T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.     

Raf regulates positive selection

T cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen-activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v-Raf under the control of the p56^lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)-mediated signaling events as assessed by the inhibition of mitogen-induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8⁺ thymocytes in female mice doubly transgenic for DN-Raf and a class I-restricted H-Y TCR. In contrast, the differentiation of double-positive thymocytes to single-positive thymocytes was enhanced in H-YTCR transgenic mice expressing constitutively active Raf (v-Raf). Thus, Raf regulates positive selection in the thymus.     

Differential contribution of Lck and Fyn protein tyrosine kinases to intraepithelial lymphocyte development

The developmental stages and the role of protein tyrosine kinases (PTK) in the maturation of CD3⁺CD8αα⁺ intraepithelial lymphocytes (IEL) have not been extensively characterized. However, comparisons of thymic and extrathymic T cell development indicate that these processes involve some distinct signaling and selection events. We used mice deficient in Lck, Fyn, or both Lck and Fyn to analyze the role that these src-family PTK play in IEL development. In contrast to thymocyte development, we found that all IEL subsets develop in mice deficient for either kinase alone. However, lck^-/- animals exhibited reduced numbers of TcRαβ⁺CD8αα⁺ IEL, indicating that Lck is important in the development of these cells. Mice which lack both Lck and Fyn fail to generate TcRαβ⁺ IEL, suggesting that signaling through the preTcR, mediated by Lck and, to a lesser extent Fyn, is required for maturation of all TcRαβ⁺ IEL lineages. Interestingly, a small population of TcRγδ⁺CD8αα⁺ cells are apparent in lck^-/-fyn^-/- animals, demonstrating that TcRαβ⁺CD8αα⁺ and TcRγδ⁺CD8αα⁺ IEL have distinct PTK requirements for their development or expansion. CD3^?-CD8α^?CD44⁺ and CD3^?CD8αα⁺CD16/32⁺B220⁺ cells comprise the majority of IEL in both lck^-/-fyn^-/- and rag^-/- mice, while they are poorly represented in wild-type controls. Comparison of the cell surface phenotype of these putative precursor IEL in lck^-/-fyn^-/- and rag^-/- animals suggests that IEL maturation in these animals is arrested at an equivalent developmental stage. Overall, the data presented demonstrate that signals mediated by Lck or Fyn direct TcRαβ⁺CD8αα⁺ IEL maturation but are dispensable for the development of TcRγδ⁺CD8αα⁺ IEL.     

The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation

The src‐family kinases p56^lck (Lck) and p59^fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre‐TCRβ selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single‐positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR‐mediated survival signals, and yet only Lck is able to trigger TCR‐mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src‐kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.     

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity

The proximal promoter of lck directs gene expression exclusivelyin T cells. To investigate the developmental regulation of thelck proximal promoter activity and its relationship to T celllineage commitment, a green fluorescence protein (GFP) transgenic(Tg) mouse in which the GFP expression is under the controlof the proximal promoter of lck was created. In the adult GFP-Tgmice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^– thymocytes, andthe majority of CD4^–CD8⁺ and CD4^–CD8^– [double-negative(DN)] thymocytes were highly positive for GFP. Slightly lowerbut substantial levels of expression of GFP was also observedin mature splenic T cells. No GFP⁺ cells was detected in non-Tlineage subsets, including mature and immature B cells, CD5⁺B cells, and NK cells, indicating a preserved tissue specificityof the promoter. The earliest GFP⁺ cells detected were foundin the CD44⁺CD25^– DN thymocyte subpopulation. The developmentalpotential of GFP^– and GFP⁺ cells in the CD44⁺CD25^–DN fraction was examined using in vitro culture systems. Thegeneration of substantial numbers of ß and T cells aswell as NK cells was demonstrated from both GFP^– and GFP⁺cells. However, no development of B cells or dendritic cellswas detected from GFP⁺ CD44⁺CD25^– DN thymocytes. Theseresults suggest that the progenitors expressing lck proximalpromoter activity in the CD44⁺CD25^– DN thymocyte subsethave lost most of the progenitor potential for the B and dendriticcell lineage. Thus, progression of T cell lineage restrictionin the earliest thymic population can be visualized by lck proximalpromoter activity, suggesting a potential role of Lck in theT cell lineage commitment.     

