T cell receptor-independent CD2 signal transduction in FcR+ cells.

CD2 subserves both adhesion and signal transduction functions in T cells, thymocytes, and natural killer (NK) cells. In mature T lymphocytes, CD2-mediated signaling function apparently requires surface expression of T cell receptors (TCRs). In contrast, in CD2+ CD3- NK cells and thymocytes, signal transduction through CD2 is TCR independent. To resolve this paradox and characterize TCR-independent triggering mechanisms, we transfected a human CD2 cDNA into a murine mast cell line, C1.MC/57 (Fc epsilon RI+, Fc gamma RII+, Fc gamma RIII+), which is known to produce interleukin 6 (IL-6) as well as release histamine in response to crosslinking of Fc epsilon RI. In the CD2 transfectant, a combination of anti-T11(2) + anti-T11(3) monoclonal antibodies (mAbs) induced a rise in intracellular free calcium [( Ca2+]i), IL-6 production, and histamine release. As expected, no activation was mediated by the same mAbs in C1.MC/57. F(ab)'s fragments of the activatory combination of anti-T11(2) + anti-T11(3) mAbs induced IL-6 in the CD2-transfected mast cells, demonstrating an Fc gamma receptor ectodomain-independent triggering mechanism. In addition, either intact anti-T11(2) or anti-T11(3) IgG alone, which failed to induce [Ca2+]i mobilization in the transfectant, was able to induce IL-6 production. A mAb directed against both Fc gamma RII (previously denoted as Fc gamma RIIb) and Fc gamma RIII (previously denoted as Fc gamma RIIa) inhibits this induction. These results indicate that: (a) Ca2+ mobilization is not essential for IL-6 production; and (b) crosslinking of CD2 and Fc gamma receptors via intact anti-CD2 IgG stimulates IL-6 production. Thus, CD2-mediated IL-6 production occurs by both Fc receptor ectodomain-independent as well as Fc receptor ectodomain-dependent mechanisms in these nonlymphoid cells. Northern blot analysis demonstrates that although the mast cells do not express CD3 zeta or CD3 eta mRNA, they express Fc epsilon RI gamma mRNA. The latter is a known component of Fc gamma RIII as well as Fc epsilon RI, has significant homology to CD3 zeta/eta, and is thought to have a signal transduction function. In these mast cells, CD2 signaling machinery does not require CD3 zeta/eta and may be linked to the Fc epsilon RI gamma subunit. We predict that this subunit or a related structure may confer a TCR-independent signal transduction pathway upon CD2 in CD3- NK cells, thymocytes, and certain B lymphocytes.

Multiple class I and class II major histocompatibility complex allospecificities are generated with T cell receptor variable (V) domains created by a single Ti beta V gene family.

10 alloreactive cytotoxic T lymphocytes using REX Ti beta variable region (V) gene segments in formation of their antigen/major histocompatibility complex (MHC) T3-Ti receptor were selected, cloned, and characterized in an effort to examine the extent of receptor diversity created by this one V gene family. Multiple and distinct class II as well as class I allospecificities were generated from the formation of different Ti beta V domains. Five allospecificities were directed at various class I epitopes whereas the other five were directed at class II MHC gene products. The following conclusions were drawn: (a) Ti beta V genes do not segregate into those that encode class I and those that encode class II allospecificities; and (b) there is no restriction on the Ti beta V gene pool available to T4+ vs. T8+ T lymphocytes.

CD2 regulates responsiveness of activated T cells to interleukin 12.

