A physiological ligand of positive selection is recognized as a weak agonist.

Positive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCR(trans)(+)) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCR(trans)(+) T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCR(trans)(+) T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.

A physiological ligand of positive selection is seen with high specificity.

Positive selection is a process that ensures that peripheral T cells express TCR that are restricted to self-MHC molecules. This process requires both self-MHC and self-peptides. We have recently established a TCR transgenic mouse model (C10.4 TCRtrans+) in which the transgenic TCR was selected on the nonclassical MHC class Ib molecule H2-M3 in conjunction with a physiologically occurring peptide derived from the mitochondrial NADH-dehydrogenase subunit 1 gene (9-mer peptide). Here, the specificity of positive selection of C10.4 TCRtrans+ T cells was examined using a fetal thymic organ culture system. We demonstrated that at low peptide concentrations, shortening the NADH-dehydrogenase subunit 1 gene 9-mer peptide or mutating its surface-exposed side chains severely impaired its ability to induce positive selection. We concluded that under physiological conditions positive selection of C10.4 TCRtrans+ T cells was highly specific and occurred at low epitope densities.

Liposome encapsulated aurothiomalate reduces collagen-induced arthritis in DBA/1J mice.

Collagen-induced arthritis (CIA) generated in rats or mice has long been a model system for the study of rheumatoid arthritis in humans. In particular, this system has been used to study the mechanisms and effects of anti-arthritic drugs in the treatment of the disease. Sodium aurothiomalate (ATM) is an agent often used to treat rheumatoid arthritis in humans; however, it possesses inherent toxicities which limits its usefulness. Liposome-encapsulated drugs are currently being developed to minimize the toxicities associated with a variety of potentially beneficial drugs. We have chosen to encapsulate ATM into small unilamellar vesicles (SUVs) to determine whether greater efficacy would be achieved in treating CIA with SUV ATM as compared to using the free drug. SUVs were prepared from hydrogenated egg phosphatidylcholine and cholesterol. These SUVs were very stable. Vesicles stored at 4 degrees C lost only 0.09% of encapsulated ATM (SUV ATM) after 14 days and were able to reduce collagen-induced arthritis in these mice. Animals treated by i.m. injections of SUV ATM exhibited a 50% reduction in symptoms. More importantly, histological examination of knee joints of the affected animals verified that SUV ATM treatment prevented cellular infiltration of lymphocytes into the synovia of the collagen-sensitized mice. Conditioned media from spleen cell cultures was assayed for the presence of inflammatory lymphokines that might be affected by SUV ATM to account for the success in suppressing collagen-induced arthritis.

Treatment of an autoimmune disease with "classical" T cell veto: a proposal.

Immune responses protect against infectious diseases and cancers. In normal circumstances, the immune system is tolerant to self. However, under certain conditions this tolerance is broken. The immune system attacks otherwise normal tissue. An autoimmune disease ensues. Strategies are now being sought that remove the pathogenic T cells without affecting other immune functions. "Classical" veto has been described as an immune suppressive mechanism able to remove T cells in a highly specific and effective manner. The present article briefly reviews the current knowledge on the development of autoreactive T cells and their regulation in the periphery. It describes "classical" veto, its mechanisms, and its novel applications. Finally, it argues that "classical" veto can be adapted to treat an autoimmune disease, such as type I diabetes mellitus.

Positive selection of an H2-M3 restricted T cell receptor.

Thymocytes are positively selected for alphabeta T cell antigen receptors (TCR) that recognize antigen in conjunction with self-major histocompatibility complex (MHC) molecules. MHC bound peptides participate in positive selection; however, their role has remained controversial. A TCR transgenic mouse was established using a TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize an NADH dehydrogenase subunit 1 (ND1)-derived peptide as the physiological ligand of positive selection. This peptide bears no apparent sequence homology to the cognate peptide, is expressed ubiquitously, and yet does not interfere with peripheral T cells. Our studies also suggest that positive selection becomes promiscuous at high epitope densities.

H2-M3 restricted presentation of a Listeria-derived leader peptide.

Protective immunity to infection by many intracellular pathogens requires recognition by cytotoxic T lymphocytes (CTLs) of antigens presented on major histocompatibility complex (MHC) class I molecules. To be presented for recognition by pathogen-specific CTLs, these antigens must gain access to the host cell class I processing pathway. In the case of intracellular bacterial pathogens, the majority of bacterial proteins are retained within the bacterial membrane and therefore remain inaccessible to the host cell for antigen processing. We have isolated a CTL clone from a C57BL/6 mouse infected with the intracellular gram-positive bacterium Listeria monocytogenes (LM) and have identified the source of the antigen. Using a genomic expression library, we determined that the clone recognizes an antigenic N-formyl peptide presented by the nonpolymorphic murine MHC class Ib molecule, H2-M3. Several lengths of this peptide were able to sensitize cells for lysis by this CTL clone. The source of this antigenic peptide is a 23-amino acid polypeptide encoded at the start of a polycistronic region. Analysis of mRNA secondary structure of this region suggests that this polypeptide may be a leader peptide encoded by a transcriptional attenuator.

