Gads‐deficient thymocytes are blocked at the transitional single positive CD4+ stage

Positive selection of T‐cell precursors is the process by which a diverse T‐cell repertoire is established. Positive selection begins at the CD4⁺CD8⁺ double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down‐regulate CD8 to become transitional single positive (TSP) CD4⁺ thymocytes. Then, cells are selected to become either mature single positive CD4⁺ or mature single positive CD8⁺ thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads‐deficient mouse line. In Gads^+/+ mice, most TSP CD4⁺ thymocytes are TCR^hiBcl‐2^hiCD69⁺, suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads^?/? mice could readily generate TSP CD4⁺ thymocytes, many Gads^?/? TSP CD4⁺ cells were TCR^loBcl‐2^loCD69^?, suggesting that Gads^?/? cells proceeded to the TSP CD4⁺ stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4⁺ thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4⁺ stage are separable events and Gads is only required for positive selection.     

Differences in Bcl‐2 expression by T‐cell subsets alter their balance after in vivo irradiation to favor CD4+Bcl‐2hi NKT cells

Although it is well known that in vivo radiation depletes immune cells via the Bcl‐2 apoptotic pathway, a more nuanced analysis of the changes in the balance of immune‐cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T‐cell subsets changes after increasing single doses of total body irradiation (TBI) or after fractionated irradiation of the lymphoid tissues (TLI) of mice due to differences in radioresistance and Bcl‐2 expression of the NKT‐cell and non‐NKT subsets to favor CD4⁺Bcl‐2^hi NKT cells. Reduction of the Bcl‐2^lo mature T‐cell subsets was at least 100‐fold greater than that of the Bcl‐2^hi subsets. CD4⁺ NKT cells upregulated Bcl‐2 after TBI and TLI and developed a Th2 bias after TLI, whereas non‐NKT cells failed to do so. Our previous studies showed TLI protects against graft versus host disease in wild‐type, but not in NKT‐cell‐deficient mice. The present study shows that NKT cells have a protective function even after TBI, and these cells are tenfold more abundant after an equal dose of TLI. In conclusion, differential expression of Bcl‐2 contributes to the changes in T‐cell subsets and immune function after irradiation.     

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus

Thymic commitment of human FOXP 3⁺ regulatory T cells begins at the double‐positive (DP ) CD 4⁺ CD 8⁺ stage. In the current study, we show that interleukin‐2 promotes the development of FOXP 3⁺ thymocytes and enhances their survival at the DP phase. IL ‐2 increases the frequency of FOXP 3⁺ cells and promotes the Treg phenotype after TCR ‐mediated positive selection at the most mature DP stage. However, it has no effect on FOXP 3⁺ cells at the earlier maturation steps before positive selection. DP FOXP 3⁺ thymocytes are highly susceptible to cell death but IL ‐2 promotes their survival. The anti‐apoptotic protein BCL ‐2 (B Cell Lymphoma 2) is also upregulated by IL ‐2 at the most mature DP stage. In addition to IL ‐2, we identify IL ‐15 to have a significant role in the upregulating FOXP 3 and survival of Tregs at the DP phase. IL ‐7 also increases the expression of BCL ‐2 in the DP FOXP 3⁺ thymocytes. Our results indicate that common gamma chain cytokines IL ‐2, IL ‐7 and IL ‐15 promote the development of regulatory T cells at the most mature DP stage after TCR ‐mediated positive selection through suppressing cell death.     

Generation of mature T cell populations in the thymus: CD4 or CD8 down-regulation occurs at different stages of thymocyte differentiation

We have studied the differentiation and repertoire selection during the maturation of CD4⁺CD8⁺ (DP) thymocytes into CD4⁺CD8^- (CD4SP) and CD8⁺CD4^- (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)-αβ restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down-regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcR^loHSA^hi thymocytes mature into a CD4⁺CD8^lo TcR^hiHSA^hi intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSA^hi. In contrast, interaction with MHC class I induces the up-regulation of TcR, which precedes the down-regulation of CD4; DPTcR^lo generate DPTcR^hi thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down-regulation and the transition from DPTcR^hi into CD8⁺CD4^lo TcR^hiHSA^lo and CD8SPTcR^hiHSA^- T cells. Since down-regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.     

