TcR-alpha/beta(+) CD4(-)CD8(-) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor alpha/beta(+) CD4(-)CD8(-) T cells (alpha/beta(+) double-negative T cells [alpha/beta(+)-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The alpha/beta(+)-DNT cells are immunophenotypically and functionally similar to alpha/beta(+)-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, alpha/beta(+)-DNT cells express the B-cell-specific CD45R isoform B220. We show that alpha/beta(+)-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4(+) T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell-cell interactions, and access to alternative apoptosis pathways.     

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.     

Proliferation of CD3+ B220- single-positive normal T cells was suppressed in B-cell-deficient lpr mice.

It is known that lpr mice develop systemic lymphadenopathy and lupus erythematosus-like autoimmune disease that are associated with the accumulation of CD4- CD8- (double-negative; DN) CD3+ B220+ abnormal T cells as well as normal mature CD4+ or CD8+ single-positive (SP) CD3+ T cells. In order to clarify the role of B cells in the lymphoproliferation and autoimmunity of lpr mice, we created B-cell-deficient C57BL/6 (B6) lpr mice (B6lpr/lpr microMT/microMT) by crossing B6lpr/lpr mice with B6 microMT/microMT mice in which the B-cell development was arrested at pre-B stage owing to a targeted disruption of the immunoglobulin mu heavy-chain gene locus. In the B-cell-deficient B6-lpr mice, both lymphadenopathy and splenomegaly were markedly suppressed. Although the accumulation of both CD3+ B220- SP normal T cells and CD3+ B220+ DN abnormal T cells was inhibited in the B-cell-deficient lpr mice, the decrease in numbers of CD3+ B220- SP normal T cells occurred more strikingly than that of the CD3+ B220+ DN abnormal T cells. Glomerulonephritis did not develop in the B-cell-deficient lpr mice over 40 weeks. The present results indicate that the B cells thus play a crucial role in the extensive proliferation of normal CD3+ B220- mature SP T cells rather than the accumulation of abnormal DN T cells.     

Intestinal intraepithelial lymphocyte T cells are resistant to lpr gene-induced T cell abnormalities.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.     

CD1d deficiency exacerbates inflammatory dermatitis in MRL-lpr/lpr mice

Mechanisms responsible for the development of autoimmune skin disease in humans and animal models with lupus remain poorly understood. In this study, we have investigated the role of CD1d, an antigen-presenting molecule known to activate natural killer T cells, in the development of inflammatory dermatitis in lupus-susceptible MRL-lpr/lpr mice. In particular, we have established MRL-lpr/lpr mice carrying a germ-line deletion of the CD1d genes. We demonstrate that CD1d-deficient MRL-lpr/lpr mice, as compared with wild-type littermates, have more frequent and more severe skin disease, with increased local infiltration with mast cells, lymphocytes and dendritic cells, including Langerhans cells. CD1d-deficient MRL-lpr/lpr mice had increased prevalence of CD4(+) T cells in the spleen and liver and of TCR alpha beta (+)B220(+) cells in lymph nodes. Furthermore, CD1d deficiency was associated with decreased T cell production of type 2 cytokines and increased or unchanged type 1 cytokines. These findings indicate a regulatory role of CD1d in inflammatory dermatitis. Understanding the mechanisms by which CD1d deficiency results in splenic T cell expansion and cytokine alterations, with increased dermal infiltration of dendritic cells and lymphocytes in MRL-lpr/lpr mice, will have implications for the pathogenesis of inflammatory skin diseases.     

Homeostatic regulation of CD8+ T cells after antigen challenge in the absence of Fas (CD95)

The role of Fas in the homeostatic regulation of CD8⁺ T cells after antigen challenge was analyzed in the murine model of lymphocytic choriomeningitis virus (LCMV) infection. Mice homozygous for the lpr mutation and carrying T cell receptor (TCR) αβ transgenes specific for the LCMV glycoprotein peptide aa 33–41 in the context of H-2D^b were used. Five main results emerged: first, development of lymphadenopathy and of CD4⁻CD8⁻ double-negative B220⁺ T cells in lpr mice was not inhibited by the αβ TCR transgenes; second, tolerance induction and peripheral deletion of CD8⁺ T cells induced by LCMV glycoprotein peptide injection was independent of Fas expression; third, clonal down-regulation of Fas-deficient TCR-transgenic CD8⁺ T cells after acute LCM virus infection was identical to the decline of transgenic T cells expressing Fas; fourth, in vivo activated CD8⁺ effector T cells from TCR transgenic and TCR-lpr/lpr mice were equally susceptible to activation-induced cell death in vitro; and fifth, transgenic effector T cells from lpr/lpr mice were cleared in the declining phase of the immune response in vivo without giving rise to CD4⁻CD8⁻ double-negative T cells. Taken together, these data suggest that the homeostatic regulation of CD8⁺ T cells after antigen challenge in vivo is regulated by mechanisms that do not require Fas.     