Specific inhibition of CD4+ T lymphocytes by a hybrid antibody.

T lymphocytes are crucial in the defense against foreign intruders and cancerous growths. Yet, in circumstances such as transplantation or autoimmunity, T-cell-mediated responses can be detrimental. Inhibition of these deleterious responses is currently achieved by drugs that induce general immune suppression. These compounds also impair the patient's defenses against infections. Strategies are now being sought that induce selective rather than generalized immune unresponsiveness. One such strategy is the ability to inhibit the activation of CD8+ T lymphocytes. As CD4+ T lymphocytes similarly participate in graft rejection and in autoimmune diseases, we have now developed a reagent to delete their activity. It comprises CD4 and an anti-MHC class II antibody. By virtue of the antibody's specificity for MHC class II molecules, this hybrid antibody (Hab) binds to class II molecules, thereby bringing CD4 accessory molecules to the surface of class II-bearing stimulator cells where they occupy CD4 binding sites on class II molecules. As a consequence CD4+ T cells with specificity to Hab-coated stimulator cells cannot engage their CD4 molecules and are no longer activated. This Hab technology provides a strategy to offer specific rather than generalized immune suppression.

Hybrid antibody mediated veto of cytotoxic T lymphocyte responses.

Strategies are being sought that allow the induction of specific tolerance to allogeneic transplants without affecting other immune functions. The so-called veto effect has been described as one such technology where CD8+ cells suppress responses of class I MHC-restricted T-lymphocyte precursors to antigens expressed by those CD8+ veto cells. Yet, veto inhibition will not be able to provide complete tolerance to allogeneic grafts since it only operates on cell populations that express CD8. Other types of cells prevalent in most organs express different tissue-specific antigens that are recognized by alloreactive T-cells. Therefore, complete tolerance to an allogeneic transplant can only be achieved if all cellular components within the graft acquire the immune-inhibitory function. Here, we studied whether the veto effect could be exploited for this purpose nevertheless. We produced a hybrid antibody (HAb) combining a mAb specific for a class I MHC molecule with a soluble CD8 molecule. We found that this HAb specifically and effectively transferred veto inhibition to different stimulator cell populations. Thus, we have developed a strategy that promises to selectively and completely tolerize graft-specific CTLs without affecting normal immune responses.

Permissive recognition during positive selection.

In the periphery alpha beta T lymphocytes recognize antigens in conjunction with major histocompatibility complex (MHC) molecules. In the thymus immature T cells are positively selected on MHC molecules in the apparent absence of cognate peptides. Thus, at different developmental stages a T cell responds to different epitopes, yet uses the identical alpha beta T cell antigen receptor (TcR). To explain this paradox it has been hypothesized that during positive selection immature T cells see peptides/ligands unique to the thymus, are selected by specific antagonists related to their cognate peptides, or are driven by lowered affinity thresholds of their TcR. Though different in detail, these theories rely on defined peptides uniquely matched to select certain TcR. However, we find that in a TcR-transgenic (TcR(trans +)) mouse severely limiting the diversity of peptides does not impair positive selection. We show that many unrelated peptides, including some naturally occurring on the cell surface, induce maturation of CD4-CD8+TcR(high) thymocytes. The same peptides when presented in conjunction with the selecting MHC molecule, are not recognized by peripheral T cells expressing the same TcR(trans). Therefore, these findings point to a promiscuous rather than discriminate recognition mode used by immature T cells.

T cell receptor V beta 8.2 gene germ-line transcription: an early event of lymphocyte differentiation.

Rearrangement of the T cell antigen receptor (TCR) beta chain genes is highly regulated in both a developmental and a tissue-specific manner. T cell precursors originate from the yolk sac or fetal liver during gestation and from the bone marrow during adulthood. They initiate the recombination of TCR genes primarily during differentiation in the thymus. It has previously been suggested that transcription of immunoglobulin genes in germ-line configuration is linked to recombination events within these loci. Here, we examine whether germ-line transcription of TCR variable genes coincides with their rearrangement or whether it marks even earlier stages of T lymphocyte development. During gestation, we found V beta 8.2 germ-line transcripts in the fetal liver and the fetal thymus, but not in the yolk sac. This transcription precedes V beta 8.2 to D beta J beta rearrangement. In adult animals, we found these transcripts in the thymus, the spleen, the liver and the bone marrow. However, in the liver, this transcription is dependent on the presence of mature lymphocytes. This transcription does not happen in non-lymphoid cells. In the B lymphocyte lineage, V beta 8.2 germ-line transcripts are detected only in the earliest stages of differentiation (pre-pro- and pro-B cells), but not in pre-B cells and mature B lymphocytes. Altogether, our results show that transcription of the unrearranged V beta 8.2 gene is an early event of lymphocyte development, taking place in lymphocyte precursors, long before V beta 8.2 rearrangement.

How are alpha beta T cells positively selected in the thymus?

