The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.

To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.

Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes.

Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.

Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.

A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell survival is described. Major histocompatibility complex class II-deficient mice were engineered to express Ealpha chains only in thymic epithelial cells in a tetracycline (tet)-controllable manner. This resulted in tet-responsive display of cell surface E complexes, positive selection of CD4(+)8(-) thymocytes, and generation of a CD4(+) T cell compartment in a class II-barren periphery. Using this system, we have addressed two unresolved issues: the half-life of naive CD4(+) T cells in the absence of class II molecules (3-4 wk) and the early signaling events associated with class II molecule engagement by naive CD4(+) T cells (partial CD3 zeta chain phosphorylation and ZAP-70 association).

The enhancer in an MHC class II gene, in vitro and in mouso.

The E alpha class II gene of the major histocompatibility complex is expressed in a variety of immunocompetent cells. Part of the control of tissue-specific expression is mediated by a block of sequences found far upstream of the transcriptional startsite; this stretch is necessary for expression in the B lymphocytes of transgenic mice, but largely dispensable elsewhere. We review the evidence for the role of this region in E alpha transcription in transgenic animals, as well as data from transfections into tissue-culture cells, which indicate that this region has non-specific enhancer activity. We discuss possible models to explain how a non-specific enhancer can participate in cell-specific control.

Prevention of nitrogen mustard-induced apoptosis in normal and transformed lymphocytes by ebselen.

The alkylating agent, nitrogen mustard (HN2), is thought to cause apoptosis through production of free oxygen radicals. To explore the mechanism of HN2-induced apoptosis, we utilized ebselen, a selenoorganic compound with potent antioxidant activity. We examined whether ebselen would inhibit apoptosis in BALB/c mouse spleen lymphocytes and human MOLT-4 leukemia cells treated with HN2 (2.5 microM) in vitro. Non-toxic concentrations (<50 microM) of ebselen were found to prevent HN2-induced apoptosis of murine lymphocytes in a dose-dependent manner, as measured by cell viability, hypodiploid DNA formation, and phosphatidylserine externalization. However, ebselen was ineffective at preventing spontaneous apoptosis in these cells, pointing to the selectivity of its action. Furthermore, pretreatment with ebselen at 1-10 microM for 72 hr protected MOLT-4 cells from HN2-induced apoptosis and maintained cell viability and proliferation as monitored by the above-mentioned parameters. This was accompanied by the preservation of mitochondrial transmembrane potential and elevated glutathione levels and by a blockage of caspase-3 and -9 activation. In vivo, ebselen also had a marked protective effect against spleen weight loss associated with lymphocyte apoptosis in mice treated by HN2. Therefore, ebselen provides an efficient protection against HN2-induced cell death in normal and tumoral lymphocytes and might prove useful as an antidote against alkylating agents.

Neonatal mouse CD4+ mature thymocytes show responsiveness to interleukin 2 and interleukin 7: growth in vitro of negatively selected V beta 6- and V beta 11-expressing CD4+ cells from (C57BL/6 x DBA/2)F1 mice.

By three colour flow microfluorimetry, we have recently shown that neonatal mouse CD4 single positive thymocytes are a population of proliferating cells. Furthermore, analysis of CD4+ thymocytes from (C57BL/6 x DBA/2)F1 mice showed that they proliferate regardless of whether they express particular V beta-encoded TCR molecules (V beta 6 and V beta 11) that are undergoing intrathymic deletion. In this report, cell culture experiments demonstrate that unstimulated neonatal CD4+ thymocytes from such mice proliferate in vitro in response to a combination of r-IL-2 and r-IL-7. Simultaneous three colour analysis of V beta TCR, CD4 expression, and DNA content of these cultured cells shows that V beta 6+, -8+, and -11+ cells grow equally well. Experiments where cells were cultured overnight in unsupplemented medium did not reveal preferential loss of negatively selected (V beta 6+ and V beta 11+) subpopulations of CD4+ cells. Taken together, these results suggest that IL-2 and IL-7 play a role in the intrathymic proliferation of developing mature T cells.

More efficient positive selection of thymocytes in mice lacking terminal deoxynucleotidyl transferase.

Mice with a drastic mutation in the terminal deoxynucleotidyl transferase (TdT) gene have recently been engineered. Igs and TCRs in these mice are essentially devoid of N-region diversity. Here we report that TdT0 mice contain elevated numbers of CD3hi single-positive (SP) thymocytes because more thymocytes make the transition from the immature double-positive to the mature SP stage. This suggests that the repertoire of TCRs encoded in the germline may be enriched for specificities capable of interacting with MHC molecules and that the loss of some of this affinity is the price paid for TdT-generated diversity.