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL-lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double-negative (DN) CD4- CD8- alpha beta T cells expressing a low density of interleukin-2 receptor beta-chain (IL-2R beta). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T-cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL-2R beta. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL-2R beta. A cell-sorting experiment for purified fractions demonstrated that only CD8- cells reconstituted TCRint cells in scid mice. Namely, DN CD4- CD8- cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.     

Effects of the lpr/lpr mutation on T and B cell populations in the lamina propria of the small intestine, a mucosal effector site.

MRL mice, which develop a lymphoproliferative disease characterized by increased numbers of alpha/beta T cell receptor+ (TCR+) B220/6B2+CD4-CD8- T cells [lymphoproliferation (lpr) T cells], were studied for the effect of the lpr/lpr mutation on the mucosal immune system in the gastrointestinal (GI) tract. We analyzed the effect of the lpr gene mutation on T and B cell populations in the Peyer's patches (PP) and the lamina propria lymphocytes (LPLs), as examples of major IgA inductive and effector tissues in the GI tract respectively. Normal mouse PP contain B cells committed to IgA (surface IgA+) but only low numbers of B cells producing IgA. However, enhanced spontaneous IgA and IgG synthesis occurs in the PP of MRL mice. Further, we have now shown that PP of MRL mice are populated by lpr T cells. Interestingly, lpr T cells were not present in significant numbers in LPLs of MRL mice, even in older animals. Of interest was the finding that the ratio of CD4+ to CD8+ T cells in the lamina propria was lower in MRL when compared with control mice, and the CD8+ T cell subset actually predominates in LPLs of autoimmune mice. In addition, the number of gamma/delta TCR+ T cells in LPL of MRL lpr/lpr mice was significantly increased, especially in MRL lpr/lpr mice at 6 and 12 weeks of age. When the isotype distribution of B cells in LPLs was analyzed, no changes were noted in MRL lpr/lpr mice in comparison with MRL +/+ or normal control mice, and the pattern was IgA much greater than IgM greater than IgG. These results show that although increased numbers of CD8+ T cells and gamma/delta TCR+ cells occur in the LPLs of MRL mice, a normal distribution of plasma cell isotypes (IgA much greater than IgM greater than IgG) is found in this mucosal compartment. Further, Ipr T cells do not develop in the lamina propria compartment of the GI tract.     

Role for T cell-independent B cell activity in the resolution of primary rotavirus infection in mice.

We examined the importance of T cell-independent B cell activity in the resolution of primary murine (EDIM) rotavirus infection in adult mice. We showed that Rag 1 (C57BL / 6 background) and Rag 2 (BALB / c background) knockout mice, which lack both T and B cells, chronically shed high levels of rotavirus Ag in stool samples following oral inoculation. However, nude mice (BALB / c and C57BL / 6 backgrounds) and alpha beta TCR knockout mice (C57BL / 6 background) chronically shed 100-fold lower levels of virus in stool samples. Thus, B cells appeared to sharply reduce the level of chronic rotavirus shedding by a T cell-independent mechanism. C57BL / 6 mice depleted of CD4(+) cells or both CD4(+) and CD8(+) cells were also unable to resolve primary rotavirus infection but chronically shed equally low levels of rotavirus Ag in stool samples, whereas mice depleted of only CD8(+) cells resolved infection. Similar results were obtained with a second rotavirus strain (EC(w)) in which virus was shed chronically in stool samples at low levels in alpha beta TCR knockout mice and at high levels in Rag 1 knockout mice. Virus-specific intestinal IgA was readily detected in mice lacking thymic T cells and alpha beta T cells and in mice depleted of CD4(+) cells but levels were 95 % reduced in comparison to immunocompetent control mice. Together, these results show that B cells lacking CD4(+) T cell help have the capacity to substantially reduce rotavirus shedding, possibly through the production of T cell-independent IgA to rotavirus, but full resolution requires alpha beta T cells.     