Interleukin (IL) 12 is a 70-kD heterodimeric cytokine produced by antigen-presenting cells (APCs) such as macrophages in response to infectious pathogens and interferon (IFN) gamma. The varied immunomodulatory effects of IL-12 include the stimulation of proliferation and IFN-gamma production by T cells, and it also has a central role in the development of the T helper cell type 1 immune phenotype. We undertook the production of antibodies capable of modulating the response of T cells to IL-12, and in the process we discovered two antibodies that inhibited the ability of IL-12 to stimulate T cell proliferation. In this report, we demonstrate that these anti-bodies recognize CD2, and we show how antibodies directed toward either the adhesion domain of CD2 or its ligand, CD58, specifically inhibit IL-12 induced proliferation and IFN-gamma production by phytohemagglutinin-activated T cells, leaving the response to IL-12 unaffected. A three-to fourfold reduction in proliferation and IFN-gamma production was observed at IL-12 concentrations as high as 1 nM, with complete inhibition occurring at < or = 1 pM. This novel effect is not directly mediated at the level of the IL-12 receptor, as shown by the inability of these antibodies to block IL-12 binding to activated T cells. Furthermore, by using activating pairs of CD2 antibodies, we show that CD2 stimulation strongly synergizes with IL-12, even at 0.1 pM, in inducing both T cell proliferation and IFN-gamma production. Cytolytic T lymphocyte-associated antigen 4-immunoglobulin-mediated inhibition of the B7/CD28 interaction did not affect the T cell response to either IL-12 or IL-2, but the removal of APCs selectively diminished the proliferative response to IL-12. Based on this data, we hypothesize that CD2 has a central role in an IL-12/IFN-gamma positive feedback loop between T cell and APC, providing the key functional link via a CD2/CD58 interaction that controls T cell responsiveness to IL-12. This model provides a basis for future investigations aimed at defining the signaling mechanisms that mediate this cytokine-specific regulatory effect of CD2, and it offers insight into how a cytokine receptor and distinct adhesion molecule can interact to modulate responsiveness to that cytokine. In addition, it underscores the possibility that the clinical potential of an immunomodulatory drug like IL-12 may be governed by the presence or absence of specific costimulation.

Lineage-specific expression of a T cell receptor variable gene promoter controlled by upstream sequences.

We have characterized the sequence contribution of DNA 5' of a functionally rearranged TCR promoter (V beta 8.1) on its T lineage-specific expression through the use of the chloramphenicol acetyl-transferase (CAT) reporter gene. A 230-bp fragment located 570 bp upstream of the determined transcription start site of the V beta 8.1 promoter confers a T lineage specificity of expression to a heterologous promoter. The inability of the V beta 8.1 promoter and its associated elements to function in B cells suggests the existence of a mechanism to prevent inappropriate V beta gene expression in B cells. Of considerable interest is the fact that both a B cell-specific and a nontissue-specific enhancer element were incapable of stimulating significant expression of this promoter in B cells. We discuss the implication of these results on the process of rearrangement of both Ig and TCR genes, and the differentiation of the lymphoid system.

Delineation of an immunoregulatory amplifier population recognizing autologous Ia molecules. Analysis with human T cell clones.

Autoreactive T lymphocytes were generated by culturing human peripheral blood mononuclear cells with an antigen-specific major histocompatibility complex (MHC)-restricted autologous inducer T cell, termed RW17C and subsequently cloned in soft agar. The majority of such clones (AC1-13) expressed the T3+T4+T8-T11+Ia+ phenotype and were directed at autologous class II MHC gene products found on B cells, macrophages, and B lymphoblastoid cells as judged by their proliferative response to the latter. For this recognition, the clones employed a T3-Ti molecular complex and a T4 structure analogous to those found on allospecific T cells. Perhaps more importantly, it was observed that the same AC1-13 autoreactive clones (AC) induced autologous B cells to produce high levels of immunoglobulin in the absence of exogenous antigen and could synergize with the RW17C clone to effect maximal B cell Ig production. These results support the notion that such autoreactive cells can function in a physiologic amplifier role by facilitating induction via an internal set of signals (i.e. autologous MHC).

One of the CD3epsilon subunits within a T cell receptor complex lies in close proximity to the Cbeta FG loop.

