IL-4-producing gamma delta T cells that express a very restricted TCR repertoire are preferentially localized in liver and spleen.

IL-4-producing gamma delta thymocytes in normal mice belong to a distinct subset of gamma delta T cells characterized by low expression of Thy-1. This gamma delta thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1dull gamma delta thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the V gamma 1 gene product mainly associated with the V delta 6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged V gamma 1J gamma 4C gamma 4 chain and a novel mAb (9D3) specific for the V delta 6.3 and V delta 6.4 murine TCR delta chains, we have analyzed the peripheral localization and functional properties of gamma delta T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral gamma delta T cells was confined to the gamma delta+9D3+ subset, which contains cells with a TCR repertoire similar to that found in Thy-1dull gamma delta thymocytes. In normal DBA/2 mice such cells represent close to half of the gamma delta T cells present in the liver and around 20% of the splenic gamma delta T cells.     

Preferential activation of an IL-2 regulatory sequence transgene in TCR gamma delta and NKT cells: subset-specific differences in IL-2 regulation

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.     

Expression of C gamma 4 T cell receptors and lack of isotype exclusion by dendritic epidermal T cell lines

Although four murine C gamma gene segments (C gamma 1, 2, 3, and 4) are known to exist, the large majority of expressed gamma-chains have been shown to be of the C gamma 1 isotype and no evidence exists for the expression of more than one receptor by gamma delta TCR-bearing cells. We investigated the nature of the TCR expressed on a number of murine dendritic epidermal T cell-derived cell lines by using both Northern blot and immunoprecipitation analyses. One of these CD3+ cell lines (T195) expresses C gamma 4, V gamma 1, and delta mRNA, and its CD3-associated TCR complex can be precipitated by both anti-C gamma 4 and anti-delta sera, indicating that this receptor is a C gamma 4/delta heterodimer. Furthermore, we show that two cell lines (Y245, Y93) express two distinct TCR gamma-chains, one derived from the C gamma 4 locus, whereas the second gamma-chain is probably derived from the C gamma 2 locus. Together with the previous demonstration of C gamma 1/delta TCR on a number of dendritic epidermal T cell lines (DETC), these results indicate that such DETC are capable of expressing a variety of gamma delta TCR and that, in some DETC, isotype exclusion of gamma-chain expression does not occur.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Expansion of myelopoietic precursors and inhibition of B-cell precursors in mice that express a T-cell receptor gamma (V gamma 1.1J gamma 4C gamma 4) transgene.

Knowledge of the genetic determinants that can affect renewal of multipotential stem cells and their commitment to specific cell lineages is essential to our understanding of multicellular development. However, despite the vast amount of accumulated knowledge in this area, genetic determinants that affect renewal and commitment of precursor cells are unknown. In this study, we demonstrate that three independently derived founder mouse strains, transgenic for the TcR V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) chain gene, differed significantly from normal mice in their development of T and B cells as well as myelopoietic precursor cells. Ontogenic programs consistent with an acceleration of T-cell development and a delayed appearance and suppressed levels of pre-B- and B-cell precursors were evident in these transgenic mice. In addition, TcR gamma 4 transgenic mice possessed a significantly elevated level of myelopoietic pluripotential precursors. 3H-thymidine cell suicide studies suggest that higher percentages of pluripotent precursors from the bone marrow of the TcR gamma 4 transgenic mice were in the S phase of the cell cycle. These modulations of the lymphoid and myelopoietic compartments, however, were not found in other T-cell receptor transgenic mice (e.g., TcR V gamma 1.2J gamma 2C gamma 2, TcR gamma 2; or V beta 8.1D beta J beta 2.4C beta 2, TcR beta) constructed with the same or similar cDNA expression vector. The results suggest that the expression of a specific T-cell receptor gamma chain gene, and/or an elevated level of particular subset of TcR gamma delta cells, may affect the proliferation and relative proportions of haemopoietic and lymphoid precursors.     

Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.     

Extrathymic positive selection of gamma delta T cells. V gamma 4J gamma 1 rearrangements with "GxYS" junctions.

Neither function nor Ag recognition properties of gamma delta T cells are well understood yet. A TCR gamma-chain family, characterized by distinct N region sequences that converge in coding for a "GxYS" VJ junctional sequence, appears late in ontogeny among highly diversified V gamma 4J gamma 1C gamma 1 chains of C57BL/6 and BALB/c mice. The glycine and serine codons are of germline V gamma 4 and J gamma 1 origin, respectively, whereas the N region consists of a variable amino acid residue x, followed by an invariant tyrosine Y. The high expression of V gamma 4 xYJ gamma 1C gamma 1 in the lung of BALB/c mice compared to that of C57BL/6 is apparently due to a novel pattern of strain-dependent positive selection which, unlike for alpha beta TCR, operates extrathymically. This type of selection seems to be determined by strain specific polymorphic ligands encoded outside of the classical H-2 region.     