Apoptosis regulators Fas and Bim synergistically control T-lymphocyte homeostatic proliferation

The size of the peripheral T-lymphocyte compartment is governed by complex homeostatic mechanisms that balance T-cell proliferation and death. Proliferation and survival signals are mediated in part by recurrent self-peptide/MHC-TCR interactions and signaling by the common γ chain-containing cytokine receptors, including those for IL-7 and IL-15. We have previously shown that the death receptor Fas (CD95/APO-1) regulates apoptosis in response to repeated TCR stimulation, whereas the Bcl-2 homology domain 3-only protein Bim mediates cytokine withdrawal-induced apoptosis. We therefore reasoned that these two molecules might cooperate in the regulation of homeostatic proliferation. In this study, we observe that the combined loss of Fas and Bim synergistically enhances the accumulation of T cells in lymphopenic host mice, and this is particularly pronounced for the unusual CD4(-) CD8(-) TCRαβ(+) T cells that are characteristic of Fas-deficient (Fas(lpr/lpr) ) mice. Our findings demonstrate that these CD4(-) CD8(-) TCRαβ(+) T cells arise from homeostatic proliferation of CD8(+) T cells. These studies also underscore the profound rate of baseline T-cell proliferation that likely occurs in wild-type mice even in the absence of foreign antigen, and the consequent need for its coordinated regulation by multiple death-signaling pathways.     

A new T cell subset expressing B220 and CD4 in lpr mice: defects in the response to mitogens and in the production of IL-2.

Autoimmune-prone mice homozygous for the lpr gene develop prominent lymphadenopathy composed mainly of Thy-1+ CD8- CD4- B220+ cells. Expression patterns of B220 vs CD4 on lymph node cells from lpr mice were analysed using two-colour flow microfluorometry. B220+CD4+ cells, which were hardly seen in lymph nodes of B6-+/+ mice, increased significantly in B6-lpr mice with ageing. Functional analysis of purified B220+ CD4+ cells from lpr mice revealed that these cells scarcely responded to T cell mitogens with or without rIL-2. Furthermore, B220+ CD4+ cells were defective in IL-2 production when cultured with Con A. On the other hand, B220-CD4+ cells from B6-lpr mice showed an ability to respond to T cell mitogens similar to that of B220- CD4+ cells from B6-+/+ mice. These results indicate that an unusual T cell subset expressing both B220 and CD4 in lpr mice is functionally defective, but the intrinsic ability of B220-CD4+ cells is almost intact as compared with the counterpart in normal mice.     

Characterization of intermediate T-cell receptor cells expanding in the liver, thymus and other organs in autoimmune lpr mice: parallel analysis with their normal counterparts.

Autoimmune MRL-lpr/lpr (lpr) mice were previously demonstrated to have an abnormal proliferation of intermediate T-cell receptor (TCR) cells of extrathymic origin in the liver. Despite this situation, thymectomy in lpr mice resulted in amelioration of autoimmune disease. To understand the underlying mechanism, we investigated associated T-cell differentiation in the thymus and other organs of these mice. When the disease was evoked, T cells with extrathymic properties, i.e. intermediate TCR-alpha beta cells expressing double-negative (DN) CD4-8- phenotype and interleukin-2 (IL-2) receptor beta-chain, became prominent not only in the liver, but also in the thymus. Such thymic T cells mainly resided in the medulla. A small-scale localization of such T cells was seen in the thymic medulla even in normal control mice. There was a heterogeneity among intermediate TCR cells in terms of the composition of DN cells and the expression of CD2 and B220 antigens, depending on the organs and the sites in the same organ. Intermediate TCR cells in the liver, thymus and autoimmune target organs (e.g. kidney) contained a high proportion of the active form (CD2+B220-), while intermediate TCR cells accumulating in peripheral organs, the spleen and lymph nodes, were mainly of the inactive form (CD2-B220+). The active form had an ability to proliferate in response to IL-2 and SEB, whereas the inactive form did not. The present results suggest that the proliferation of intermediate TCR cells occur at multiple sites; this may explain the effect of thymectomy, namely, the retarded onset of disease, in lpr mice.     

