Mature Αβ Research Articles

Unexpected Role for the B Cell-Specific Src Family Kinase B Lymphoid Kinase in the Development of IL-17–Producing γδ T Cells

The Ag receptors on αβ and γδ T cells differ not only in the nature of the ligands that they recognize but also in their signaling potential. We hypothesized that the differences in αβ- and γδTCR signal transduction were due to differences in the intracellular signaling pathways coupled to these two TCRs. To investigate this, we used transcriptional profiling to identify genes encoding signaling molecules that are differentially expressed in mature αβ and γδ T cell populations. Unexpectedly, we found that B lymphoid kinase (Blk), a Src family kinase expressed primarily in B cells, is expressed in γδ T cells but not in αβ T cells. Analysis of Blk-deficient mice revealed that Blk is required for the development of IL-17-producing γδ T cells. Furthermore, Blk is expressed in lymphoid precursors and, in this capacity, plays a role in regulating thymus cellularity during ontogeny.

The Importance of LAT in the Activation, Homeostasis, and Regulatory Function of T Cells

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T(reg) cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.

Position-Dependent Silencing of Germline Vβ Segments on TCRβ Alleles Containing Preassembled VβDJβCβ1 Genes

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

Expanded γδ T cell populations in absence of transcription factor PU.1 (36.49)

Abstract T cell development is characterized by a succession of developmental stages that lead to the generation of mature αβ and γδ T cells. γδ T cells are considered innate cells that critically contribute to host defense. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi-1) is essential for early stages of T cell development and is down regulated during the pro T-cell stage. PU.1 is expressed in Th2 cells but not Th1 cells. Expression of PU.1 in other T cell subsets has not been studied extensively. In this study, we show that PU.1 is expressed in γδ T cells, and in mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpilck-/-) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild type mice. The increase in γδ T cell numbers in PU.1 deficient mice is reflected in several subsets examined (Vγ2T, Vγ3T and Vδ4T). In mice where PU.1 was deleted using a CD4-Cre transgene, the expansion of γδ T cell numbers was restricted to the intestinal population. PU.1-deficient γδ T cells secrete higher levels of IL-17 than wild type cells, but similar levels of IFN-γ. Thus, our data show that PU.1 is expressed in γδ T cells and that deletion of PU.1 results in γδ T cell expansion. This suggests that PU.1 has a negative influence on γδ T cell development or expansion.

CD4-CD8 Lineage Differentiation: Thpok-ing into the Nucleus

The mature alphabeta T cell population is divided into two main lineages that are defined by the mutually exclusive expression of CD4 and CD8 surface molecules (coreceptors) and that differ in their MHC restriction and function. CD4 T cells are typically MHC-II restricted and helper (or regulatory), whereas CD8 T cells are typically cytotoxic. Several transcription factors are known to control the emergence of CD4 and CD8 lineages, including the zinc finger proteins Thpok and Gata3, which are required for CD4 lineage differentiation, and the Runx factors Runx1 and Runx3, which contribute to CD8 lineage differentiation. This review summarizes recent advances on the function of these transcription factors in lineage differentiation. We also discuss how the "circuitry" connecting these factors could operate to match the expression of the lineage-committing factors Thpok and Runx3, and therefore lineage differentiation, to MHC specificity.

Changes in bulk properties and molecular compositions within New Zealand Coal Band solvent extracts from early diagenetic to catagenetic maturity levels

The bulk properties and bitumen molecular compositions of a rank-series of 38humic coals from the New Zealand Coal Band (Cretaceous–Cenozoic) have been analysed to investigate early maturation processes affecting coaly organic matter through diagenesis to moderate catagenesis (Rank(Sr) 0.0–11.8, Ro 0.23–0.81%). The samples comprise a relatively restricted range of vitrinite rich coal types formed largely from higher land plant material under relatively oxic conditions, but with a significant contribution from microbial biomass. With increasing rank, total organic carbon contents show a general increase, whereas moisture and asphaltene contents decrease. Bitumen yields also decrease through the stages of diagenesis and early catagenesis (Rank(Sr)<9, Ro<0.55%), indicating partial loss of initial bitumen during early maturation. Thermal generation of hydrocarbons begins slowly at Rank(Sr)∼5–6 (Ro∼0.40%) as indicated by the constant occurrence and gradual increase of isoprenoids (e.g., pristane and phytane) and hopanoids in their more mature αβ configuration. This early phase of catagenesis, not previously recognised in New Zealand coals, is followed at Rank(Sr)∼9 (Ro∼0.55%) by the main catagenesis phase characterised by a more rapid increase in the generation of hydrocarbons, including total n-alkanes, isoprenoids and αβ-hopanes. Changes in the maturity of New Zealand coals can be traced by the Carbon Preference Index and several hopane maturity parameters, including 22S/(22S+22R), αβ/(αβ+βα) and ββ/(αβ+βα+ββ).

