RAG reexpression and DNA recombination at T cell receptor loci in peripheral CD4+ T cells

Under most circumstances, allelic exclusion at the T cell receptor (TCR)beta locus is tightly regulated. Here, we describe a system in which TCRbeta allelic exclusion is overcome as a result of V(D)J recombination in peripheral CD4+ T cells. In TCRbeta chain transgenic mice, tolerogen-mediated chronic peripheral selection against cells expressing the transgene leads to surface expression of endogenous TCRbeta chains. Peripheral CD4+ T cells reexpress the recombination activating genes, RAG1 and RAG2, and contain signal end intermediates indicative of ongoing V(D)J recombination. The rescue from deletion of mature T cells expressing newly generated TCRbeta chains suggests that receptor revision plays a role in the maintenance of peripheral T cell tolerance.     

Phenotypes and invariant alpha beta TCR expression of peripheral V alpha 14+ NK T cells

A novel subset of peripheral T cells, peripheral NK T cells, is found to be a major population comprising 5% of splenic T and 40% of bone marrow T cells. The majority of peripheral NK T cells are characterized by the expression of an invariant TCR-alpha encoded by V alpha 14/J alpha 281 with a one nucleotide N region. Moreover, a specific reduction of V alpha 14+ NK T cells has been demonstrated to be tightly associated with various autoimmune diseases, indicating their decisive role in autoimmune disease development. In this study, we investigated the phenotypes of peripheral V alpha 14+ NK T cells and their TCR-beta repertoire. Peripheral V alpha 14+ NK T cells, comprise two populations, i.e., small and large sized cells, at an equal frequency, belonged to the CD4- CD8- fraction, and are heat stable antigen(bright), macrophage-1bright, B220bright, CD45RBdim, and Mel-14dim, but CD5-, distinct from thymic NK T cells. TCR-beta analysis clearly showed that peripheral V alpha 14+ NK T cells utilized two to three dominant invariant TCR-beta, such as V beta 8.2 D beta J beta 2.5/V beta 7 D beta J beta 2.1 in the spleen and liver, V beta 8.2 D beta J beta 2.5/V beta 8.3 D beta J beta 2.2/V beta 7 D beta J beta 2.6 in the bone marrow, and V beta 7 D beta J beta 2.1/V beta 3 D beta J beta 1.2 in intestinal intraepithelial lymphocytes. Judging from the unusual surface phenotypes, such as heat stable antigen, macrophage-1, B220, CD45RBdim, and Mel-14dim, which are known to be T cell activation markers, peripheral V alpha 14+ NK T cells may always be activated under physiologic conditions, resulting in the oligoclonal expansion of V alpha 14+ NK T cells with different invariant TCR-beta in different peripheral organs. The unique features of V alpha 14+ NK T cells are discussed.     

T cell development in mice lacking all T cell receptor zeta family members (Zeta, eta, and FcepsilonRIgamma)

The zeta family includes zeta, eta, and FcepsilonRIgamma (Fcgamma). Dimers of the zeta family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking zeta/eta chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a zeta family dimer can promote T cell maturation, or that in the absence of zeta/eta, Fcgamma serves as a subunit in TCR complexes. To elucidate the role of zeta family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only zeta/eta or Fcgamma. The data reveal that surface complexes that are expressed in the absence of zeta family dimers are capable of transducing signals required for alpha/beta-T cell development. Strikingly, T cells generated in both zeta/eta-/- and zeta/eta-/--Fcgamma-/- mice exhibit a memory phenotype and elaborate interferon gamma. Finally, examination of different T cell populations reveals that zeta/eta and Fcgamma have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of zeta family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.     

Exit of the pre-TCR from the ER/cis-Golgi is necessary for signaling differentiation, proliferation, and allelic exclusion in immature thymocytes

A major issue is whether surface expression of the pre-TCR is necessary for signaling the development of immature thymocytes. To address this question, we generated transgenic mice expressing a TCRbeta chain that had a strong endoplasmic reticulum (ER) retrieval signal (TCRbetaER) and that was expressed intracellularly but failed to reach the cell surface. In TCRbetaER transgenic mice, there was a failure of allelic exclusion. Also, the transgene failed to rescue the developmental defects observed in TCRbeta-null mice. In contrast, TCRbeta transgenes with a mutant ER retrieval sequence or lacking this sequence signaled efficient allelic exclusion and suppressed the TCRbeta-/- defect. These data show that exit of the pre-TCR from the ER/cis-Golgi is required for progression through the double-negative thymocyte checkpoint.     