Impact of early expression of TCR alpha chain on thymocyte development

CD4(-)CD8(-) thymocytes expressing a transgenic T cell receptor (TCR) alpha chain have decreased capacity to give rise to CD4(+)CD8(+) thymocytes when compared with wild-type thymocytes. This inefficient CD4(-)CD8(-) to CD4(+)CD8(+) maturation is mediated by the transgenic TCR alpha chain pairing with endogenous TCR beta chain but not with endogenous TCR gamma chain. Comparison between TCR alpha chain-transgenic mice with or without a functional pre-TCR alpha (pT alpha ) chain reveals that the formation of transgenic alpha/endogenous beta TCR on CD4(-)CD8(-) thymocytes inhibits the formation of pre-TCR, but at the same time mediates CD4(-)CD8(-) to CD4(+)CD8(+) maturation in the absence of pre-TCR, albeit inefficiently. These results indicate that alpha beta TCR and pre-TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR beta and alpha loci and the cellular expansion induced by the pre-TCR may both be evolved to ensure the efficient generation of mature alpha beta T cells.     

CD45 congenic bone marrow transplantation: evidence for T cell-mediated immunity

CD45 congenic mice have been used to study stem cell engraftment in the absence of alloreactivity. Recently, impaired engraftment was reported in this model and attributed to weak immune reactivity. We have confirmed that there is indeed low-level reactivity mediated by CD8(+) cells and alpha beta-TCR(+) T cells. B6 (CD45.2) recipients were conditioned with total body irradiation (TBI) and transplanted with increasing doses of B6 (CD45.1) bone marrow cells (BMCs). Although chimerism was present at 1 month in all recipients, durable engraftment did not occur with <150 cGy of TBI, emphasizing the importance of long-term follow-up in evaluating nonmyeloablative conditioning approaches. A single dose of cyclophosphamide on day 2 also significantly enhanced engraftment. When B6 TCR beta(-/-), TCR delta(-/-), or TCR beta(-/-)/delta(-/-) (CD45.2) mice were transplanted with CD45.1 bone marrow, significantly enhanced engraftment occurred in recipients lacking alpha beta-TCR(+) T cells (p < .00005). Similarly, removal of alpha beta-TCR(+) host T cells in wild-type recipients resulted in enhanced engraftment. Engraftment was also significantly increased in CD8(-/-) and CD4(-/-)/8(-/-) recipients (p < .0005). Taken together, these results demonstrate that alpha beta-TCR(+) and CD8(+) T cells play a critical role in regulating engraftment of CD45 congenic marrow and suggest that these cells are the main effector cells in low-level alloreactivity to the CD45 disparity.     

Subpopulations of CD4+ cells in lpr/lpr mice: differences in expression of T cell receptor/CD3 complex and proliferative responses.

CD4+ cells from autoimmune-prone C57BL/6 lpr/lpr mice contain two subpopulations, B220-CD4+ and B220+CD4+ cells. Highly purified B220-CD4+ cells from C57BL/6 +/+ and lpr/lpr mice were examined by comparing functional characteristics and expression of cell surface antigens and T cell receptor (TcR)/CD3 complex. Both lpr B220+CD4+ and B220+CD4-CD8- cells, most of which were PgP-1 positive, expressed TcR/CD3 complex on the cell surface at lower level as compared with B220-CD4+ cells of age-matched normal mice. In addition, the B2200-CD4+ cells were heterogeneous on the basis of surface expression of PgP-1 and CD3 antigens. Normal levels of TcR C alpha-, C beta- and V beta 8-specific mRNA were found in the B220-CD4+ cells and B220+CD4+ cells as compared with normal B220-CD4+ cells, while V beta 8-specific mRNA was preferentially expressed only by B220+CD4-CD8- cells. Either B220+CD4+ cells and B220+CD4-CD8- cells failed to respond to anti-CD3 monoclonal antibody (MoAb) as assessed by proliferative responses and production of interleukin-2 (IL-2). However, appreciable levels of reactivity to anti-CD3 MoAb were detected in the B220-CD4+ cells, although the responsiveness of this subset to such stimuli were reduced, compared with those of normal control. These results indicate that the B220-CD4+ cells in lpr mice are phenotypically and functionally distinct from normal B220-CD4+ cells.     

Influence of the lpr environment on the lymph node cell phenotypes in C57BL/6 nubg and nulpr chimeras.

Mice homozygous for the lpr gene show a marked lymphoproliferative syndrome. Most T cells which accumulate in their lymphoid organs belong to a fairly unusual subpopulation. Although being CD44+ T cells expressing neither CD4 nor CD8, they are CD3 T-cell receptor (TCR) alpha beta positive and express both Thy-1 and B220, the B-cell form of the CD45 marker. To support engraftment and development of transferred lpr lymphomyeloid cells, athymic recipients must be genetically lpr. While nude/beige (nubg) recipients do not allow the development of any lymphoproliferative syndrome, this is variable in nude/lpr (nulpr) recipients, and the genotypic origin of the proliferating lymphocytes in nulpr recipients is unclear. In this study, the surface phenotype of lymph node cells from nulpr recipients of lpr grafts ([lpr-->nulpr] chimeras) was analysed by flow cytometry, and compared with various chimeras and parental (donor and recipient) strains as controls. Abnormal cells of the lpr type were not detectable either in [lpr-->nubg] chimeras or in [wild-->nubg] controls. Absence of lpr cells was also seen in neonatal lpr thymus-grafted nubg mice engrafted previously with lpr haematopoietic cells. In contrast, a substantial emergence of double-positive B220+ Thy-1+ cells occurred in [lpr-->nulpr] chimeras, together with high levels of CD4+ cells, a substantial fraction of which might express B220. Finally, in thymus-grafted nulpr mice, the levels of B220+ Thy-1+ cells were as high as in lpr mice and there was again an expansion of CD4+ (potentially B220+) cells. Abnormality of the nulpr haemopoietic environment was also shown by the low percentages of T cells, particularly CD8+ cells, in short-lived [wild-->nulpr] chimeras. Taken together, our results underline the differences between the nubg and nulpr environments.     

Expression and selection of productively rearranged TCR beta VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1(+) alpha beta T cells

The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta VDJ rearrangements and pre-T alpha mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3 zeta/Lck double-deficient mice fail to express TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCR beta VDJ genes but before expression of TCR beta proteins. Most NKT cells of TCR alpha-deficient mice exhibit cell surface gamma delta TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.     

Role of endogenous 4-1BB in the development of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies directed against nuclear antigens including nucleosomes and DNA. To determine the role of T-cell costimulatory molecule 4-1BB in the regulation of SLE, MRL-Fas(lpr) (lpr) mice deficient in 4-1BB (lpr/4-1BB(-/-)) were generated and their disease phenotype was compared to that of control lpr mice. The main finding of this study is that the lpr/4-1BB(-/-) mice had more pronounced skin lesions which appeared earlier, increased lymphadenopathy, increased renal damage, and higher mortality than 4-1BB-intact control lpr mice. The increased severity of lesions in lpr/4-1BB(-/-) mice was closely associated with increases in CD4(+) T, CD3(+) B220(+) double-negative T cells, serum immunoglobulin, anti-dsDNA autoantibodies, and tissue immunoglobulin deposits. These data suggest that the 4-1BB-4-1BB ligand signalling pathway plays an important role in SLE and that deletion of 4-1BB confers susceptibility to lpr mice, leading to accelerated induction of disease and early mortality.     

Marked increase in number of dendritic cells in autoimmune-prone (NZW x BXSB)F1 mice with age.

Here, we report that the number of CD11c(+)CD3(-) B220(-) cells increases in autoimmune-prone male (NZW x BXSB)F1 (W/BF1) mice with age. The CD11c(+)CD3(-)B220(-) cells from W/BF1 mice show a typical stellate shape and induce the proliferation of T cells. In the CD11c(+)CD3(-)B220(-) cells from W/BF1 mice, CD11b (Mac-1alpha), NK 1.1, and CD95 (Fas) are upregulated in comparison with normal mice, while the expression of CD8alpha, CD117 (c-kit), CD135 (Flk-2/Flt-3), and Sca-1 decreases. There is a significant increase in Flt-3L (FL) mRNA in the bone marrow of W/BF1 mice with age. Moreover, activated hemopoietic cells express high levels of FL. The injection of CD11c(+)CD3(-)B220(-) cells from old W/BF1 mice to young W/BF1 mice transiently induces autoimmune disease (thrombocytopenia). These results suggest that hyperproduction of FL from activated hemopoietic cells induces a dramatic increase in the number of dendritic cells in aged W/BF1 mice, followed by the acceleration of autoimmunity.