Central memory CD8+ T cells induce graft-versus-host disease and mediate graft-versus-leukemia.

In allogeneic hemopoietic stem cell transplantation, mature donor alphabeta T cells in the allograft promote T cell reconstitution in the recipient and mediate the graft-vs-leukemia (GVL) effect. Unfortunately, donor T cells can attack nonmalignant host tissues and cause graft-vs-host disease (GVHD). It has previously been shown that effector memory T cells not primed to alloantigen do not cause GVHD yet transfer functional T cell memory and mediate GVL. Recently, central memory T cells (T(CM)) have also been reported to not cause GVHD. In contrast, in this study, we demonstrate that purified CD8(+) T(CM) not specifically primed to alloantigens mediate GVHD in the MHC-mismatched C57BL/6 (B6)-->BALB/c and the MHC-matched, multiple minor histocompatibility Ag-mismatched C3H.SW-->B6 strain pairings. CD8(+) T(CM) and naive T cells (T(N)) caused similar histological disease in liver, skin, and bowel. B6 CD8(+) T(CM) and T(N) similarly expanded in BALB/c recipients, and the majority of their progeny produced IFN-gamma upon restimulation. However, in both models, CD8(+) T(CM) induced milder clinical GVHD than did CD8(+) T(N). Nonetheless, CD8(+) T(CM) and T(N) were similarly potent mediators of GVL against a mouse model of chronic-phase chronic myelogenous leukemia. Thus, in contrast to what was previously thought, CD8(+) T(CM) are capable of inducing GVHD and are substantially different from T(EM) but only subtly so from T(N).

Overlapping functions of human CD3δ and mouse CD3γ in αβ T-cell development revealed in a humanized CD3γ-deficient mouse

AbstractHumans lacking the CD3γ subunit of the pre-TCR and TCR complexes exhibit a mild αβ T lymphopenia, but have normal T cells. By contrast, CD3γ-deficient mice are almost devoid of mature αβ T cells due to an early block of intrathymic development at the CD4–CD8– double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3δ chain replaces CD3γ during αβ T-cell development. To determine whether human CD3δ (hCD3δ) functions in a similar manner in the mouse in the absence of CD3γ, we introduced an hCD3δ transgene in mice that were deficient for both CD3δ and CD3γ, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3δ efficiently supported pre-TCR–mediated progression from the DN to the CD4+CD8+ double-positive (DP) stage. However, αβTCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3γ-deficient TCR complexes that had incorporated hCD3δ displayed abnormalities in structural stability resembling those of T cells from CD3γ-deficient humans. Taken together, these data demonstrate that CD3δ and CD3γ play a different role in humans and mice in pre-TCR and TCR function during αβ T-cell development.

Crystal structure of plant asparaginase

Abstract In plants, specialized enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. In addition, K + -independent plant asparaginases are also active in splitting the aberrant isoaspartyl peptide bonds, which makes these proteins important for seed viability and germination. Here, we present the crystal structure of potassium-independent l -asparaginase from yellow lupine (LlA) and confirm the classification of this group of enzymes in the family of Ntn-hydrolases. The α- and β-subunits that form the mature (αβ) 2 enzyme arise from autoproteolytic cleavage of two copies of a precursor protein. In common with other Ntn-hydrolases, the (αβ) heterodimer has a sandwich-like fold with two β-sheets flanked by two layers of α-helices (αββα). The nucleophilic Thr193 residue, which is liberated in the autocatalytic event at the N terminus of subunit β, is part of an active site that is similar to that observed in a homologous bacterial enzyme. An unusual sodium-binding loop of the bacterial protein, necessary for proper positioning of all components of the active site, shows strictly conserved conformation and metal coordination in the plant enzyme. A chloride anion complexed in the LlA structure marks the position of the α-carboxylate group of the l -aspartyl substrate/product moiety. Detailed analysis of the active site suggests why the plant enzyme hydrolyzes asparagine and its β-peptides but is inactive towards substrates accepted by similar Ntn-hydrolases, such as taspase1, an enzyme implicated in some human leukemias. Structural comparisons of LlA and taspase1 provide interesting insights into the role of small inorganic ions in the latter enzyme.