Caring for patients with HIV disease: the experience of a generic hospice

Following the recent realization that TCR beta transgenes can severely inhibit the rearrangement of endogenous Vbeta gene segments in the absence of pre-TCR alpha (pT alpha) chains, we tested whether the pre-TCR has an essential role in TCR beta allelic exclusion under more physiological conditions by analyzing TCR rearrangement in immature thymocytes by single-cell PCR. Our results in pT alpha+ mice are consistent with an ordered model of TCR beta rearrangement beginning on one allele and continuing on the other only when the first attempt is unsuccessful. By contrast, a higher proportion of thymocytes from pT alpha-/- mice exhibited two productive TCR beta alleles. Thus, the pre-TCR-independent suppression of rearrangement by TCR beta transgenes represents a transgene artifact, whereas under physiological conditions the pre-TCR is essential for allelic exclusion.     

T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2)-producing, T cells results in impaired ZAP70/CD3-zeta association

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.     

Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis

The development of T cell-mediated autoimmune diseases hinges on the balance between effector and regulatory mechanisms. Using two transgenic mouse lines expressing identical myelin basic protein (MBP)-specific T cell receptor (TCR) genes, we have previously shown that mice bearing exclusively MBP-specific T cells (designated T/R-) spontaneously develop experimental autoimmune encephalomyelitis (EAE), whereas mice bearing MBP-specific T cells as well as other lymphocytes (designated T/R+) did not. Here we demonstrate that T/R- mice can be protected from EAE by the early transfer of total splenocytes or purified CD4(+) T cells from normal donors. Moreover, whereas T/R+ mice crossed with B cell-deficient, gamma/delta T cell-deficient, or major histocompatibility complex class I-deficient mice did not develop EAE spontaneously, T/R+ mice crossed with TCR-alpha and -beta knockout mice developed EAE with the same incidence and severity as T/R- mice. In addition, MBP-specific transgenic mice that lack only endogenous TCR-alpha chains developed EAE with high incidence but reduced severity. Surprisingly, two-thirds of MBP-specific transgenic mice lacking only endogenous TCR-beta chains also developed EAE, suggesting that in T/R+ mice, cells with high protective activity escape TCR-beta chain allelic exclusion. Our study identifies CD4(+) T cells bearing endogenous alpha and beta TCR chains as the lymphocytes that prevent spontaneous EAE in T/R+ mice.     

Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation

T cell activation by specific antigen results in a rapid and long-lasting downregulation of triggered T cell receptors (TCRs). In this work, we investigated the fate of downregulated TCR- CD3-zeta complexes. T cells stimulated by peptide-pulsed antigen-presenting cells (APCs) undergo an antigen dose-dependent decrease of the total cellular content of TCR-beta, CD3-epsilon, and zeta chains, as detected by FACS(R) analysis on fixed and permeabilized T-APC conjugates and by Western blot analysis on cell lysates. The time course of CD3-zeta chain consumption overlaps with that of TCR downregulation, indicating that internalized TCR-CD3 complexes are promptly degraded. Inhibitors of lysosomal function (bafilomycin A1, folimycin) markedly reduced zeta chain degradation, leading to the accumulation of zeta chain in large Lamp1(+) vesicles. These results indicate that in T cell-APC conjugates, triggered TCRs are rapidly removed from the cell surface and are degraded in the lysosomal compartment.     

Receptor-specific allelic exclusion of TCRV alpha-chains during development

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.     

Extensive junctional diversity of Ig heavy chain rearrangements generated in the progeny of single fetal multipotent hematopoietic cells in the absence of selection

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.     

Receptor editing occurs frequently during normal B cell development

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that lambda light chain genes usually rearrange after kappa genes. This allowed us to analyze kappa loci in IgMlambda+ cells to determine how frequently in-frame kappa genes fail to suppress lambda gene rearrangements. To do this, we analyzed recombined VkappaJkappa genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the kappa locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged kappa loci, as nearly half (47%) of the RS-inactivated VkappaJkappa joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.     

Styryl-pyrones from Goniothalamus arvensis

CD3gamma and CD3delta are two highly related components of the T cell receptor (TCR)-CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3delta or CD3gamma, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-alphabeta+ T cells were detected in the periphery of both mice. gammadelta T cell development was either normal in CD3delta-/- mice or partially blocked in CD3gamma-/- mice. To examine the collective role of CD3gamma and CD3delta in the assembly and function of pre-TCR and in the development of gammadelta T cells, we generated a mouse strain with a disruption in both CD3gamma and CD3delta genes (CD3gammadelta-/-). In contrast to mice deficient in either CD3gamma or CD3delta chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-alphabeta+ or TCR-gammadelta+ T cells were absent in the CD3gammadelta-/- mice. Taken together, these studies demonstrated that CD3gamma and CD3delta play an essential, yet partially overlapping, role in the development of both alphabeta and gammadelta T cell lineages.     

Thymocyte differentiation in gamma-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor alpha locus

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR alpha locus. We found that J alpha-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+ CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR alpha is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of gamma-irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.     

Specific requirement for CD3epsilon in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3gamma, -delta, -epsilon, and zeta). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3gamma, -delta, or zeta results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3epsilon-/- mice, and thymocyte development is arrested at the early CD4(-)CD8(-) stage. Although these results suggest that CD3epsilon is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3gamma and CD3delta genes also is reduced in CD3epsilon-/- mice. Thus, it is unclear whether the phenotype of CD3epsilon-/- mice reflects the collective effects of CD3gamma, -delta, and -epsilon deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3epsilon gene via Cre/loxP-mediated recombination, we generated mice that lack CD3epsilon yet retain normal expression of the closely linked CD3gamma and CD3delta genes. These (CD3epsilonDelta/Delta) mice exhibited an early arrest in T cell development, similar to that of CD3epsilon-/- mice. Moreover, the developmental defect could be rescued by expression of a CD3epsilon transgene. These results identify an essential role for CD3epsilon in T cell development not shared by the CD3gamma, CD3delta, or zeta-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.     

TCR selection and allelic exclusion in RAG transgenic mice that exhibit abnormal T cell localization in lymph nodes and lymphatics

RAG-1 and RAG-2 are developmentally regulated genes that are essential for V(D)J recombination and lymphocyte development. Expression of RAG-1 and RAG-2 by thymocytes is normally limited to cells that have not completed selection. We have previously documented that persistent expression of the recombinase activating genes (RAG) in transgenic mice results in aberrant thymic development, altered lymphatic microanatomy, and a profound immunodeficiency. Here we further document the pathologic changes found in TG.RAG-1,2 mice and examine the role of TCR recombination and positive and negative thymic selection, as well as allelic exclusion, in the etiology of the phenotype. We find that neither selection nor TCR allelic exclusion can be overcome by transgenic expression RAG-1 and RAG-2 under the control of an lck promoter.     

Enzyme-linked immunosorbent assay of neocarzinostatin chromophore (NCS-chr) by use of a monoclonal antibody against NCS-chr analog

Allelic exclusion at the IgH locus was examined in B lineage cells of wild-type mice and mice unable to express the surrogate light chain molecule lambda 5 using a single-cell PCR approach. By analyzing B precursor cells containing two VHDHJH rearrangements, we found that in wild-type animals, cells are allelically excluded as soon as mu chains are expressed. Furthermore, we provide evidence that in cells expressing D mu proteins VH-->DHJH rearrangement is inhibited. In contrast, in the absence of lambda 5 protein, B precursor cells were allelically "included", indicating that allelic exclusion at the IgH locus requires expression of the pre-B cell receptor either containing a mu chain or a D mu chain. However, although mu chain double-producing B precursor cells are generated in lambda 5-deficient mice, such cells were not detected among surface immunoglobulin positive B cells.     

Kappa chain monoallelic demethylation and the establishment of allelic exclusion

Allelic exclusion in kappa light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional kappa light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two kappa alleles for rearrangement. Analysis of kappa+ B-cell populations and of individual kappa+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3' kappa enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single kappa allele for rearrangement.     

Junctional diversity in signal joints from T cell receptor beta and delta loci via terminal deoxynucleotidyl transferase and exonucleolytic activity

The site-specific V(D)J recombination reaction necessary to assemble the genes coding for immunoglobulin (Ig) and T cell receptor (TCR) variable regions is initiated by a precise double strand cut at the border of the recombination signals flanking the genes. Extensive processing of the coding ends before their ligation accounts for most of the Ig and TCR repertoire diversity. This processing includes both base additions to and loss from the coding ends. On the other hand, it has generally been thought that signal ends are not modified before they are fused, and that signal joints consist of a perfect head-to-head ligation of the recombination signals. In this study, we analyzed signal joints created during the rearrangement of different TCR-beta and TCR-delta genes in thymocytes. We show that a significant fraction (up to 24%) of these signal joints exhibits junctional diversity. This diversity results from N nucleotide additions for TCR-beta signal joints, and from N additions and exonucleolytic digestion for TCR-delta joints. Altogether, our findings suggest that: (a) signal ends can undergo some of the same modifications as coding ends, (b) inversional rearrangement generates more diversity than deletional events, and (c) fine differences exist in the recombinase/DNA complexes formed at each rearranging locus.