Thymus-derived (T) lymphocytes have the potential to express antigen receptors (TCR) that can recognize both self, as well as foreign antigens as they appear on the cell surface. In the thymus, positive selective allows the maturation of T cells that are able to see foreign antigens in conjunction with molecules encoded by genes of the major histocompatibility complex (MHC), whereas negative selection deletes auto-aggressive T cells. Control of T cell development is the only known function of the thymus. Therefore, it has been argued that recognition events responsible for selection of the T cell repertoire are guided by unique cellular interactions in the thymus. Here, we will show that positive selection can also occur on non-thymic cells. We will also argue that positive selection is not dependent on unique thymic accessory cells (AC) function. In other words, restricted recognition is not taught rather thymocytes learn it by themselves.

Thymic education--T cells do it for themselves.

The phenomenon of positive selection of developing T cells is well established. However, the cellular interactions that are responsible for selecting the restriction element are still ill defined. Here, Tomasz Pawlowski and Uwe Staerz discuss the latest developments in the field, as well as some new evidence suggesting that cells of epithelial or hematopoietic origin are not the only ones capable of selecting the restriction element for T cells expressing the alpha beta T-cell receptor.

Positive selection of T lymphocytes on fibroblasts.

Thymocytes are selected for expression of alpha beta T-cell antigen receptors (TCR) which recognize antigen in conjunction with self-major histocompatibility complex (MHC) molecules. In the thymus the restriction element is imprinted on radioresistant stromal elements and on cells of haematopoietic origin. In mice negative for beta 2-microglobulin that are devoid of mature cytotoxic T lymphocytes, we find that intrathymic injection of different fibroblasts causes the maturation of CD4-CD8+TCRhigh thymocytes with distinct patterns of TCR V beta distribution. Here we show that in TCR-transgenic mice, intrathymic injection of L cells expressing the selecting H-2Kb molecule (L-Kb cells) reconstitutes the maturation of thymocytes bearing the transgenic TCR, and that in normal B10.BR (H-2k) mice, H-2Kb molecules expressed on L-Kb cells lead to the development of T lymphocytes with recognition restricted to H-2Kb. A class I MHC restriction element can thus be selected by interaction with fibroblasts, that is, cells of other than epithelial or haematopoietic origin.

[Monoclonal hybrid antibodies. New perspectives for basic research and tumor therapy]. / Monoklonale Hybridantikörper. Neue Perspektiven für Grundlagenforschung und Tumortherapie.

Monoclonal hybrid antibodies are bi-specific constructs of two monoclonal antibodies with defined specificity. Hybrid antibodies can be used to force interactions between cell populations that under normal circumstances would not react with one another. Applications include the field of basic research as well as employment in therapy of malignancies and infectious diseases. The first part of this review describes the characteristic properties of hybrid antibodies and gives a short introduction into basic techniques for the production of these molecules. In the second part we present our observations on T-lymphocyte development studied with hybrid antibodies in fetal thymic organ cultures. Finally, we show that hybrid antibodies encompassing a binding site to the T-cell receptor and a binding site to a surface marker on tumor cells can be used to recruit cytotoxic T-cells to eliminate efficiently the malignant cells. Our data demonstrate that monoclonal hybrid antibodies are useful in the future development of new therapeutical principles, through facilitation of the immune response.

Macrophages as accessory cells for class I MHC-restricted immune responses.

Ag do not elicit T lymphocyte responses unless they are presented in conjunction with MHC molecules on the surface of an appropriate APC. In the case of CD4+ T lymphocytes dendritic cells can deliver all signals required for complete induction as can macrophages and (activated) B cells. The function of CTL also depends on the presence of specialized accessory cells. Here we show that these accessory cells can behave like scavenger cells. They use foreign Ag in the form of cellular debris as immunogen. They are also crucial for CTL induction because in vivo depletion of phagocytotic cells completely inhibits CTL responses. In these animals CTL activity could be restored by transfer of macrophages. All of the reappearing CTL used MHC restriction elements expressed by the infused macrophages. These experiments suggest that a cognate interaction between macrophages and CTL precursors initiates class I MHC-restricted immune responses.

Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide.

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16-residue nucleoprotein peptide (NPP; 365-380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H-2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP-conjugated to ovalbumin as a carrier. However, in contrast to NPP-specific CTL lines raised from virus-primed animals none of the peptide-induced CTL lines were able to lyse virus-infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100-fold lower than that of CTL from virus-primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide-induced CTL was very sensitive to blocking by anti-CD8 antibodies as compared to virus-specific CTL. Furthermore, all peptide-induced CTL showed a high second reactivity for allogeneic H-2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.

Redirecting the cellular immune response.

A technology is described which combines the power of T lymphocytes in eliminating unwanted cells and causing beneficial inflammatory reactions with the great advantages of monoclonal antibodies. We show that heteroconjugate antibodies and hybrid antibodies, in which one of the component binding sites is anti-T-cell receptor and the other component binding is directed against any chosen target antigen, can focus T cells to act at the targeted site. We present experimental evidence for the in vitro efficacy of this system in viral infections and in the elimination of tumor cells. In an in vivo tumor model pilot experiments have been undertaken that resulted in a significant higher rate of survival of animals treated with hybrid antibodies compared to untreated animals.

Thymic selection process induced by hybrid antibodies.

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.