Crystal Structure of Plant Asparaginase

In plants, specialized enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. In addition, K +-independent plant asparaginases are also active in splitting the aberrant isoaspartyl peptide bonds, which makes these proteins important for seed viability and germination. Here, we present the crystal structure of potassium-independent l-asparaginase from yellow lupine (LlA) and confirm the classification of this group of enzymes in the family of Ntn-hydrolases. The α- and β-subunits that form the mature (αβ) 2 enzyme arise from autoproteolytic cleavage of two copies of a precursor protein. In common with other Ntn-hydrolases, the (αβ) heterodimer has a sandwich-like fold with two β-sheets flanked by two layers of α-helices (αββα). The nucleophilic Thr193 residue, which is liberated in the autocatalytic event at the N terminus of subunit β, is part of an active site that is similar to that observed in a homologous bacterial enzyme. An unusual sodium-binding loop of the bacterial protein, necessary for proper positioning of all components of the active site, shows strictly conserved conformation and metal coordination in the plant enzyme. A chloride anion complexed in the LlA structure marks the position of the α-carboxylate group of the l-aspartyl substrate/product moiety. Detailed analysis of the active site suggests why the plant enzyme hydrolyzes asparagine and its β-peptides but is inactive towards substrates accepted by similar Ntn-hydrolases, such as taspase1, an enzyme implicated in some human leukemias. Structural comparisons of LlA and taspase1 provide interesting insights into the role of small inorganic ions in the latter enzyme.

Revision of T cell receptor α chain genes is required for normal T lymphocyte development

To become mature alphabeta T cells, developing thymocytes must first assemble a T cell receptor (TCR) beta chain gene encoding a TCRbeta chain that forms a pre-TCR. These cells then need to generate a TCRalpha chain gene encoding a TCRalpha chain, which, when paired with the TCRbeta chain, forms a selectable alphabeta TCR. Newly generated VJalpha rearrangements that do not encode TCRalpha chains capable of forming selectable alphabeta TCRs can be excised from the chromosome and replaced with new VJalpha rearrangements. Such replacement occurs through the process of TCRalpha chain gene revision whereby a Valpha gene segment upstream of the VJalpha rearrangement is appended to a downstream Jalpha gene segment. A multistep, gene-targeting approach was used to generate a modified TCRalpha locus (TCRalpha(sJ)) with a limited capacity to undergo revision of TCRalpha chain genes. Thymocytes from mice homozygous for the TCRalpha(sJ) allele are defective in their ability to generate an alphabeta TCR. Furthermore, those thymocytes that do generate an alphabeta TCR have a diminished capacity to be positively selected, and TCRalpha(sJ/sJ) mice have significantly reduced numbers of mature alphabeta T cells. Together, these findings demonstrate that normal T cell development relies on the ability of developing thymocytes to revise their TCRalpha chain genes.

Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection

The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR–mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4−CD8−CD44−CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in αβ TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the αβ TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an αβ TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature αβ TCR in primary cells of the T lineage.

The Spectrum of Human T Cell Receptor (TCR)-Vβ Frequencies Are Established Prior to Thymic Selection.

Human T cell development is characterized by three phases of clonal selection and expansion: (1) β-selection which occurs independent of ligand recognition; (2) Positive thymic selection resulting in the outgrowth of T cell clones with an intermediate affinity for self-MHC/ self-peptide complexes; (3) Post-thymic antigen-driven expansion. The T cell receptor (TCR) Vβ frequencies in the peripheral blood could therefore be determined by T cell selection and expansion at one or all phases. To determine whether Vβselection patterns originate at the first phase of thymic proliferation we used flow cytometric analysis to quantitate 21 TCRVβ proteins in peripheral blood T cells of 23 HLA-disparate healthy adults. A common rank order of TCRVβ allele frequencies was found in both CD4 and CD8 T cell subsets (figure A). We found the same spectrum of HLA-independent TCR Vβ frequencies in three published studies of healthy adults. Furthermore, the percentages of CD4 cells expressing a particular Vβ were almost identical in a total of 151 data points, and was significantly correlated in CD8 cells (R2 = 0.79; slope 0.61; p < 0.00001). A significant correlation in TCRVβ allele frequencies in CD4 cells and CD8 cells was identified in all data sets (R2 = 0.73, slope 0.54, p < 0.00001)(figure B). Individuals over 50 years of age showed a 5-fold increase in variability of Vβ frequency in CD4 cells than younger subjects. The difference in the median values was statistically significant (p = 0.03) using a permutation test, indicating that post-thymic antigen recognition skewed the mature αβ T cell repertoire predominantly in elderly individuals. Our data therefore indicate that the patterns of TCRVβ protein frequencies are shaped during β-selection, conserved throughout life and are only weakly modified by antigen-driven thymic and post-thymic selection and expansion. A genetic basis for this underlying TCR Vβ frequency pattern remains undefined since there was no association of TCRVβ expression frequencies with chromosomal (5′-3′) organization. However, the reproducible pattern of TCR Vβ frequencies between individuals of diverse HLA types and distinct origins suggests that they are determined by a common genetic mechanism. [Display omitted]

Early ontogeny of thymocytes in pigs: sequential colonization of the thymus by T cell progenitors.

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3epsilon-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3epsilonhigh and belonged to the gammadelta T cell lineage. Mature CD3epsilonhigh alphabeta thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3epsilonlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Ralpha (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRgammadelta+ thymocytes that were cycling late during their development in the thymus.

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM.

Major histocompatibility complex (MHC) class II molecules bind and present to CD4(+) T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II-Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide-binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii-class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H-2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H-2DM. The Ii-free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide-loaded complexes.

CD3 IMMUNODEFICIENCIES

CD3 chains associated with calnexin or with TCR molecules are first expressed and used by T cells early during their intrathymic development. Accordingly, CD3 deficiencies strongly influence early T-cell differentiation events, including the up-regulation of the TCR/ CD3 complex itself. Available data from human and murine CD3 deficiencies indicate that CD3 chains play specialized roles in the support of TCR chains and the transduction of signals that emanate from them. Because expression (or aggregation) is a requirement for signaling, the chains that are crucial supporters also strongly affect signaling. CD3ε and ζ, which occur in pairs in the minimal TCR/CD3 complex (see Fig. 1), seem to be important supporters, because inactivating their genes virtually abolishes TCR/CD3 expression. CD3-γ and δ, in contrast, are less crucial for TCR/CD3 expression, particularly by CD4 + cells, but they do have dissimilar signaling roles; CD38, but not CD37, is apparently dispensable for the development and TCR expression of the γδ T-cell lineage. T lymphocytes cannot predict the isotype (αβ, γδ and surrogates thereof) or the specificity (CD1, MHC class I or class II) of the particular TCR with which they are endowed. They do not know whether their TCR has to transduce maturation/activation, anergy or apoptosis signals, or whether it has to aggregate, for instance, with CD4 or CD8 or with CD45RA or RO for antigen recognition. They may carry a range of adaptor CD3 chains capable of supporting and signalling for different TCR ensembles (αβ, γδ, pTαβ, calnexin) in different phenotypic backgrounds (CD4, CD8) and with different functional outcomes (maturation/activation, anergy, apoptosis). Because of the large extracellular domains of CD3γ, δ, and ε, the authors favor the interpretation that these CD3 chains fulfil selective extracellular docking or bumper roles in the macromolecular ensemble that recognizes antigens. Intracellularly, they may be linked to selective downstream signaling pathways. CD3γ, for instance, seems to be dispensable for inducing a proliferative or cytolytic response and TNF-α, but not IL-2 synthesis, in mature αβ T cells. Testing for a broader range of TCR-induced events in human and murine CD3-deficient mature T cells may shed light on the common and selective roles of CD3 chains in the TCR/CD3 complex.

In vivo roles of receptor tyrosine kinases and cytokine receptors in early thymocyte development

The early phases of T-cell development require both cell—cell interactions and soluble factors provided by stromal cells within the thymic microenvironment. Still, the precise nature of the signals delivered in vivo by cytokines (resulting in survival, proliferation or differentiation) remains unclear. Recent studies using mice deficient in cytokines or in their receptors have helped to identify essential signaling pathways required for the development of intrathymic precursors to mature αβ and γβ T cells. In addition, cytokine requirements for the development of natural killer cells were revealed in such mutants. The results obtained demonstrate that the development of all classes of lymphocytes (natural killer, γδ T cells and αβ T cells) is cytokine dependent, but the specific requirements differ for each lineage.

