Antigen-specific CD8+ T cell subset distribution in lymph nodes draining the site of herpes simplex virus infection.

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis.

Prevention of vascular and neural dysfunction in diabetic rats by C-peptide.

C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects.

Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.

In this report, we show that cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8(+) T cells. We had previously shown that transfer of ovalbumin (OVA)-specific CD8(+) T cells (OT-I cells) into rat insulin promoter-membrane-bound form of OVA transgenic mice, which express the model autoantigen OVA in the proximal tubular cells of the kidneys, the beta cells of the pancreas, the thymus, and the testis of male mice, led to the activation of OT-I cells in the draining lymph nodes. This was due to class I-restricted cross-presentation of exogenous OVA on a bone marrow-derived antigen presenting cell (APC) population.

Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help.

Class I-restricted presentation is usually associated with cytoplasmic degradation of cellular proteins and is often considered inaccessible to exogenous antigens. Nonetheless, certain exogenous elements can gain entry into this so-called endogenous pathway by a mechanism termed cross-presentation. This is known to be effective for class I-restricted cytotoxic T lymphocyte (CTL) cross-priming directed against a variety of exogenous tumor, viral, and minor transplantation antigens.

Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection.

Mice expressing a Kb-restricted transgenic T cell receptor (TCR) and a naturally occurring MHC class I variant molecule, Kbm8, were used to study thymic selection. The transgenic TCR was specific for the major peptide determinant from ovalbumin (OVA(257-264)), while Kbm8 has a mutation that alters the position 2 binding pocket of the Kb molecule, abolishing antigenic peptide presentation and positive selection of transgenic T cells. Peptide presentation was restored by identifying a position 2 analog peptide with Kbm8-binding capacity.

T cell tolerance and autoimmunity.

Many T cells with auto-aggressive potential are deleted in the thymus. Although some of these escape to the general circulation, they do not usually damage organs such as the pancreas. To investigate the mechanisms preventing autoimmunity, we generated transgenic mice expressing known genes under the control of various promoters.

Induction of autoimmune diabetes by oral administration of autoantigen.

An antigen administered orally can induce immunological tolerance to a subsequent challenge with the same antigen. Evidence has been provided for the efficacy of this approach in the treatment of human autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. However, oral administration of autoantigen in mice was found to induce a cytotoxic T lymphocyte response that could lead to the onset of autoimmune diabetes.

A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production.

Previous work indicated that a subclass of mouse spleen dendritic cells (DC), those bearing CD8alpha, expresses the Fas ligand and restricts peripheral CD4 T cell responses by initiating Fas-mediated apoptosis. To determine whether a similar regulation applies to CD8 T cells, they were purified from normal or from TCR-transgenic mice, and then cultured with purified splenic CD8+ DC or CD8- DC presenting either alloantigens or the specific Ag for the TCR transgene. In all systems studied, the proliferative response of CD8 T cells was markedly less on stimulation with CD8+ DC compared with conventional CD8- DC.

Constitutive class I-restricted exogenous presentation of self antigens in vivo.

Ovalbumin (OVA)-specific CD8+ T cells from the T cell receptor-transgenic line OT-I (OT-I cells) were injected into unirradiated transgenic RIP-mOVA mice, which express a membrane-bound form of OVA (mOVA) in the pancreatic islet beta cells and the renal proximal tubular cells. OT-I cells accumulated in the draining lymph nodes (LN) of the kidneys and pancreas and in no other LN. They displayed an activated phenotype and a proportion entered cell cycle.

(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta.

Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy.

Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor.

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

LY290181, an inhibitor of diabetes-induced vascular dysfunction, blocks protein kinase C-stimulated transcriptional activation through inhibition of transcription factor binding to a phorbol response element.

Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181.

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice.

Autoimmunity caused by ignorant CD8+ T cells is transient and depends on avidity.

RIP-Kb mice, which express H-2Kb (Kb) molecules on their pancreatic beta cells, were used to examine the requirements for induction of autoimmune diabetes caused by CD8+ T cells. Previous studies showed that when these mice were crossed to mice expressing a Kb-specific TCR transgene, those CD8+ cells expressing the highest density of the transgenic TCR (presumably the highest avidity cells) were deleted intrathymically due to aberrant expression of Kb at this site. The remaining low avidity cells ignored Kb-bearing beta cells, even after priming, but were able to cause autoimmune diabetes when supplied with Il-2.