A recent crystal structure of the N15 alpha/beta-T cell receptor (TCR) in complex with an Fab derived from the H57 Cbeta-specific monoclonal antibody (mAb) shows the mAb fragment interacting with the elongated FG loop of the Cbeta domain. This loop creates one side wall of a cavity within the TCR Ti-alpha/beta constant region module (CalphaCbeta) while the CD and EF loops of the Calpha domain form another wall. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3epsilon ectodomain. By using specific mAbs to mouse TCR-beta (H57) and CD3epsilon (2C11) subunits, we herein provide evidence that only one of the two CD3epsilon chains within the TCR complex is located in close proximity to the TCR Cbeta FG loop, in support of the above notion. Moreover, analysis of T cells isolated from transgenic mice expressing both human and mouse CD3epsilon genes shows that the heterologous human CD3epsilon component can replace the mouse CD3epsilon at this site. The location of one CD3epsilon subunit within the rigid constant domain module has implications for the mechanism of signal transduction throughout T cell development.

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex.

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) showed rearrangement of the TiC beta 1 gene on Southern blot analysis with or without deletion of the other TiC beta 1 allele. In contrast, TiC beta 2 always remained in germline configuration. Moreover, the finding that one additional suppressor clone deleted both TiC beta 1 alleles, maintained a germline TiC beta 2 configuration, and yet actively transcribed TiC beta 2 message suggested that TiC beta 2 is not a pseudogene. Rather, it appeared to be used less frequently than the TiC beta 1 gene and in the absence of detectable DNA rearrangements. Together, these results demonstrate that the functional repertoire (or isotype) of a given subclass of T cells is not encoded within the Ti beta genes.

Structural and binding analysis of a two domain extracellular CD2 molecule.

The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.

Expression of a functional CD3-Ti antigen/MHC receptor in the absence of surface CD2. Analysis with clonal Jurkat cell mutants.

To investigate the requirement for CD2 expression in activation of T lymphocytes via the CD3-Ti antigen/MHC receptor complex, we produced and characterized a series of CD2- Jurkat variants. These mutants lack detectable surface CD2 as determined by indirect immunofluorescence, immunoprecipitation analysis, and specific radiolabeled antibody binding assay, but nevertheless, expressed normal numbers of CD3-Ti receptors. As expected, the combination of anti-CD2 antibodies, termed anti-T112 and anti-T113, which are mitogenic for resting T lymphocytes, failed to stimulate activation of these variants. In contrast, triggering of their CD3-Ti components resulted in the normal set of T lymphocyte-associated activation events, including phosphoinositide turnover, elevation in intracellular free calcium, early gene-induction events, and IL-2 production. Assuming that the Jurkat cell line is representative of normal cycling human T lymphocytes, we conclude that the presence of the CD2 molecule on the plasma membrane is not in itself a requirement for an operational CD3-Ti-alpha/beta receptor.

The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure.

The first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, Lys46, and Arg59, on the C, C', C", and D strands, respectively. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gp120. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.

Dissection of the human CD2 intracellular domain. Identification of a segment required for signal transduction and interleukin 2 production.

To evaluate those residues in the 117 amino acids of the CD2 cytoplasmic domain required for transduction of T lymphocyte activation signals, a full-length human CD2 cDNA and a series of deletion and substitution mutants were inserted into the ovalbumin-specific, I-Ad-restricted murine T cell hybridoma 3DO54.8 using a retroviral system. The resulting cells express surface CD2 protein and unlike the parental murine line, are reactive with murine anti-human CD2 antibodies. Anti-T11(2) plus anti-T11(3) antibody stimulation of cells expressing a full-length CD2 cDNA results in a characteristic rise in cytosolic-free calcium [( Ca2+]i), and subsequent IL-2 secretion that accompany CD2 stimulation in human T lymphocytes. Transfectants expressing CD2 delta C98 and CD2 delta C77, partially deleted CD2 molecules containing the entire extracellular and transmembrane CD2 segments but only 98 and 77 amino acids of the cytoplasmic domain, respectively, are also activated by anti-CD2 mAbs. In contrast, clones expressing more severely truncated CD2 structures, CD2 delta C43 and CD2 delta C18, are not stimulated. These data show that the cytoplasmic domain plays an essential role in transduction of activation signals via CD2, and that the segment between amino acid residues 253 and 278 is necessary for activation. This region contains two tandem repeats of the sequence PPPGHR, thought to form part of a putative cationic site. Disruption of the latter by site-directed mutagenesis does not affect IL-2 gene induction, suggesting that only one of the repeats is required for activating this function of the CD2 molecule.

