Gammadelta Thymocytes Research Articles

In vivo administration of a novel synthetic GMCSF and IL7 fusion cytokine (GIFT7) leads to transient thymic hyperplasia by driving double-negative (DN), single-positive CD8 (SPCD8) and gamma-delta (γδ) thymocyte proliferation (165.7)

Abstract T-cell deficiency secondary to cytotoxic agents, viral assaults or ageing predisposes to defective adaptive immunity. Therefore, therapeutic approaches to enhance thymic output leading to T cell reconstitution are of clinical importance. We develop a novel synthetic fusion cytokine between Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin 7 Fusion Transgene (GIFT7). In vitro, GIFT7 acts as a hyperagonist for the IL7 receptor (IL7R) leading to hyperphosphorylation of STAT5. To test the effect of GIFT7 on IL7R-expressing primary thymocytes, we dissociated normal thymii from C57BL/6 mice and exposed unfractionated thymic cells in vitro to GIFT7 for 5 days. We observed a marked anti-apoptotic and proliferative response in CD3+CD4-CD8+ single-positive CD8 (SPCD8) and CD4-CD8-CD44+CD25- double negative 1 (DN1) T cell precursors in both αβ and γδ T cell lineage. In vivo, intravenous administration of recombinant GIFT7 at 5 µg/kg daily for 2 days into non-irradiated mice leads to a significant 2 fold increase in thymic DN1 population after 7 days. After 14 days, there was a subsequent 100% increase in total thymocyte number which returned to baseline at day 35. We propose the use of GIFT7 as a novel IL7R targeted thymopoietic growth factor for the treatment of catastrophic human immune deficient ailments characterized by thymic involution such as congenital or HIV-mediated acquired immunodeficiency, post-chemotherapy/radiation therapy and ageing.

TCR-mediated ThPOK induction promotes development of mature (CD24−) γδ thymocytes

T lymphocytes develop into two major lineages characterized by expression of the alphabeta and gammadelta T cell receptor (TCR) heterodimers. Within each major lineage, further specialization occurs, resulting in distinct subsets that differ in TCR specificity, phenotype and functional attributes. Thus, in the murine thymus, two distinct subsets of mature (CD24-) gammadelta cells have been identified, that is NK1.1+ cells, which are enriched for Vgamma1.1 usage and selectively produce IFNgamma on stimulation, and CCR6+ cells, which are enriched for Vgamma2 usage produce IL17. The upstream signals and transcriptional pathways that promote development of these distinct gammadelta subsets remain relatively poorly understood. Here, we show that the Zn-finger transcription factor ThPOK has a critical function in the development of gammadelta thymocytes. Thus, lack of functional ThPOK causes a marked reduction in the percentage and absolute number of mature gammadelta thymocytes, and a particularly severe reduction of NK1.1+ cells. Conversely, constitutive ThPOK expression leads to a striking increase in mature NK1.1+ gammadelta thymocytes. Further, we show that ThPOK induction in gammadelta thymocytes is induced by strong TCR signals mediated by engagement with antibody or high-affinity endogenous ligands, and that an important ThPOK cis-acting element, the distal regulatory element (DRE), is sufficient for this TCR-dependent induction. These results show that ThPOK expression in gammadelta thymocytes is regulated in part by the strength of TCR signalling, identify ThPOK as an important mediator of gammadelta T cell development/maturation, and lend strong support to the view that development of a significant fraction of gammadelta T cells depends on TCR engagement/signalling.

Roles of the Src Tyrosine Kinases Lck and Fyn in Regulating γδTCR Signal Strength

Lck and Fyn, members of the Src family of tyrosine kinases, are key components of the αβTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the αβTCR, recent studies have shown that Lck and Fyn have distinct functions in this signaling pathway, with Lck being a positive regulator and Fyn being a negative regulator of αβTCR signal transduction. To determine whether Lck and Fyn also differentially regulate γδTCR signal transduction, we analyzed γδ T cell development and function in mice with reduced Lck or Fyn expression levels. We found that reducing Lck or Fyn levels altered the strength of the γδTCR signaling response, with low levels of Lck weakening γδTCR signal strength and low levels of Fyn augmenting γδTCR signal strength. These alterations in γδTCR signal strength had profound effects not only on αβ/γδ lineage choice, but also on γδ thymocyte maturation and γδ T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the γδTCR signaling response at different stages in the life of the γδ T cell.

Development of γδ thymocyte subsets during prenatal and postnatal ontogeny

In this report, we describe 12 subpopulations of porcine gammadelta thymocytes based on their expression of CD1, CD2, CD4, CD8- isoforms and CD45RC. Our data suggest that gammadelta thymocytes can be divided into two major families: (a) one large family of CD4-gammadelta thymocytes that could be further subdivided according to the CD2/CD8alphaalpha phenotype and (b) a small family of CD4+ gammadelta thymocytes bearing CD8alphabeta and possessing certain unusual features in comparison with other gammadelta thymocytes. Maturation of gammadelta thymocytes within the CD4- family begins with proliferation of the CD2+ CD8- CD1+ CD45RC- gammadelta common precursor. This developmental stage is followed by diversification into the CD2+ CD8alphaalpha+, CD2+ CD8- and CD2- CD8- subsets. Their further maturation is accompanied by a loss of expression of CD1 and by increased expression of CD45RC. Therefore, individual subsets develop from CD1+ CD45RC- through CD1- CD45RC- into CD1- CD45RC+ cells. On the other hand, gammadelta thymocytes within the CD4+ family bear exclusively CD8alphabeta, always express CD1, but may coexpress CD45RC. These cells have no counterpart in the periphery. Our observations suggest that all peripheral CD8+ gammadelta T cells express CD8alphaalpha and that two subsets of these cells differing in major histocompatibility complex II expression, occur. We propose that one subset acquires CD8alphaalpha in the thymus while the second acquires CD8alphaalpha as a result of stimulation in the periphery.

Early expression of a functional TCRbeta chain inhibits TCRgamma gene rearrangements without altering the frequency of TCRgammadelta lineage cells.

To investigate the consequences of the simultaneous expression in progenitor cells of a TCRgammadelta and a pre-TCR on alphabeta/gammadelta lineage commitment, we have forced expression of functionally rearranged TCRbeta, TCRgamma, and TCRdelta chains by means of transgenes. Mice transgenic for the three TCR chains contain numbers of gammadelta thymocytes comparable to those of mice transgenic for both TCRgamma and TCRdelta chains, and numbers of alphabeta thymocytes similar to those found in mice solely transgenic for a rearranged TCRbeta chain gene. gammadelta T cells from the triple transgenic mice express the transgenic TCRbeta chain, but do not express a TCRalpha chain, and, by a number of phenotypic and molecular parameters, appear to be bona fide gammadelta thymocytes. Our results reveal a remarkable degree of independence in the generation of alphabeta and gammadelta lineage cells from progenitor cells that, in theory, could simultaneously express a TCRgammadelta and a pre-TCR.

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors.

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.

Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull gamma delta thymocytes.

Thy-1 dull gamma delta thymocytes constitute an unusual subset of mature TCR gamma delta cells which share with NK T cells the expression of cell surface markers usually associated with activated or memory cells and the simultaneous production of high levels of IL-4 and IFN-gamma upon activation. In DBA / 2 mice, Thy-1 dull gamma delta thymocytes express a restricted repertoire of TCR that are composed of the V1 gene product mainly associated with V6.4 chains exhibiting very limited junctional sequence diversity. In this study we have characterized this gamma delta T cell population in different strains of mice and show that Thy-1 dull gamma delta thymocytes are present in every strain tested, albeit at different frequencies. Moreover IL-4 production by gamma delta thymocytes is mainly confined to the Thy-1 dull population in every strain tested. Finally, the repertoire of TCR expressed by Thy-1 dull gamma delta thymocytes varies in different strain of mice, although a biased expression of Vgamma1 and Vdelta6 chains was observed in all strains studied. However, the extent of junctional diversity of the V1 and V6 chains expressed by Thy-1 dull gamma delta thymocytes varied from oligoclonal in DBA/2 mice to polyclonal in FVB/N mice. Thy-1 dull gamma delta thymocytes from mouse strains such as C3H/HeJ and BALB/c contain cells with diverse Vdelta6(D)Jdelta junctions together with cells with relatively homogeneous Vdelta6(D)Jdelta junctions, similar to those found in DBA/2. Thus, the Thy-1 dull gamma delta population appears to contain two subsets of cells which differ in the diversity of their TCR.

Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice.

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.

The homogeneity of the TCRdelta repertoire expressed by the Thy-1dull gammadelta T cell population is due to cellular selection.

Thy-1dull gammadelta T cells are an unusual subset of mature TCRgammadelta T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vgamma1 gene together with that of a member of the Vdelta6 subfamily (the Vdelta6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRgammadelta repertoire, we have cloned all Vdelta6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1dull and Thy-1bright gammadelta thymocyte populations. Furthermore, we have also cloned non-functional Vdelta6DdeltaJdelta1 rearrangements present in the Thy-1dull gammadelta T cell population and compared their Vdelta6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRdelta repertoire expressed by the Thy-1dull gammadelta thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRgammadelta repertoire analysis of Thy-1dull gammadelta T cells in beta2-microglobulin (beta2m)-deficient mice indicated that these putative ligands are not beta2m-dependent major histocompatibility complex class I or class I-like molecules.

TCR-delta gene rearrangement and selection during fetal thymocyte development.

TCR delta-chain gene rearrangements were analyzed at different stages of thymic ontogeny. The VDJ delta junctional sequences of the dominantly expressed TCR delta-chain V7 subfamily are highly conserved in fetal and neonatal thymocytes. They are equally conserved in C delta-targeted mice lacking cell surface expression of gamma delta receptors, indicating evolutionary selection acting at the level of DNA rearrangement. Yet, in C delta-mutant mice, the frequency of in-frame transcripts is reduced to 61%, from 88% in wild-type mice, suggesting cellular selection of this DV7-overexpressing gamma delta thymocyte subset. In contrast, in genomic DNA rearrangements, no difference was found in the frequency of productive rearrangements between mutant (26%) and wild-type mice (31%). This in-frame rate, characteristic of random rearrangements, indicates that positive selection is not required for thymic maturation of gamma delta T cells. The results are discussed with regard to models of alpha beta/gamma delta lineage commitment.

Concurrent or sequential delta and beta TCR gene rearrangement during thymocyte development: individual thymi follow distinct pathways.

Analysis of TCR rearrangement profiles of well-defined thymocyte populations in a number of individual thymi provides evidence for a new pathway of lineage commitment. In all of the thymi analyzed, alphabeta thymocytes have rearrangements in the delta locus that are enhanced for out-of-frame rearrangements. Thus, not only did alphabeta thymocytes pass through a stage in differentiation that included delta rearrangement, but they also constitute a population that was relatively unsuccessful at these rearrangements. Interestingly, in some thymi, gammadelta thymocytes have out-of-frame beta rearrangements. This represents a novel pathway in which delta and beta rearrangement happens concurrently. In this pathway, selection favors whichever gene is in frame, thus improving the chances of generating useful T cells. In other thymi, gammadelta cells showed no obvious beta gene rearrangement, indicating a sequential rearrangement. Thus, alphabeta/gammadelta lineage commitment can follow at least two distinct pathways in different individuals.

Phenotypic and functional maturation of TCR gammadelta cells in the human thymus.

In contrast to thymic differentiation of TCR alphabeta cells, differentiation stages of TCR gammadelta cells are largely unknown. This report shows that CD1, a known marker of immature TCR alphabeta thymocytes, was expressed on some postnatal TCR gammadelta thymocytes. Only CD1+ TCR gammadelta thymocytes expressed recombination-activating gene-1 mRNA, and they were shown to differentiate into CD1- TCR gammadelta thymocytes. Functionally, sorted CD1- TCR gammadelta thymocytes proliferated in the presence of immobilized anti-CD3 Ab plus exogenous rIL-2 or rIL-15. Interestingly, in contrast to CD1- TCR alphabeta cells, CD1- TCR gammadelta thymocytes also proliferated extensively when cultured with exogenous rIL-2 or rIL-15 alone. FACS analysis as well as reverse transcription-PCR analysis showed that only CD1- TCR gammadelta thymocytes expressed IL-2Rbeta protein and mRNA. The differential expression of maturation markers, such as CD27, CD45RO, and CD45RA, as a function of expression of CD1 was similar in TCR gammadelta and TCR alphabeta thymocytes. An important exception is the expression of CD4 and CD8. Whereas TCR alphabeta thymocytes are mainly CD4-CD8 double positive at the immature CD1+ stage and CD4 or CD8 single positive at the mature CD1- stage, CD1(bright) TCR gammadelta thymocytes all expressed CD4, but only some of them expressed CD8. Some CD1- TCR gammadelta thymocytes also expressed CD8, but were negative for CD4. Collectively, our data clearly show that CD1 is a useful marker to distinguish immature human TCR gammadelta thymocytes from functional mature gammadelta cells based on recombination-activating gene-1 expression, in vitro differentiation, and phenotypic and functional characteristics.

A novel subset of adult gamma delta thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire.

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of gamma delta thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5% and 30% of total gamma delta thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1dull gamma delta thymocytes from DBA/2 mice with anti-gamma delta monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-gamma (IFN-gamma), IL-4, IL-10, and IL-3. In contrast, only IFN-gamma was detected in parallel cultures of Thy-1bright gamma delta thymocytes. Virtually all Thy-1dull gamma delta thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1dull gamma delta thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1dull gamma delta thymocytes from DBA/2 mice express TCR encoded by the V gamma 1 gene and a novel V delta 6 gene named V delta 6.4. Sequence analysis of these functionally rearranged gamma and delta genes revealed highly restricted V delta-D delta-J delta junctions, and somewhat more diverse V gamma-J gamma junctions. We conclude that Thy-1dull gamma delta thymocytes exhibit properties that are equivalent to those of natural killer TCR alpha beta T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.

Alloreactive gamma delta thymocytes utilize distinct costimulatory signals from peripheral T cells.

Interactions between CD28/CTLA-4 on T cells and CD80 (B7.1) and CD86 (B7.2) counter receptors provide crucial costimulatory signals for TCR-alpha beta+ lymphocytes. To test the role of CD28 in thymic development and activation of TCR-gamma delta+ T cells, we introduced the alloreactive V gamma 2V alpha 11.3 TCR into CD28-deficient mice (CD28-/-). We show that positive and negative selection of gamma delta Tg thymocytes proceeded normally in the absence of CD28. Although mature Tg gamma delta+ thymocytes required a second costimulatory signal for proliferation, gamma delta+ thymocytes from CD28-/- and CD28+/- littermates responded equally well to the alloantigen Tlab. Alloreactivity of CD28-/- and CD28+/- Tg gamma delta+ thymocytes could not be blocked with mAbs against CD80 and CD86 ligands. Thus gamma delta thymocytes utilize a costimulatory system during development and alloresponses that is independent of CD28/CD80 and CD28/CD86 interactions. By contrast to V gamma 2V alpha 11.3+ thymocytes, alloreactivity of V gamma 2V alpha 11.3+ lymph node T cells depended on CD28 costimulation and was severely impaired in CD28-/- mice. These data provide functional evidence that maturation and selection of gamma delta cells is independent of CD28. These results also indicate that distinct costimulatory pathways are operational in mature thymocytes and peripheral T cells.

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Little is known about the role of apoptosis in the regulation of gamma delta T cell development and function. We have used chicken as a model to study apoptosis of gamma delta T cells at different stages of their development. Apoptosis was measured with electrophoretic analysis of DNA fragmentation and flow cytometric determination of DNA content combined with immunofluorescence staining of cell surface molecules. In vitro culture, dexamethasone, and gamma-irradiation induced apoptosis of both gamma delta TCR+ thymocytes and peripheral gamma delta T cells. Apoptosis could be induced even in the earliest thymic gamma delta thymocytes on embryonic day 13. Resting peripheral blood gamma delta T cells were more resistant to apoptosis than thymocytes and spleen cells. Following polyclonal activation of splenic gamma delta T cells by Con A, the proportion of the CD8+ gamma delta T cell blasts decreased significantly when recultured without further stimulation. These results indicate that gamma delta T cells are susceptible to apoptosis in a manner similar to alpha beta T cells, and suggest that apoptosis plays an important role in the regulation of the development and function of both thymic and peripheral gamma delta T cells.

New monoclonal antibody (23D12) recognizing three different V gamma elements of the human gamma delta T cell receptor. 23D12+ cells comprise a major subpopulation of gamma delta T cells in postnatal thymus.

The gamma delta TCR is expressed on 1 to 5% of CD3+ human peripheral blood T lymphocytes. The majority of peripheral blood gamma delta T cells expresses V gamma 9 paired with V delta 2; this subset strongly responds to certain microbial ligands. Other gamma delta T cell subsets with unknown Ag specificity expressing different V gamma elements are present in peripheral blood and lymphoid tissue. We describe a new anti-human V gamma mAb termed 23D12 with unusual specificity. As revealed by analysis of a large number of T cell clones and transfectants expressing molecularly well-defined gamma delta TCR, mAb 23D12 recognized several, but not all, members of the human V gamma 1 family, specifically V gamma 2, V gamma 3, and V gamma 4 but not V gamma 5 or V gamma 8. In combination with available mAb against V gamma 4, mAb 23D12 was used to identify V gamma 2- or V gamma 3-bearing cells. On average, 23D12+ cells accounted for 18% of peripheral blood gamma delta T cells and 56% of postnatal gamma delta thymocytes. In combination with anti-V gamma 9 mAb, mAb 23D12 23D12 identified gamma delta cells expressing V elements other than V gamma 2, V gamma 3, V gamma 4, or V gamma 9. Such cells are detectable in peripheral blood and postnatal thymus. Using mAb 23D12, we also confirmed the appearance of two distinct TCR gamma-chains on the surface of some gamma delta T cells.

CD45RA expression of gamma delta and alpha beta T cells emigrating from the fetal and postnatal thymus.

We have compared the expression of CD45RA on alpha beta and gamma delta T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA+ and CD45RA- T cells. The number of gamma delta +CD45RA+ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5-8% of gamma delta thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to gamma delta T cells, up to one quarter of both fetal and postnatal alpha beta emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression of alpha beta emigrants, which occurred both before and after birth, appeared to be antigen independent.

Evidence for programmed cell death of self‐reactive γδ T cell receptor‐positive thymocytes

The negative selection of T cells expressing the gamma delta T cell antigen receptor (gamma delta T cells) was studied using transgenic mice expressing a gamma delta receptor with specificity for an H-2T-linked class I major histocompatibility complex molecule from H-2b mice. The potentially self-reactive gamma delta thymocytes in H-2b/d transgenic mice are larger and have lower levels of gamma delta T cell receptor expression than gamma delta thymocytes from H-2d mice. H-2b/d gamma delta thymocytes do not respond to H-2b antigen-presenting cells, and thus are inactive compared to H-2d gamma delta thymocytes. However, the H-2b/d gamma delta thymocyte population, but not the H-2d gamma delta thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self-reactive gamma delta thymocytes undergo programmed cell death. This in vitro programmed cell death of self-reactive gamma delta thymocytes may reflect the clonal deletion process that results in a depletion of gamma delta T cells in the peripheral lymphoid organs of adult H-2b/d mice. We also present evidence that self-reactive gamma delta T cells, similarly to alpha beta T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.

Emigration of selected subsets of gamma delta + T cells from the adult murine thymus.

Cells bearing the gamma delta form of the TCR make up only 1-3% of T cells in the adult murine thymus and peripheral lymphoid organs. Evidence from studies of nude mice suggests that the development of at least some gamma delta T cells is thymus dependent; however, until now it has not been directly demonstrated that gamma delta cells are exported from the thymus. In this paper we have used the technique of labelling thymocytes in vivo with FITC, followed by flow cytometric analysis to trace cells emigrating from the thymus to the spleen. Using this approach we have been able to demonstrate for the first time that gamma delta T cells are exported from the adult murine thymus to the spleen. We also demonstrate that the cells emigrating to the spleen are a selected subset of gamma delta thymocytes being heat stable antigen positive, Thy-1+, and expressing low levels of CD44 (Pgp-1). In addition, investigation of TCR V gamma gene usage among adult gamma delta + thymocytes, recent emigrants, and spleen cells, indicated a selective emigration of gamma delta cells expressing certain V gamma genes.

Selective proliferation of gamma delta T lymphocytes exposed to high doses of ionomycin.

The alpha beta T cell repertoire is primarily shaped in the thymus. However, extrathymic positive selection has been demonstrated for many gamma delta T cell clonotypes. This latter type of selection is the result of a peripheral clonal expansion which could be facilitated by special physiological properties of gamma delta T cells, distinguishing them from most alpha beta T cells. In studying the behavior of T cells under conditions of polyclonal activation, we noticed a differential sensitivity between alpha beta and gamma delta T cells to strong stimulatory signals. When induced with high doses of ionomycin, a large fraction of peripheral gamma delta T cells and a small fraction of alpha beta T cells are able to proliferate exponentially while most alpha beta T cells die. This phenomenon appears to be related to intracellular regulation of high concentrations of cytosolic Ca2+. The ability to proliferate under strong stimulatory conditions is a striking feature of many peripheral gamma delta T cells but not of gamma delta thymocytes. In general, T cells selected in the periphery by clonal expansion might be characterized by resistance to strong stimuli and typically, by their ability to "handle" higher concentrations of free cytoplasmic calcium.