Positive selection of thymocytes induced by gene transfer: MHC class II-mediated selection of CD8 lineage cells.

Recombinant adenovirus vectors are powerful tools for inducing de novo gene expression in vivo. Here we have exploited them to study the specificity of CD4/CD8 lineage commitment during thymocyte positive selection, transferring MHC class II genes directly into thymi of mice deficient in both class I and II molecules. Expression of class II molecules was induced on cortical stroma, provoking the selection of a large population of mature CD4(+)CD8(-) cells, as expected, but also of a significant number of CD4(-)CD8(+) cells. The latter constituted a diverse population, containing both immature precursors and, though less frequent, cells that were mature according to several criteria. CD4(-)CD8(+) cells appeared with the same kinetics as their CD4(+)CD8(-) counterparts, but tended to be more prevalent at early times or when thymocyte reconstitution was only modest. These observations, derived from a dynamic selection system, indicate that CD4/CD8 lineage commitment is not irredeemably linked to the class of MHC molecule driving positive selection, a conclusion most compatible with selective models of commitment.

Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.

De novo differentiation of CD4+ T cells was provoked in mice lacking major histocompatibility complex (MHC) class II molecules by intrathymic injection of adenovirus vectors carrying class II genes. This permits a new approach to questions concerning the dynamics of CD4+ T cell compartments in the thymus and peripheral lymphoid organs. Here two issues are explored. First, we show that mature CD4+ CD8- cells reside in the thymus for a protracted period before emigrating to the periphery, highlighting the potential importance of, and our ignorance of, the postselection maturation period. Second, we demonstrate that the survival of CD4+ cells in peripheral lymphoid organs is markedly curtailed when class II molecules are absent and is not further reduced in the absence of both class II and class I molecules, raising the possibility that MHC-mediated selection may continue in the periphery.

The shaping of the T cell repertoire.

By combining a TCRbeta transgene with a TCRalpha minilocus comprised of a single V and two J gene segments, we engineered a mouse line exhibiting ample but focused TCR diversity, restricted to CDR3alpha. Using single-cell PCR and high-throughput sequencing, we have exploited this system to scrutinize T cell repertoire selection and evolution. Some striking observations emerged: (1) thymic selection produces a repertoire that is very "bumpy," with marked overrepresentation of a subset of sequences; (2) MHC class I- and class II-restricted TCRs can be distinguished by minute, single-residue changes in CDR3alpha; and (3) homeostatic expansion and survival in the periphery can markedly remold the postselection repertoire, likely reflecting variability in the potential of cells displaying different TCRs to respond to homeostatic cues.

Selection of murine T cell receptor alpha beta and gamma delta cells in organ cultures established from 14-day embryos.

The expression of minor lymphocyte stimulatory locus (Mls) determinants in combination with murine major histocompatibility complex (MHC) class II molecules, leads to the destruction of lymphocytes bearing specific V region-encoded T cell receptor (TcR) products. A much studied example is the elimination of V beta 6+ cells in IE+/Mls-1a mice, in which deletion can be detected 7-10 days after birth but is not fully operational earlier in embryonic life. Here we investigate this transitional period in development and show that selective deletion of V beta 6 occurs in vitro, approximately 1 week after organ cultures are established from 14 day embryos. These unmanipulated organ cultures receive no additional cell immigrants after day 14, suggesting that the cellular elements mediating negative selection (or their direct precursors), are already resident in the fetal thymus by day 14 of gestation. Hence, the developmental timing of the outset of rigorous negative selection of V beta 6 is not dictated by the postnatal entry of deleting elements into the thymus, but perhaps by the maturation of the pre-existing environment. Using a parallel organ-culture approach we have looked at the development of V delta 4 and V gamma 3, TcR gamma delta+ cells in a variety of mouse strains. These receptors have recently been reported to be subject of MHC and non-MHC linked selection, respectively. We find that after an initial period of expansion, the number of V gamma 3-expressing cells dramatically declines. However, this selective loss of V gamma 3 cells is not contingent on the C57BL/6 mouse strain (in contrast to a previous report). These findings are discussed in the context of current models of ontogeny and repertoire selection.

Role of coreceptors in positive selection and lineage commitment.

Recent experiments have re-awakened interest in a stochastic/selective model of positive selection of T lymphocytes. A revised version of the model has been proposed whereby commitment of double-positive thymocytes to either the CD4 or CD8 lineage requires two engagements with MHC molecules: the first, initiating the differentiation program, signals down-regulation of one or the other coreceptor, regardless of the T cell receptor's specificity for MHC class I or II molecules; the second, leading to terminal differentiation, screens the choice of coreceptor by permitting only those cells with matched receptors and coreceptors to proceed. Here we explore the role of coreceptors in the two stages of positive selection by manipulating CD8 expression in MHC class II-deficient mice, crossing them with either CD8-negative animals or animals carrying combinations of CD8 alpha and CD8 beta transgenes. We find that coreceptors are required at both stages of positive selection and that artificial expression of the down-modulated CD8 molecule can quite efficiently rescue cells that have made a 'mistake' in their choice of coreceptor. We also establish that commitment to the CD4 pathway and to the helper phenotype can be linked.

An influence of CD5 on the selection of CD4-lineage T cells.

Combining CD5-null, MHC-deficient and lineage-specific reporter animals, we have investigated the influence of CD5 on positive selection and the choice of CD4- versus CD8-lineage commitment on broad populations of thymocytes. CD5 has no obvious quantitative effect in wild-type mice. In mice lacking MHC class II molecules, however, increased numbers of transitional, class I-selected CD4+ CD8(int) CD3(hi) cells were positively selected in the absence of CD5. Importantly, they were committed to the CD4 lineage. Our results indicate that CD5 negatively regulates the differentiation of CD4-committed cells in suboptimal conditions, thus perhaps serving to tighten the correlation between restriction of the TCR and lineage choice.

Studies on the immunological properties of oxysterols: in vivo actions of 7,25-dihydroxycholesterol upon murine peritoneal cells.

Oxygenated derivatives of cholesterol are potent immunosuppressors. It has been reported previously that 7,25-dihydroxycholesterol (7,25-OHC), synthesized in the URA31, strongly inhibits the early steps of T-cell activation. So far, the mechanisms underlying this type of effect have been mainly investigated in vitro, and the activity of these substances on the immune system has been poorly studied. This study describes that a single i.p. injection of 7,25-OHC induces a strong inflammatory response, consisting of a massive influx of macrophages and neutrophils into the abdominal cavity. Macrophages harvested from 7,25-OHC-treated mice express class II antigen to a lesser extent. Moreover, the 7,25-OHC treatment abolishes the class II induction by bacillus Calmette-Guérin (BCG) or interferon-gamma (IFN-gamma). The inflammatory process triggered by the oxysterol is not the consequence of a non-specific effect due, for instance, to the presence of crystals in the abdominal cavity. Moreover, treatments by inhibitors of the acid arachidonic cascade do not affect the peritoneal exudate cell (PEC) influx induced by these substances. This study could be an important contribution to the mechanism determining the oxysterol-induced immunosuppression.

The thymic compartment responsible for positive selection of CD4+ T cells.

Our aim was to assess the generality of the observation that positive selection of CD4+ T cells is mediated by MHC class II molecules on epithelial cells of the thymic cortex. By appropriate matings of previously established transgenic and mutant mouse lines, we were able to produce animals that lacked MHC class II molecules; individuals expressing only the class II E complex, but in all the usual thymic compartments; animals that had E molecules in the thymic medulla but not in the cortex; and, reciprocally, individuals expressing the E complex in the thymic cortex but essentially not in the medulla. Those mice which displayed class II molecules in the cortex had normal numbers of CD4+CD8- T cells in the thymus and CD4+ T cells in the periphery, while 'bare' cortex mice were almost devoid of mature CD4 single positive cells. This finding serves to generalize observations from previous studies of similar design but limited to assaying positive selection of T cells which expressed a single transgenic E-restricted TCR or a subset of V beta 6+ TCRs.

How much TCR does a T cell need?

Kinetic features of TCR:MHC/peptide interactions dictate their outcome in vitro, some important parameters of which include the number of molecules engaged and the duration of engagement. We explored the in vivo significance of these findings in transgenic mice expressing TCRs in a quantitatively and temporally controlled manner. As anticipated, reduced TCR levels resulted in attenuated reactivity, but response thresholds were substantially lower than expected-at as low as 1/20th the normal TCR numbers and with no indication of phenotypic skewing at suboptimal levels. We also studied survival of T lymphocytes stripped of their TCRs. Unlike B cells, T cells lacking antigen receptors did not die precipitously; instead, populations decayed gradually, just as previously reported in the absence of MHC molecules.

Positive and negative regulation of granulopoiesis by endogenous RARalpha.

