-like factor 2
- KO
knockout
- M1
mature 1
- M2
mature 2
- MNT
mature naive T cell
- Mst
mammalian sterile twenty-like
- PAK2
P21-activated kinase 2
- RTE
recent thymic emigrant
- SM
semimature
- SP
single positive
- TAK1
TGF-β–activated kinase 1
- WT
wild-type.
References
Disclosures
The authors have no financial conflicts of interest.