Double-negative thymocyte subsets in CD3′ chain-deficient mice: Absence of HSA+CD44−CD25− cells

Double-negative (DN) thymocyte subsets were examined in mice deficient in the CD3′ chain (ζ −/−). The HSA ⁺CD44⁻CD25⁻ subset was found to be missing, and DN thymocytes seemed to differentiate directly from HSA⁺CD25⁺CD44⁻cells to double-positive (DP) cells. When fetal thymic ontogeny was examined, we found a marked difference between ζ −/− embryos and heterozygous littermates from embryonic day 17.5, in terms of CD25, CD4 and CD8 expression, and thymus size. The ζ −/− thymocytes failed to down-regulate CD25 and to expand exponentially. The cell cycle status of adult thymocyte subsets indicated that although the HSA ⁺CD25⁻CD44⁻ subset was missing, the CD25⁺ DN population contained normal numbers of cycling cells, and the CD25⁺ DP cells (which were not detectable in normal mice) contained 5–10% cells in G2/M + S. Taken together these data suggest that the CD3′ chain might have a specific role in the control of proliferation of DN thymocytes during T cell development. Our data clearly show that one can dissociate the signal for a CD25⁺ DN cell to differentiate (which occurs in the absence of CD3′), from a signal to proliferate and from loss of cell surface CD25.     

Murine CD8+ Invariant Natural Killer T Cells are Negatively Selected by CD1d Expressed on Thymic Epithelial Cells and Dendritic Cells

Background: CD1d-dependent invariant natural killer (iNKT) cells are found as either CD4 single positive (SP) or CD4/CD8 double negative (DN) cells in mice. The size of the CD8⁺ iNKT population is extremely small. It is known that CD1d expression on developing thymocytes is sufficient for iNKT development and co-receptor choice, which is driven by Th-POK expression. This study aimed to examine the factors involved in the CD4/CD8 co-receptor choice of iNKT cells in addition to Th-POK-driven silencing of CD8 expression. Methods: In this study, we compared iNKT cells of wild-type (WT) mice with those of transgenic mice in which CD1d expression is restricted to developing thymocytes by the proximal Lck (pLCK) promoter. CD8 positive iNKT cell population were analyzed by flow cytometry. Results: We found that there was a substantial population of CD8⁺ iNKT cells in the thymus and spleen of transgenic mice, and these cells are negatively selected in between Stage 2 and Stage 3 of their developmental program by the CD1d expressed on Thymic epithelial cell (TEC) and Dendritic cells in WT mice. Conclusion: We conclude that TEC expression of CD1d in the murine thymus contributed to co-receptor choice of iNKT cells, in addition to Th-POK-driven silencing of CD8. Therefore, mostly CD4 SP and DN iNKT cells are produced under normal physiological conditions in mice.     

Comparative analysis of thymic subpopulations during different modes of atrophy identifies the reactive oxygen species scavenger,N-acetyl cysteine,to increase the survival of thymocytes during infection-induced and lipopolysaccharide-induced thymic atrophy

The development of immunocompetent T cells entails a complex pathway of differentiation in the thymus. Thymic atrophy occurs with ageing and during conditions such as malnutrition, infections and cancer chemotherapy. The comparative changes in thymic subsets under different modes of thymic atrophy and the mechanisms involved are not well characterized. These aspects were investigated, using mice infected with Salmonella Typhimurium, injection with lipopolysaccharide (LPS), an inflammatory but non-infectious stimulus, etoposide (Eto), a drug used to treat some cancers, and dexamethasone (Dex), a steroid used in some inflammatory diseases. The effects on the major subpopulations of thymocytes based on multicolour flow cytometry studies were, first, the CD4⁻ CD8⁻ double-negative (DN) cells, mainly DN2–4, were reduced with infection, LPS and Eto treatment, but not with Dex. Second, the CD8⁺ CD3^lo immature single-positive cells (ISPs) were highly sensitive to infection, LPS and Eto, but not Dex. Third, treatment with LPS, Eto and Dex reduced all three subpopulations of CD4⁺ CD8⁺ double-positive (DP) thymocytes, i.e. DP1, DP2 and DP3, but the DP3 subset was relatively more resistant during infection. Fourth, both CD4⁺ and CD8⁺ single-positive (SP) thymocytes were lowered by Eto and Dex, but not during infection. Notably, LPS lowered CD4⁺ SP subsets, whereas the CD8⁺ SP subsets were relatively more resistant. Interestingly, the reactive oxygen species quencher, N-acetyl cysteine, greatly improved the survival of thymocytes, especially DNs, ISPs and DPs, during infection and LPS treatment. The implications of these observations for the development of potential thymopoietic drugs are discussed.     

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1^−/−, TCRβ^−/−, and p56^lck−/− mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRβ and p56^lck gene mutations but partially decreased in RAG-1^−/− mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRβ chain. Genetic blocks in CD8 lineage commitment (CD8^−/− and IFN regulatory factor-1^−/− mice) and positive and negative T cell selection (CD45^−/−, TCRα^−/−, and CD30^−/− mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4^−/− mice, but not MHC class II^−/− mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4^−/− block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.     

CD4− CD8− C.B-17 SCID Thymocytes Enter the CD4+ CD8+ Stage in the Presence of Neonatally Grafted T Cells

The aim of this work was to study the selection of donor T cells and their influence on thymic development in C.B-17 scid/scid (severe combined immunodeficient; SCID) mice during chronic graft-versus-host disease (GVHD). Recipient SCID mice (H-2^d), neonatally grafted with allogeneic peripheral T cells from CBA/J strain (H-2^k) of mice, only developed a mild acute GVHD, and were, at the chronic stage, devoid of pathological symptoms. Thymic cell numbers of injected mice differed from 10⁵ to 1.2 × 10⁷ at 2–3 weeks post-injection (p.i.), and from 4 × 10⁵ to 8.5 × 10⁷ at 2 months p.i. In these mice, the thymus size was correlated to the CD4^? CD8^? (double negative; DN) to CD4⁺ CD8⁺ (double positive; DP) cell ratio, where at 2 months p.i, 8 out of 16 treated SCID mice contained 5 × 10⁶ cells or more and also possessed the highest frequencies of endogenous DP cells (25–95%). In contrast to previous findings, peripheral donor T cells from allogeneic and syngeneic mice, infiltrating the host thymus, had a positive effect on the development of endogenous DP thymocytes. Furthermore, these thymocytes were developmentally blocked at the DP stage, occasionally in combination with the expression of CD25, CD44 and CD117 but in the absence of T-cell receptor (TCR) expression. Also, at this time-point, the CBA/J donor TCR Vβ repertoire was equal to that of normal CBA/J mice, but purified responding donor cells were proliferatively inhibited against H-2^d stimulators in ex vivo mixed lymphocyte cultures. In contrast, the same responders showed a pronounced proliferation against syngeneic H-2K^k stimulators, suggesting either a reversion from anergy of autoreactive CBA/J T cells or a vast expansion of multiple self-reactive T-cell clones, when parked in a milieu with a lower concentration of self-antigens.     

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.     

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck^−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3⁺ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck^−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L⁺ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.     

Metalloproteinase-dependent control of thymocyte differentiation and proliferation

The development of T cells in the thymus is dependent on interactions between thymocytes and thymic stromal cells, on stimulation by growth factors, and on the binding to and migration along extracellular matrix (ECM) components. As metalloproteinases (MP) are involved in processes such as growth factor release and ECM modelling, we assessed the effect of MP inhibitors on T-cell development using fetal thymic organ culture systems. MP inhibitors significantly reduced the numbers of CD4/CD8 double-positive (DP) and mature single-positive thymocytes generated, correlated with a reduced number of cell cycles between the double-negative (DN)3 and DP stages. The progression of early thymocyte progenitors through the DN1-4 stages of development was also severely affected, including incomplete upregulation of CD25, decreased DN3 cell numbers, reduced rearrangement of the T-cell receptor (TCR)-beta locus and expression of intracellular TCR-beta by fewer DN3 cells. When purified DN1 cells were utilized as donor cells in reaggregate thymic organ cultures, essentially no DP thymocytes were produced in the presence of MP inhibitors. The results suggest that MP inhibitors affect the differentiation of developing thymocytes before, and reduce proliferation after, pre-TCR-mediated selection.     

Unusual selection and peripheral homeostasis for immunoregulatory CD4− CD8− T cells

Immunoregulatory CD4^? CD8^? (double‐negative; DN) T cells exhibit a unique antigen‐specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T‐cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2^k MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2^b MHC haplotype. Herein, we take advantage of this well‐defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC‐selecting haplotype. By defining DN T‐cell differentiation in 3A9 TCR transgenic CD47‐deficient mice as well as in mice bearing the NOD.H2^k genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.