Acute promyelocytic leukemia (APL) is always associated with chromosomal translocations that disrupt the retinoic acid receptor alpha (RARalpha) gene. Whether these translocations relate to a role for endogenous RARalpha in normal granulopoiesis remains uncertain because most studies addressing this question have used non-physiological overexpression systems. Granulocyte differentiation in cells derived from RARalpha-deficient (RARalpha(-/-)) mice was studied and evaluated in the context of agonist-bound and ligand-free RARalpha. Our results demonstrate that RARalpha is dispensable for granulopoiesis, as RARalpha(-/-) mice have a normal granulocyte population despite an impaired ability to respond to retinoids. However, although it is not absolutely required, RARalpha can bidirectionally modulate granulopoiesis. RARalpha stimulates differentiation in response to exogenous retinoic acid. Furthermore, endogenous retinoids control granulopoiesis in vivo, as either vitamin A-deficient mice or animals treated with an RAR antagonist accumulate more immature granulocytes in their bone marrow. Conversely, RARalpha acts to limit differentiation in the absence of ligand because granulocyte precursors from RARalpha(-/-) mice differentiate earlier in culture. Thus, the block in granulopoiesis exerted by RARalpha fusion proteins expressed in APL cells may correspond to an amplification of a normal function of unliganded RARalpha.

Amino acids specifying MHC class preference in TCR V alpha 2 regions.

Some TCR variable regions are preferentially expressed in CD4+ or CD8+ T cells, reflecting a predilection for interacting with MHC class II or class I molecules. The molecular basis for MHC class bias has been studied previously, in particular for V alpha 3 family members, pointing to a dominant role for two amino acid positions in complementary-determining regions (CDRs) 1 and 2. We have evaluated the generality of these findings by examining the MHC class bias of V alpha 2 family members, an attractive system because it shows more variability within the CDR1 and -2, exhibits variation in the framework regions, and includes a member for which the crystal structure has been determined. We find that preferential recognition of MHC class I or II molecules does not always depend on residues at the same positions of CDR1 and -2; rules for one family may be reversed in another. Instead, there are multiple influences exerted by various CDR1/2 positions as well as the CDR3s of both the TCR alpha- and TCR beta-chains.

Slot-machine mutagenesis of a polymorphic residue on the A kappa alpha-chain.

This study explores the limitations on variability at a polymorphic position of an MHC class II molecule. Using a convenient and rapid method termed "slot-machine mutagenesis," we have converted Glu75 on the A kappa-chain to 15 alternative amino acids. This residue is of interest because it is an immunodominant site on the A kappa alpha chain and because it participates in certain T cell epitopes. The wild-type and mutant A kappa alpha cDNA were transfected into L cells (together with the A kappa beta cDNA and a selection marker), and transfectants displaying high surface levels of the A kappa complex were selected and expanded. We sought to examine three questions: what is the effect of these mutations on the expression and overall conformation of the A alpha: A beta complex? How do these diverse mutations influence mAb epitopes for which Glu75 makes a direct contribution to specificity? Do such substitutions affect T cell recognition of the A kappa alpha:A kappa beta complex? The answers to these three questions are quite different. Position 75 of the A alpha chain can accommodate essentially all chemically divergent amino acids without major consequences for expression and overall A alpha:A beta structure. In contrast, mAb that recognize Glu75-dependent epitopes are extremely particular about the amino acid residing at this position. T cells are less fastidious: those that are affected by the mutations still recognize a number of substitutions. These data emphasize the tolerance of MHC molecules to evolutionary tampering.

In vitro modulation of leucocyte-function-associated antigen-1 expression on two leukemic cell lines.

Leukocyte-function-associated antigen-1 (LFA-1) expression on two widespread tumor cell lines: K562 (an erythroleukemia) and MOLT-4 (a T leukemia), was investigated using two monoclonal antibodies specific for the alpha chain of this surface antigen, and flow cytometry analysis. When K562 cells are in the exponential phase of growth, they display very low levels of LFA-1. By contrast, cells from the plateau phase exhibit a strong labelling, which disappears rapidly when they are allowed to resume division by changing the culture medium. Using the same experimental conditions, we failed to detect any LFA-1 expression on MOLT-4 cells. However, after stimulation of these cells by phorbol myristate acetate, we observed a significant labelling, which occurred within 2 days of treatment. The LFA-1 expression disappears progressively after removal of the phorbol ester. From these results it may be concluded that (a) LFA-1 expression can vary considerably according to the culture conditions, (b) the expression of this antigen on the surface of non-expressing variants can be induced by phorbol ester, and (c) in both cases, the change in expression can be reversed completely by replacing the culture medium or by removing phorbol myristate acetate from it.