Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

NZB mice exhibit a primary T cell defect in fetal thymic organ culture

Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.     

Altered T cell differentiation and Notch signaling induced by the ectopic expression of keratin K10 in the epithelial cells of the thymus

Transgenic mice expressing hK10 under the keratin K5 promoter display several alterations in the epidermis including decreased cell proliferation, and reduced susceptibility to tumor development. Given that K5 promoter is also active in the epithelial cells of the thymus, we explored the possible alterations of the thymus because of K10 transgene expression. We found severe thymic alterations, which affect not only the thymic epithelial cells (TEC), but also thymocytes. We observed altered architecture and premature thymus involution in the transgenic mice associated with increased apoptosis and reduced proliferation of the thymocytes. Interestingly, prior to the development of this detrimental phenotype, thymocytes of the transgenic mice also displayed altered differentiation, which is aggravated later on. Molecular characterization of this phenotype indicated that Akt activity is reduced in TEC, but not in thymocytes. In addition, we also observed altered expression of Notch family members and some of their ligands both in TEC and T cells. This produces reduced Notch activity in TEC but increased Notch activity in thymocytes, which is detectable prior to the disruption of the thymic architecture. In addition, we also detect altered Notch expression in the epidermis of bK5hK10 transgenic mice. Collectively the present data indicate that keratin K10 may induce severe alterations not only in a cell autonomous manner, but also in neighboring cells by the modulation of signals involved in cell-cell interactions.     

IL-12 inhibits thymic involution by enhancing IL-7- and IL-2-induced thymocyte proliferation

IL-12 has been reported to affect thymic T cell selection, but the role of IL-12 in thymic involution has not been studied. We found that in vivo, IL-12b knockout (IL-12b(-/-)) mice exhibited accelerated thymic involution compared with wild-type (WT) B6 mice. This is characterized by an increase in thymocytes with the early development stage phenotype of CD25(-)CD44(+)CD4(-)CD8(-) in aged IL-12b(-/-) mice. Histologically, there were accelerated degeneration of thymic extracellular matrix and blood vessels, a significantly decreased thymic cortex/medulla ratio, and increased apoptotic cells in aged IL-12b(-/-) mice compared with WT mice. There was, however, no apparent defect in thymic structure and thymocyte development in young IL-12(-/-) mice. These results suggest the importance of IL-12 in maintaining thymic integrity and function during the aging process. Surprisingly, in WT B6 mice, there was no age-related decrease in the levels of IL-12 produced from thymic dendritic cells. Stimulation of thymocytes with IL-12 alone also did not enhance the thymocyte proliferative response in vitro. IL-12, however, provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in aged WT and IL-12b(-/-) mice. Our data strongly support the role of IL-12 as an enhancement cytokine, which acts through its interactions with other cytokines to maintain thymic T cell function and development during aging.     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Impaired development of T lymphoid precursors from pluripotent hematopoietic stem cells in New Zealand Black mice.

Bone marrow cells from autoimmune-prone New Zealand Black (NZB) mice are less efficient at colonizing fetal thymic lobes than cells from normal strains. This study demonstrates that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Rather, the T lymphopoietic defect of NZB mice is due to an impaired ability of pluripotent hematopoietic stem cells (PHSCs) to generate more committed lymphoid progeny, which could include common lymphoid precursors and/or other T cell-committed progenitors. Although PHSCs from NZB mice were not as efficient at thymic repopulation as comparable numbers of PHSCs from control strains, the ability of common lymphoid precursors from NZB mice to repopulate the thymus was not defective. Similarly, more differentiated NZB T cell precursors included in the intrathymic pool of CD4(-)CD8(-) cells also exhibited normal T lymphopoietic potential. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that generation of lymphoid progeny from the PHSCs is a regulated event.     

Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Atypical memory phenotype T cells with low homeostatic potential and impaired TCR signaling and regulatory T cell function in Foxn1Delta/Delta mutant mice

Foxn1Delta/Delta mutants have a block in thymic epithelial cell differentiation at an intermediate progenitor stage, resulting in reduced thymocyte cellularity and blocks at the double-negative and double-positive stages. Whereas naive single-positive thymocytes were reduced >500-fold in the adult Foxn1Delta/Delta thymus, peripheral T cell numbers were reduced only 10-fold. The current data shows that Foxn1Delta/Delta peripheral T cells had increased expression of activation markers and the ability to produce IL-2 and IFN-gamma. These cells acquired this profile immediately after leaving the thymus as early as the newborn stage and maintained high steady-state proliferation in vivo but decreased proliferation in response to TCR stimulation in vitro. Single-positive thymocytes and naive T cells also had constitutively low alphabetaTCR and IL7R expression. These cells also displayed reduced ability to undergo homeostatic proliferation and increased rates of apoptosis. Although the frequency of Foxp3+CD4+CD25+ T cells was normal in Foxn1Delta/Delta mutant mice, these cells failed to have suppressor function, resulting in reduced regulatory T cell activity. Recent data from our laboratory suggest that T cells in the Foxn1Delta/Delta thymus develop from atypical progenitor cells via a noncanonical pathway. Our results suggest that the phenotype of peripheral T cells in Foxn1Delta/Delta mutant mice is the result of atypical progenitor cells developing in an abnormal thymic microenvironment with a deficient TCR and IL7 signaling system.     

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.     

T cell tolerance to self antigens in New Zealand hybrid mice with lupus-like disease

We determined if self-reactive T cells are able to escape thymic tolerance in autoimmune New Zealand mice. T cells utilizing V beta 17a and V beta 11 encoded receptors have been shown to be clonally eliminated in nonautoimmune mice expressing I-E because of their potential self-reactivity. Similarly, V beta 8.1+ and V beta 6+ T cells are tolerized in the thymus of nonautoimmune mice that express Mls-1a. These T cell subsets were quantitated in the lymph nodes and spleens of (NZB x NZW)F1 and (NZB x SWR)F1 mice. In young mice from both autoimmune strains, deletion was similar to that observed in control animals matched for I-Ed and Mls-1a expression. Furthermore, older female autoimmune mice with elevated levels of IgG antinuclear antibodies and severe lupus-like renal disease did not demonstrate evidence of a global tolerance defect. We also found that the levels of residual V beta 17a+ cells in MHC-matched control F1 strains were further reduced by up to 80% in autoimmune (NZB x SWR)F1 mice. The greater in vivo elimination corresponded to an enhanced ability of NZB spleen cells, compared with other H-2d spleen cells, to stimulate V beta 17a+ hybridomas in vitro. The increased stimulation in culture could not be attributed to quantitative differences in I-E Ag expression. The results suggest that autoreactive T cells have been eliminated in these autoimmune mice by normal mechanisms of self-tolerance. Furthermore, the data demonstrate the existence of an NZB minor locus not present in other H-2d strains that influences T cell repertoire and enhances stimulation of T cells potentially reactive to self class II MHC Ag.     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Glucocorticoid resistance in thymocytes from mice expressing a T cell receptor transgene

A majority of thymocytes undergo apoptosis during differentiation due to lack of survival signals provided by T cell receptor (TCR) activation. As glucocorticoids (GC) have been suggested to be involved in this process, we have investigated the GC sensitivity in thymocytes from mice expressing a transgenic selecting TCR. We now report that immature CD4(+)CD8(+) double-positive thymocytes from these mice are comparatively more resistant to corticosterone-induced apoptosis. This is associated with reduced glucocorticoid receptor (GR) expression, increased levels of membrane CD28, increased NF-kappaB DNA binding activity, and increased binding to the CD28 response element in the interleukin-2 gene promoter. Analysis of NF-kappaB/Rel proteins from nuclear extracts demonstrated altered levels of some of these proteins. Our results suggest that TCR recognition of self major histocompatibility antigens generates intracellular signals which alter the thymocyte GC sensitivity and thereby protect them against apoptosis induced by endogenous GC.