CD28 controls the development of innate‐like CD8+ T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger⁺ IL‐4⁺ NKT cells and upregulation of LFA‐1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28‐deficient mice is cell autonomous. Moreover, we show in both wild‐type C57BL/6 mice and in downstream of tyrosine kinase‐1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28‐mediated regulation of thymic IL‐4⁺ NKT cells promotes the differentiation of eomesodermin⁺ CD44^high innate‐like CD8⁺ T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT‐cell homeostasis and the size of the innate‐like CD8⁺ T‐cell pool.     

In vitro differentiation and commitment of CD4+ thymocytes to the CD4+ lineage without TCR engagement

Thymocyte positive selection is based on protection of immatureCD4/CD8 double-positive (DP) thymocytes from apoptosis and theirdifferentiation into CD4 or CD8 single-positive (SP) cells.Intracellular signals essential for positive selection appearto be induced through the TCR and some of the accessory moleculesincluding LFA-1, CD4 and CD8 upon Interaction with thymic stromalcells. The signals, however, still remain to be identified.Since physiological levels of glucocorticoids potentially induceor enhance thymocyte apoptosis even in vivo, the signals arelikely to inhibit the apoptotic effect of glucocorticoids. Wehave previously shown that proper cross-linking of TCR-CD3 withLFA-1, CD4 or CD8 inhibited glucocortlcold-lnduced thymocyteapoptosis in vitro, and that a proper combination of the calciumionophore, ionomycin and the protein kinase C (PKC) activator,phorbol 12-myrlstate 13-acetate (PMA), mimicked the inhibitoryeffect. Here we determined whether this combination of ionomycinand PMA induces differentiation of isolated DP thymocytes fromnormal and TCR transgenic mice. We found that pretreatment ofDP thymocytes with ionomycin and PMA followed by 1 day cultureof the cells without the reagents resulted in the differentiationof the cells into CD4 SP and CD4⁺ CD8^lo T cells that have mostlycommitted to the CD4 lineage. The changes in expression of otherdifferentiation markers were also in good accordance with thoseassociated with positive selection, except the final maturation.The results indicate that moderate and transient increases inintracellular Ca²⁺ level and PKC activity induce differentiationand commitment of DP thymocytes to the CD4 lineage, and suggestedthat the biochemical pathway leading to positive selection isbased on a similar mechanism.     

Aire controls the recirculation of murine Foxp3+ regulatory T‐cells back to the thymus

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4⁺ and CD8⁺ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self‐antigen availability for negative selection. Interestingly, alterations in both Foxp3⁺ Regulatory T‐cells (T‐Reg) and conventional SP thymocytes in Aire^?/? mice suggest additional, yet poorly understood, roles for Aire during intrathymic T‐cell development. To examine this, we analysed thymocytes from Aire^?/? mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post‐selection CD4⁺ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT‐cells, it plays a key role in controlling the intrathymic T‐Reg pool. Surprisingly, a decline in intrathymic T‐Reg in Aire^?/? mice maps to a reduction in mature recirculating Rag2GFP^? T‐Reg that express CCR6 and re‐enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire^?/? mice, and that CCR6 is required for T‐Reg recirculation back to the thymus. Collectively, our study re‐defines requirements for late stage intrathymic αβT‐cell development, and demonstrates that Aire controls a CCR6‐CCL20 axis that determines the developmental makeup of the intrathymic T‐Reg pool.     

Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widelyused in transplantation medicine. A major effect of CsA is inhibitionof the differentiation of immature double-positive (DP) CD4⁺CD8⁺thymocytes into mature single-positive (SP) CD4⁺CD8^– orCD4^–CD8⁺thymocytes. The mechanisms underlying the changesin CD4/CD8 expression during normal differentiation of thymocytesand the way CsA interferes with this differentiation processare still unknown. Here we show that protein kinase C (PKC)activation by phorbol 12-myristate 13-acetate (PMA) causes adecrease of both CD4 and CD8 expression at the cell surfacelevel and at the mRNA level in a CD4⁺CD8⁺ T cell line and infreshly isolated thymocytes. A PKC inhibitor, staurosporin,interferes with the differentiation from DP to SP in fetal thymusorgan culture system. These data suggest that the alternationof CD4/CD8 expression from DP to SP is dependent on PKC activation.CsA blocks this decrease of CD4/CD8 expression by PMA in vitro.Moreover, this PMA effect is also blocked by treatment withcycloheximlde. These results suggest that the reduction of CD4/CD8expression requires de novo synthesis of a protein(s) inducedin response to a signal conveyed by activated PKC. CsA may blockthe transition from DP to SP by inhibition of CD4/CD8 down-regulationinduced by PKC activation.     

c‐Jun NH2‐terminal kinase is a critical node in the death of CD4+CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy, due to the depletion of CD4⁺CD8⁺ thymocytes, is observed during infections with numerous pathogens. Several mechanisms, such as glucocorticoids and inflammatory cytokines, are known to be involved in this process; however, the roles of intracellular signaling molecules have not been investigated. In this study, the functional role of c‐Jun NH₂‐terminal kinase (JNK) during infection‐induced thymic atrophy was addressed. The levels of phosphorylated JNK in immature CD4⁺CD8⁺ thymocytes from C57BL/6 (Nramp‐deficient) and 129/SvJ (Nramp‐sufficient) mice were increased upon oral infection of mice with Salmonella enterica serovar Typhimurium (S. typhimurium). Furthermore, inhibition of JNK signaling, but not ERK or p38 MAPK, prevented the in vitro death of infected thymocytes. Importantly, the in vivo inhibition of JNK signaling with SP600125 protected C57BL/6 CD4⁺CD8⁺ thymocytes from depletion via multiple mechanisms as follows: lower intracellular ROS, inflammatory cytokines, Bax and caspase 3 activity, increase in Bcl‐xL amounts, and prevention of the loss in mitochondrial membrane potential. Notably, thymic architecture was preserved in infected mice treated with SP600125. Overall, this study identifies a novel role for JNK as a crucial regulator of the death of CD4⁺CD8⁺ thymocytes during S. typhimurium infection.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

FBXL12 regulates T‐cell differentiation in a cell‐autonomous manner

Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells including embryonic, adult tissue and cancer stem cells. The SCF^FBXL12 complex is an authentic ubiquitin ligase that targets ALDH3 for degradation. FBXL12 is essential for the differentiation of trophoblast stem cells into specific cell types in the placenta during mouse embryogenesis, but its physiological functions in adult tissues have remained unknown. We have now investigated the role of the FBXL12‐ALDH3 axis in the thymus, in which FBXL12 was most abundant among adult mouse tissues examined. During T‐cell differentiation, FBXL12 is most abundant in CD4⁺CD8⁺ (DP) cells, with its expression declining as these cells differentiate into CD4⁺CD8^? or CD4^?CD8⁺ (SP) cells. T cells of FBXL12‐null mice manifested a differentiation block at the DP–SP transition that was associated with ALDH3 accumulation in DP cells. This differentiation block was also apparent in wild‐type mouse recipients of FBXL12‐null bone marrow transplants as well as in FBXL12‐null fetal thymic organ culture, suggesting that it is a cell‐autonomous phenomenon in the thymus rather than an indirect effect of altered systemic conditions. Our results thus indicate that, in addition to its role in placental development, the FBXL12‐ALDH3 axis is required for maturation of undifferentiated thymocytes.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.