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

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2Kb (Kb) under the control of the sheep metallothionein promoter (Met-Kb mice). To follow the fate of Kb-specific T cells, and to prevent any aberrant thymic expression of the Kb transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a Kb-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although Kb-specific CD8+ T cells were produced in the thymus grafts of these manipulated Met-Kb mice, only small numbers of such cells could be detected in the spleen and lymph nodes.

Expression of two T cell receptor alpha chains on the surface of normal murine T cells.

We have previously reported that a subset of T cells in T cell receptor (TCR)-transgenic mice may express two different alpha chains on their surface. The expression of two functional alpha chains has also been demonstrated for human peripheral blood T cells. In this report, we show that a proportion of normal murine lymph node T cells express two functional alpha chains on their surface.

Intermediate steps in positive selection: differentiation of CD4+8int TCRint thymocytes into CD4-8+TCRhi thymocytes.

The differentiation potential of putative intermediates between CD4+8+ thymocytes and mature T cells has been examined. Such intermediate populations were sorted, in parallel with CD4+8+ thymocytes, from three types of C57BL/6 mice: major histocompatibility complex (MHC) class II-deficient mice, mice transgenic for an alpha/beta T cell receptor (TCR) restricted by class I MHC and normal mice. The sorted populations were then transferred into the thymus of nonirradiated C57BL/Ka mice differing in Thy 1 allotype, and the progeny of the transferred cells were analyzed 2 d later.

Split tolerance in spleen chimeras.

Transferring small doses of T cells to heavily irradiated F1 mice expressing isolated MHC class I or class II differences invariably leads to rapid death from graft-vs-host disease (GVHD). Paradoxically, GVHD is mild or absent when irradiated F1 mice are reconstituted with large doses of unseparated parental strain spleen cells. This applies when bulk populations of B6 spleen cells are transferred to irradiated class II-different (B6 x bm12)F1 mice or class I-different (B6 x bm1)F1 mice.

Peptide antagonists that promote positive selection are inefficient at T cell activation and thymocyte deletion.

We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I-restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen-driven deletion in single-cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the Kb-restricted peptide OVA257-264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with Kb. OVA257-264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double-positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse.

Profound atrophy of the bone marrow reflecting major histocompatibility complex class II-restricted destruction of stem cells by CD4+ cells.

The effector functions of CD4+ cells in vivo are presumed to reflect a combination of lymphokine-mediated bystander reactions and direct cytotoxic T lymphocyte activity. To assess the relative importance of these two mechanisms, we studied the effects of transferring small doses of purified unprimed CD4+ cells to lightly irradiated (600 cGy) recipients expressing major histocompatibility complex class II (Ia) differences. Within the first week after transfer, the host marrow was rapidly repopulated with hemopoietic cells.

T cell receptor antagonist peptides induce positive selection.

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified.

Avidity for antigen can influence the helper dependence of CD8+ T lymphocytes.

To examine the influence of avidity on the helper dependence of CD8+ T cells, purified CD8+ T cells from transgenic mice expressing a TCR specific for H-2Kb were tested for their responsiveness to spleen cells expressing different densities of H-2Kb. High avidity interactions resulted in the induction of strong proliferative responses by purified CD8+ T cells. These cells synthesized their own IL-2 and IL-2R.

Failure of a protective major histocompatibility complex class II molecule to delete autoreactive T cells in autoimmune diabetes.

The association of major histocompatibility complex genes with autoimmune diseases is firmly established, but the mechanisms by which these genes confer resistance or susceptibility remain controversial. The controversy extends to the nonobese diabetic (NOD) mouse that develops disease similar to human insulin-dependent diabetes mellitus. The transgenic incorporation of certain class II major histocompatibility complex genes protects NOD mice from diabetes, and clonal deletion or functional silencing of autoreactive T cells has been proposed as the mechanism by which these molecules provide protection.

Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation.

The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats.