CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of {alpha}{beta} TCR+CD4 CD8 thymocytes

We have examined CD38 expression on mouse lymphocytes usingthe rat mAb NIM-R5 and demonstrate that CD38 expression is restrictedto {small tilde}8% of thymocytes. Although CD38 is absent fromthe majority of CD4+ CD8^– and CD4^–CD8⁺ T cells,we detected a strong correlation between CD36 expression andß⁺CD4^–CD8^– T cells in the thymus, withnearly 80% of ß TCR⁺CD4^–CD8^– thymocytesbeing CD38⁺. Using heat stable antigen (HSA) and CD38, we dividedß⁺CD4⁺CD8^– thymocytes into four subsets: HSA⁺CD38^–,HSA^– CD38^hi, HSA^–CD3810^low and HSA^– CD38^–.Two established characteristics of ß TCR⁺CD4CD8^–cells, bias towards Vß 8.2 TCR expression and highlevels of IL-4 production, were used to establish a possiblerelationship between the above thymocyte subsets. Our presentdata show that the HSA⁺CD38^– subset is not biased towardsVß8.2 TCR expression whereas the HSA^– CD38^–subset does show this bias (–47%). Neither of these subsetsmake IL-4 upon CD3 mediated stimulation. In contrast, the CD38⁺subsets are heavily biased toward Vß8.2 expressionand produce large amounts of IL-4 upon stimulation, particularlythe CD38^low cells. Taken together, these data suggest that thesefour subsets represent various stages of a possible differentiationpathway for ß TCR⁺ CD4^–CD8^– cells, withthe HSA⁺CD38^– subset being the most Immature while theHSA^–CD38^low subset is the most functionally mature. Thesecharacteristics support the view that ap TCR⁺CD4^–CD8^–T cells represent an independent lineage with a distinct, butas yet obscure, role in immunity     

CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2 / mice in the absence of TCR {beta} chain expression

Recent studies have shown that TCR ß chain expressioncan effect the differentiation of CD4^–CD8^– double-negative(DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes.The TCR ß chain is expressed on the surface of DPthymocytes in association with CD3, and chains, suggestinga potential role for CD3 components in this signaling process.We now report detection of a very tow level of surface expressionof CD3 on adult DN RAG-2^–/–; thymocytes. This surfaceCD3 was associated with CD3 and chains, as detected by anti-CD3immunoprecipitation analyses. Significantly, injection of anti-CD3mAb into RAG-2^–/– mice led to the accumulation ofan IL-2R^– CD2⁺ DP cell population and a nearly 100-foldincrease in thymic cellularity to essentially normal levels.Together, these data strongly indicate that TCR ßchain-mediated developmental signals are transduced by CD3 componentsand provide potential insights into mechanisms by which TCRß chain expression may effect this process.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2K^b (K^b) under the control of the sheep metallothionein promoter (Met-K^b mice). To follow the fate of K^b-specific T cells, and to prevent any aberrant thymic expression of the K^b transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a K^b-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although K^b-specific CD8⁺ T cells were produced in the thymus grafts of these manipulated Met-K^b mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8⁺ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the K^b-specific CD8⁺ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.     

A distinct role for ICOS-mediated co-stimulatory signaling in CD4+ and CD8+ T cell subsets

While the ligand of inducible co-stimulator (ICOS), B7 homologous protein, is widely expressed in somatic cells, B7-1 and B7-2 expression is mainly limited to lymphoid organs. Thus, the activation of T cells through ICOS without a CD28-mediated signal may occur in physiological situations. In order to gain a better understanding of the role of the ICOS co-stimulatory signal in immune responses, we studied the cellular response of T cells to beads coated with anti-ICOS or anti-CD28, plus sub-optimal anti-CD3 mAb. We demonstrate that while CD28 ligation induced expansion of both CD4+ and CD8+ populations, ICOS ligation only resulted in the expansion of CD8+ T cells, and induced apoptosis in the CD4+ T cell population. It was found that IL-2 is critically required for CD8+ T cell expansion triggered by ICOS ligation, whereas it had only a limited effect on the expansion of CD4+ T cells. This distinct reactivity of CD4+ and CD8+ T cell populations to exogenous IL-2 strongly correlates with the expression level of IL-2 receptor beta-chain, CD122, on T cells. Furthermore, we defined a small but distinct population of memory phenotype CD4+ T cells that constitutively express ICOS. Interestingly, while naive CD4+ T cells were unable to produce IL-2, ICOS-expressing T cells produced a substantial amount of IL-2 by stimulation with anti-ICOS/CD3 beads, suggesting that IL-2, which is indispensable for CD8+ T cell expansion, is produced by this ICOS-expressing T cell population. These results provide evidence indicating that the ICOS co-stimulatory signal plays a distinct role in the development of CD4+ and CD8+ T cell-mediated immune responses.     