Donor γδ T Lymphocytes Promote Allogeneic Engraftment Across the Major Histocompatibility Barrier in Mice

AbstractT cells that express the αβ T-cell receptor are thought to be the T-cell population primarily responsible for facilitating alloengraftment. The role of γδ+ T cells that comprise only a minority of mature T cells in promoting allogeneic engraftment, however, has not been extensively studied. The purpose of this study was to determine whether γδ T cells were capable of facilitating alloengraftment in murine recipients of major histocompatibility complex-mismatched marrow grafts. We developed a model where engraftment of C57BL/6 × 129/F2 (H-2b) marrow in sublethally irradiated (800 cGy) recipients (AKR/J, H-2k) is dependent on the presence of mature donor T cells in the marrow graft. In this model, donor T-cell engraftment was significantly augmented by as few as 1 × 105 αβ T cells. The role of γδ T cells was then investigated using transgenic donors (C57BL/6 × 129 background) in which a portion of the T-cell receptor–β chain gene was deleted by gene targeting so that these mice lack αβ T cells. Addition of 10 × 106 naive γδ T cells to T-cell depleted marrow grafts was required to significantly increase alloengraftment, although donor T cells averaged <50% of total splenic T cells. To determine whether higher doses of γδ T cells would improve donor engraftment and eradicate residual host T cells, γδ T cells were ex vivo expanded with a γδ T-cell–specific monoclonal antibody and interleukin-2 and then transplanted into irradiated recipients. Transplantation of ≥ 160 × 106 activated γδ T cells was necessary to consistently and significantly augment donor cell chimerism and enhance hematopoietic reconstitution when compared to control mice, but host T cells persisted in these chimeras. Addition of 2.5 × 104 mature αβ T cells, which alone were incapable of facilitating engraftment, to T-cell depleted marrow grafts containing 160 × 106 activated γδ T cells resulted in long-term (<100 day) complete donor engraftment, indicating that limiting numbers of αβ T cells were required in the marrow graft for the eradication of residual host T cells. Using serial weight curves and B-cell reconstitution as end points, clinically significant graft-versus-host disease was not observed in these chimeras under these experimental conditions. These data show that, whereas less potent than αβ T cells, γδ T cells are able to promote engraftment and enhance hematopoietic reconstitution in allogeneic marrow transplant recipients.

Human γδ Cells Are Resistant to Induction of Anergy but Not to Induction of Cell Death in Vitro

A condition of hyporesponsiveness can be induced in certain mature human αβ (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component or immune responsiveness. Little is known about whether the same condition applies to γδ (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-Ig, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (>50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.

Inhibition of peptide binding to DR molecules by a leupeptin-induced invariant chain fragment

Loading of peptides onto DR molecules was studied by characterizing precursors of the mature peptide-DR complexes expressed at the surface of B cells. Since invariant chain (Ii) prevents binding of peptides by DR molecules, it was speculated that analysis of complexes between DR heterodimers and proteolytic fragments of Ii offers the possibility to examine how DR molecules and peptides assemble. Using a procedure combining a two-step affinity chromatography and gel filtration, we isolated from leupeptin-treated B cells complexes between DR molecules and N-terminal Ii fragments previously called “leupeptin-induced polypeptides” (LIP; Blum and Cresswell, 1988, Proc. natn. Acad. Sci. U.S.A. 85, 3975–3979). It was observed that the most prominent LIP fragment has a relative molecular mass (M r) of 16 kDa. In addition, we show that this polypeptide species does not bear N-linked glycans, indicating that this fragment does not extend beyond residue 129 of Ii. Similarly to DR αβ heterodimers associated with the full length 33 and 35 kDa Ii forms, DR αβ heterodimers associated with LIP fragments are unstable in sodium dodecyl sulfate (SDS) at ambient temperature, whereas mature DR αβ heterodimers are resistant to dissociation with SDS. These results are indirect evidence that LIP-DR complexes are devoid of bound peptides. This possibility was supported by showing that LIP-DR complexes fail to bind a radioiodinated tetanus toxin peptide ( 125I-p2), while DR molecules, which are spontaneously released from complexes with LIP fragments, bind the labeled peptide. These results demonstrate that association with LIP fragments is sufficient to prevent binding of peptides by DR molecules. This notion was further documented by showing that binding of 125I-p2 on DR heterodimers is inhibited by preparations of LIP fragment. By contrast, a soluble recombinant fragment corresponding to the extracytoplasmic region of Ii did not block 125I-p2 binding. The results presented in this study indicate that the cytoplasmic and/or transmembrane region of Ii is required to prevent peptide binding by DR molecules, while the extracytoplasmic portion of Ii, though capable of associating with DR molecules, lacks the capacity to block peptide binding.