T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation.

A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.     

Identification of the transgenic integration site in 2C T cell receptor transgenic mice

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3′ region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.     

Modulation of gamma delta T cells in mouse buccal epithelium following antigen priming

T cells using the gamma delta T cell receptor (TCR) are abundant in mucosal and epidermal tissues in mice. Most studies of mucosal gamma delta T cells, however, have examined cells from the intestinal mucosa, whereas little is known about the presence or function of gamma delta T cells in the oral cavity. To better understand the involvement of oral gamma delta T cells in immunity, we have characterized TCR variable gamma-gene usage in the buccal epithelium from normal mice, and from mice challenged locally with a non-replicating antigen (bovine serum albumin [BSA]) or by influenza-virus infection as a replicating antigen. Our findings demonstrate a restricted use of V gamma genes by buccal gamma delta T cells, consisting primarily of V gamma 1.2, V gamma 3, and V gamma 5, with minimal use of V gamma 2 and V gamma 4 genes. Of particular interest, 3-4 days post-antigen challenge with BSA, there was a precipitous drop in the level of expression of V gamma 1.2, V gamma 3, and V gamma 5 genes, and to a lesser extent for the V gamma 2 gene, whereas V gamma 4 gene expression increased between days 1 and 2 post-priming. In influenza-infected mice, a similar pattern was observed for the V gamma 2 and V gamma 5 genes, but not other V gamma genes. The immune-modulating effects of oral antigen exposure on buccal gamma delta T cells suggest that these cells are functionally involved in the local immune response to both replicating and non-replicating antigens in oral mucosal surfaces.     

Blockade of transgenic gamma delta T cell development in beta 2-microglobulin deficient mice.

The gamma delta T cell receptor (TCR) of the hybridoma KN6 recognizes the self molecule encoded by a class I gene which maps within the TL region of the major histocompatibility complex (MHC) of H-2b mice. Mice transgenic (Tg) for this TCR were crossed with mice genetically deficient in beta 2-microglobulin (beta 2m). No mature Tg gamma delta T cells were detected in the thymus or the spleen of the beta 2m- gamma delta Tg mice. We conclude that interaction between the Tg gamma delta TCR and a beta 2m-associated molecule (probably an MHC class I molecule) is required for the generation of mature Tg gamma delta T cells.     

T cell receptor gamma delta expression of Thy-1+ dendritic epidermal cells--an update

Thy-1+ dendritic epidermal cells (Thy-1+DEC) are present in the murine epidermis. They are morphologically dendritic and express Thy-1, CD3 and asialoGM1, but not CD4 or CD8. T cell receptor (TCR) of Thy-1+DEC is TCR gamma delta. Allison et al and Tonegawa et al recently found that TCR of Thy-1+DEC is V gamma 5 J gamma C gamma -V delta 1D2J2C delta and has no junctional diversity. This TCR gamma delta of Thy-1+DEC is identical to TCR expressed on the earliest fetal thymocytes. It is distinct from that of other epithelial associated lymphocytes or other thymocytes. The ligand of Thy-1+DEC is not known, although TCR gamma delta of adult type could recognize allogenic major histocompatibility complex(MHC) class I or class II and mycobacterium antigen, especially heat shock protein. The TCR of Thy-1+DEC may not be the homing receptor to epidermis. The further studies are needed to elucidate the ligands or functions of Thy-1+DEC.     

Peripheral murine CD3+, CD4-, CD8- T lymphocytes express novel T cell receptor gamma delta structures

A mAb directed against the CD3 molecule was used to identify a subset of CD3+, CD4-, CD8- T cells previously undefined in the peripheral lymphoid organs of the mouse. Biochemical analysis of CD3+, CD4-, CD8- splenocytes revealed that the vast majority of these cells express one of at least two distinct CD3-associated TCR gamma delta heterodimeric structures, but no detectable TCR alpha beta. One disulfide-linked heterodimer (77 kDa) is composed of two chains of 45 to 46 and 32 kDa. The latter chain was immunoprecipitated with an anti-TCR C gamma 1/C gamma 2 antiserum and was not glycosylated. An antiserum produced against a peptide corresponding to the C-terminal region of the predicted C gamma 4 gene product immunoprecipitated additional heterodimers (80 to 90 kDa). One heterodimer, composed of disulfide-linked 41- to 45-kDa protein (including a V gamma/C gamma 4 component), is expressed on a T cell hybridoma, DN-1.21, which was derived from fused splenic CD3+, CD4-, CD8- T cells. Another V gamma/C gamma 4-containing heterodimer is composed of disulfide-linked 46- to 47-kDa glycoproteins. These findings demonstrate that CD3+, CD4-, CD8- T cells present in the peripheral lymphoid organs express a variety of paired TCR gamma delta proteins. Unlike CD3+, CD4-, CD8- thymocytes, these cells express high levels of C gamma 4, but little, if any TCR alpha beta.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.     

T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.