Interleukin 4 induces CD8 alpha expression on T cell receptor V gamma 5 thymocytes

It is generally accepted that most T cell receptor (TcR) gamma/delta cells are CD4-CD8-. After in vitro culture; however, a low percentage of these cells express the CD8 alpha subunit. We show here that addition of recombinant interleukin (IL) 4 to IL 2-cultured murine TcR V gamma 5 thymocytes induces the expression of CD8 alpha; CD8 beta is not expressed. Co-addition of the anti-IL 4 mAb 11B11 abrogates the induction of CD8 alpha expression, ruling out the possibility of a contaminant. Furthermore, we demonstrate that a substantial part of freshly prepared TcR V gamma 5 thymocytes express CD8 alpha.     

Resident CD4+ {alpha}{beta} T cells of the murine female genital tract: a phenotypically distinct T cell lineage that rapidly proliferates in response to systemic T cell activation stimuli

A population of CD4⁺ cells has been identified in the murinefemale genital tract (FGT). Phenotypic studies of FGT CD4⁺ cellsdemonstrate that they express CD3 and that the majority of thesecells are ßTCR⁺Thy-1⁺. Most of the Thy-1⁺CD4⁺ßTCR⁺ cells resemble memory T cells based on their expressionof CD44, L-selectin and CD45RB antigens. The vast majority ofThy-1⁺CD4⁺ßTCR⁺ FGT cells are CD5⁺ and all of themare B220^–. Systemic stimuli including infection with Trypanosomabrucel brucel, injection with anti-CD3, or bacterial superantigensstaphylococcal enterotoxin A or B cause a rapid accumulationof CD4+cells in the FGT exceeding that observed for CD4⁺ cellsin spleen and lymph nodes (LN). Expansion of the FGT CD4⁺ cells,which are phenotypically distinct from the splenic and LN CD4⁺T cells, is due to local proliferation rather than an influxof cells from the circulation. The CD4⁺ population in the FGTof adult nu/nu mice is dramatically reduced, indicating itsthymic dependency. In lpr/lpr mice, FGT CD4 cells do not displaychanges characteristic of splenic or LN CD4 cells in the sameanimals. These findings demonstrate that the CD4⁺ cells of themurine FGT are thymic dependent, but that they constitute aT cell lineage that phenotypically and, probably functionally,is distinct from other peripheral CD4⁺ T cell populations.     

Heterogeneity within medullary-type TCR{alpha}{beta}+CD3+CD4-CD8+ thymocytes in normal mouse thymus

Tian Tian, Jun Zhang,     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Frequent deletion of the transgene in T cell receptor {beta} chain transgenic mice

TCR V_ß8.1 transgenic mice were generated using a genomicTCR V_ß gene construct under the control of its promoterand enhancer. Among three lines of transgenic mice, one lineexpressed the transgenic TCR on only 70% of peripheral T cells,while the other two lines expressed it on almost all matureT cells. T cells which lacked expression of the transgenic TCRß chain expressed endogenous TCR ß chains.The molecular basis underlying the lack of transgene expressionin T cells of this line of transgenic mice was investigated.The transgenic TCR^– cells were isolated by two methods.First, Thy-1⁺ V_ß8.1/8.2^– cells were purifiedfrom peripheral T cells using cell sorting. Second, transgenicTCR^– T cell clones were established. In both cases, Southernblotting indicated that V_ß8.1^– T cells had deletedthe transgenic TCR gene. Thus, deletion of the transgenic TCRcan occur in a high proportion of T cells, which allows rearrangementand expression of endogenous TCR ß chains.     

Activation of functionally distinct subsets of CD4+ T lymphocytes.

The studies summarized in this review have established that cloned lines of CD4+ T cells that produce distinct cytokines differ markedly in their responses to different forms of antigenic stimulation. Furthermore, we are beginning to develop experimental systems for better defining the signals that stimulate the differentiation of resting T cells into functionally distinct subsets. From these studies it is possible to construct the following hypothetical model for the differentiation of mature CD4+ T cells. Resting cells produce IL2 as the principal growth factor, IFN gamma, and little or no IL4. Antigenic stimulation in the presence of IL4 (which may be produced by non-T cells) leads to the preferential expansion of IL4-producing cells. These cells secrete their cytokines maximally when stimulated with antigens presented by B cells, which are also the principal targets of these cytokines. Continued expansion of IL4-producing T cells may require antigen exposures that also stimulate the production of IL1 by macrophages. In the absence of IL4 and IL1 (and in the presence of costimulators that are not yet defined) the T cells that are preferentially expanded belong to the IL2-producing subset. In addition, each subset may produce cytokines that stimulate the expansion of that subset and inhibit the other (Fiorentino et al., 1989). It is apparent that a number of assays and reagents need to be developed if these results are to be extended to physiologic immune responses. First, it is important to identify surface molecules that may serve as phenotypic markers for functionally distinct subsets of CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors

The majority of thymocytes are immature cells co-expressing the surface markers CD4 and CD8. About two thirds of these cells also express the T cell antigen receptor (TcR), though at a level distinctly lower than found on mature T cells. The direct precursors of these "double-positive" thymocytes are cycling cortical blast cells of the CD4-8+ phenotype. Using a new monoclonal antibody to a constant determinant of the rat TcR alpha/beta, it is shown here that (a) about 50% of these CD8 "single-positive" committed precursor cells already express the TcR alpha/beta, though at very low levels, (b) during short-term suspension culture in medium supplemented only with fetal calf serum they not only acquire CD4 but also TcR alpha/beta levels characteristic of CD4,8 "double-positive" thymocytes, and (c) cross-linking of the TcR during culture inhibits the acquisition of the CD4 antigen in the majority of these cells.     

IL-4 producing CD4+ TCR{alpha} {beta}int liver lymphocytes: influence of thymus, {beta}2-microglobulin and NK1.1 expression

The present report describes developmental, phenotypic and functionalfeatures of unconventional CD4⁺ TCRß lymphocytes.In C57BL/6 mice, the majority of liver lymphocytes expressingintermediate intensity of TCRß (TCRß^int)are CD4⁺NK1.1⁺ and express a highly restricted TCR V_ßrepertoire, dominated by V_ß8 with some contributionby V^ß7 and V^ß2. Although these cells expressthe CD4 co-receptor, they are present in H2-l Aß (Aß)^+/–gene disruption mutants but are markedly reduced in ß₂-microglobulin(ß₂m)^–/– mutant mice and hence are ß₂mdependent. Thymocytes expressing the CD4⁺NK1.1⁺ TCR ßphenotype are also (ß₂m) contingent, suggesting thatthese two T lymphocyte populations are related. The CD4⁺NK1.1⁺TCRßlymphocytes in liver and thymus share several markers such asLFA-1⁺, CD44⁺, CD5⁺, LECAM-1^– and IL-2Rßa. TheCD4⁺NK1.1 ⁺ TCRß^int liver lymphocytes were not detectedin athymic nuinu mice. We conclude that ß₂m expressionis crucial for development of the CD4⁺NK1.1⁺ TCRß^intliver lymphocytes and that thymus plays a major role. CD4⁺ TCRß^intliver lymphocytes were also identified in NK1.1⁺ mouse strains,there lacking the NK1.1 marker. We assume that the NK1.1 moleculeis a characteristic marker of the CD4⁺TCR"^int liver lymphocytesin NK1.1⁺ mouse strains, although its expression is not obligatoryfor their development. The liver lymphocytes from +₂m^+/–,but not from +₂m^–/–mice are potent IL-4 producersin response to CD3 or TCRß engagement and the IL-4production by liver lymphocytes was markedly reduced by treatmentwith anti-NK1.1 mAb. We conclude that the CD4+NK1.1⁺ TCRß^intliver lymphocytes are capable of producing IL-4 in responseto TCR stimulation.     

Proliferation and apoptosis of B220+ CD4 CD8 TCR{alpha}{beta}intermediate T cells in the liver of normal adult mice: implication for Ipr pathogenesis

Small numbers of T cells have been isolated from the normalmouse liver and many of these are of the CD4^–CD8^–TCRß⁺phenotype. Larger numbers of such cells are present in the liversof mice homozygous for the Ipr mutation and the liver has beenproposed to be the site of an extrathymlc T cell developmentpathway that is expanded in Ipr/lpr mice. Using a modified separationprocedure that increases the liver T cell yield, we have beenable to characterize a subset of CD4^–CD8^–TCRß^intermediateT cells that express the B220 epltope of the CD45 molecule,and resemble in this and many other ways the accumulating Tcells in Ipr lymph nodes. These cells are an actively dividingpopulation and even in healthy, unmanipulated mice a large proportionof them are undergoing apoptosis. We propose the model thatthe normal liver is a major site for T cell destruction andthat the Ipr defect results in failure of this process withleakage of B220⁺CD4^–CD8^–TCRß⁺ cells fromthe liver to peripheral lymphoid tissues, particularly lymphnodes.     

Positive selection of V beta 2+ CD8+ T cells.

T cells bearing V beta 4, V beta 6, V beta 10, V beta 14, and V beta 17a are positively selected by MHC class I and/or class II molecules with poorly elucidated mechanisms. In this paper levels of V beta 2+ CD4+ and V beta 2+ CD8+ T cells from 33 inbred, five F1 hybrid, and 48 [(C58 x DBA/2)F1 x DBA/2] backcross mice have been examined. The results show that (i) V beta 2+ CD8+ T cells are positively selected by MHC class I H-2k molecules, (ii) this positive selection might be mediated by a non-H-2 ligand(s) in association with the Kk molecule, and (iii) inbred strains of mice, so far examined, do not have endogenous superantigens for deletion of V beta 2+ T cells.     

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells

To evaluate directly the developmental potential of cortical CD4⁺8⁺ thymocytes, highly purified populations of small, nondividing CD4⁺8⁺TCR^low and large, dividing CD4⁺8⁺TCR^high thymocytes from H-2^d mice expressing a transgenic T cell receptor restricted by H-2D^b (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2^b recipient mice. The results show that both populations generate CD4^?8⁺ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.