Peptide variability exists within alpha and beta subunits of the T cell receptor for antigen.

The T cell receptor for antigen (Ti) has recently been identified as a 90-kdalton T3-associated clonotypic structure composed of one 49-51-kdalton alpha and one 43-kdalton beta subunit, which are disulfide linked. Here, Ti molecules from two alloreactive CTL clones derived from the same donor but of differing specificities (CT8III and CT4II) are directly compared following isolation with anticlonotypic monoclonal antibodies. Isoelectric focusing shows that the alpha subunits (pI 4.4-4.7) are more acidic than the beta subunits (pI 6.0-6.2) but that each glycoprotein species is distinctive. More importantly, two-dimensional peptide maps of 125I-labeled surface receptors indicate that the beta chains of Ti1 and Ti2 appear unique and share only two peptides in common. In contrast, peptide maps of Ti1 and Ti2 alpha chains are more related although not identical. These results suggest that the human T cell receptor is composed of constant as well as variable regions and that at least one of the latter is located within the beta subunit.

Location of T cell and major histocompatibility complex antigens in the human thymus.

A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2-microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in sections stained with the immunoperoxidase technique. Many medullary cells reacted with anti-T4--and a smaller fraction with anti-T5, anti-T6, anti-T8, and anti-T10 antibodies. In addition, T1 and T3 antibodies, which react with all peripheral T cells, stained a majority of medullary cells. The medullary cells were also more intensely stained with antibodies directed against beta 2-microglobulin than the majority of cortical cells. Hence, the staining profile of medulla approximates the staining pattern of peripheral T cells, with large numbers of cells bearing T1+, T3+, and T4+ antigens (helper/inducer cells) and a small number of cells bearing T1+, T3+, and T5+/T8+ antigens (suppressor/cytotoxic cells). This supports the conclusion that mature cells present in the medulla are derived from immature cells in the cortex. However, a small number of cells scattered throughout the cortex stained with T1 and T3 antibodies, which suggests that maturation of thymocytes can also occur in the cortex. Antibody directed against Ia antigens resulted in a characteristic patchy pattern of staining in the cortex and in diffuse staining in the medulla, which was interpreted as resulting from staining of epithelial reticulum. The majority of thymocytes did not stain. The staining pattern suggests a close relationship between epithelial cells and thymocytes.

Distribution of T cell subsets in human lymph nodes.

A series of T cell-specific monoclonal antibodies was used to determine the location of T lymphocyte subpopulations in frozen sections of human lymph nodes by means of an immunoperoxidase technique. The majority of cells in the paracortical regions were reactive with anti-T1 and anti-T3 antibodies, which define all mature peripheral T cells. In contrast, the majority of cells within primary follicles were unreactive with anti-T1 and anti-T3 antibodies, but were reactive with anti-Ia and anti-IgM antibodies. In addition, a substantial number of T1+, T3+ cells were found in the germinal centers of secondary follicles on the capsular side. The vast majority of T1+, T3+ cells in the paracortex and the follicles were reactive with anti-T4 antibody, which defines inducer/helper T cells. Only a minority of cells in these areas were reactive with anti-T5 and anti-T8 antibodies, which define cytotoxic/suppressor cells. No lymphocytes were stained with anti-T6 antibody, which reacts with a majority of thymocytes but not with peripheral T cells. Scattered cells in the paracortex showed staining for Ia antigen in an irregular dendritic pattern. The findings demonstrate that the major T cell population found within human lymph node bears the mature T1+, T3+, T4+ phenotype characteristic of inducer T cells. Moreover, the location of this population indicates that they play a role in the induction of B cell differentiation in vivo.

A naturally processed mitochondrial self-peptide in complex with thymic MHC molecules functions as a selecting ligand for a viral-specific T cell receptor.

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4(+)CD8(+) double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K(b) molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K(b)-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K(b). The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68-75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO(68-75)). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2(-/)- transporter associated with antigen processing 1(-/)- fetal thymic organ culture, NUbO(68-75) induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO(68-75) to support differentiation of a second K(b)-restricted TCR indicates that its inductive effects are not general.

Involvement of the PPPGHR motif in T cell activation via CD2.

Prior studies identified a segment of the CD2 cytoplasmic domain between amino acid (aa) residues 253 and 287 as important in T lymphocyte signal transduction. This region contains two repeats of the sequence motif PPPGHR, thought to form a "cage" structure involved in CD2-mediated signaling. To evaluate this segment, a series of mutant human CD2 molecules were produced by oligonucleotide-directed mutagenesis and inserted into the ovalbumin-specific, I-Ad-restricted murine T-T hybridoma 3DO54.8 using the DOL retroviral system. CD2 M1 (271-272), CD2 M2 (278-279), and CD2 M4 (264-265) mutants replaced the positively charged adjacent aa histidine and arginine (HR) in the wild-type CD2 sequence with aspartic and glutamic acid (DE) at positions 271-272, 278-279, and 264-265, respectively. In addition, a truncation mutant, CD2 M3 (268), containing only 57 of the 117 cytoplasmic aa and terminating before the second PPPGHR sequence, was generated. Stimulation of transfectants CD2 FL, CD2 M1 (271-272), and CD2 M2 (278-279) with anti-T11(2) + anti-T11(3) antibodies resulted in a rise in cytosolic-free calcium [( Ca2+]i) and subsequent interleukin 2 (IL-2) secretion. In contrast, CD2 M4 (264-265) transfectants could not be activated in either assay. Thus, alteration of histidine 264 and/or arginine 265 within the first PPPGHR motif affects the process of signal transduction via CD2, whereas identical mutations in residues at 271-272 or 278-279 were individually without effect. Consistent with these data, CD2 M3 (268) transfectants were able to generate a detectable amount of IL-2 via CD2 triggering. These data support the notion that the PPPGHR motif at aa 260-265 is important for activation of T lymphocytes via the CD2 molecule.

Regional location of T cell receptor gene Ti alpha on human chromosome 14.

The chromosomal location of Ti alpha was determined by hybridization of a radiolabeled cDNA for the alpha chain of human T cell receptor with 12 human X mouse cell hybrid DNAs cleaved with BamHI. Seven hybrids contained human Ti alpha, while the remaining five lacked it. Only human chromosome 14 matched the distribution of human Ti alpha signal across the mapping panel. Hybrids segregating a chromosome 14 translocation were used to demonstrate that Ti alpha is in the region 14pter greater than 14q21. Thus, the alpha and beta chain genes that contribute structural components to the Ti moiety of the human T cell receptor lie on different chromosomes. In humans, the immunoglobulin heavy chain locus and Ti alpha are in different regions of chromosome 14, with Ti alpha more proximal and the immunoglobulin heavy chain locus more distal.

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.

A lymphokine that activates the cytolytic program of both cytotoxic T lymphocyte and natural killer clones.

A 10-12 kD lymphokine, herein termed TCAF, was recently shown to be secreted from Th after crosslinking of their antigen/MHC (T3-Ti) receptors. TCAF stimulates resting T lymphocyte proliferation via binding to surface components of the T11 pathway. To determine whether TCAF could induce antigen-independent activation of the lytic machinery of cytotoxic cells, the present studies were conducted. In the presence of TCAF, both T8+ class I MHC-specific and T4+ class II MHC-specific cytotoxic T cell clones were induced to kill targets, including those lacking the appropriate MHC molecules. This effect was unique to TCAF, since IL-1, IL-2, IFN-gamma could not stimulate lytic activity. Furthermore, both T3+T11+ and T3-T11+ NK clones were triggered to lyse NK-resistant target cells. These findings suggest that TCAF can function in an antigen-independent fashion to amplify cytotoxic effector responses.

T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of CD3 zeta and CD3 eta components: a novel isoform expressed on large granular lymphocytes.

CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha-beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets.