Control of Transcription and Cell Cycle

Barbara Murphy, MD
Medical Writer: Peggy Keen, RN, PhD, FNP

Speakers at this session provided an in-depth look at critical events in T-cell triggering and signal transduction. Presenters focused on the role of proteins in T-cell signaling, discussing a newly discovered protein called T-bet (pronounced “Tibet”) and the known effects of c-MAf. Current concepts of cytotoxic T-cell mechanisms in transplantation also were reviewed, with a brief description offered of an as-yet-unnamed cell surface receptor for granzyme B, which facilitates the internalization of this enzyme. Deficient expression of this factor has resulted in decreased apoptosis in laboratory studies. The session concluded with an update on dendritic cells in antigen presentation, in which the potential role that enhanced tolerogenicity might play in transplantation was described.

T HELPER CELL DEVELOPMENT: THE ROLE OF T-BET AND C-MAF

It has long been recognized that T-helper cells influence the immune system through secretion of cytokines, which in turn have dramatic effects on other immune regulatory cells. But as of a year ago, no tissue-specific regulator of T-helper cell production had been identified.

Laurie Glimcher, MD, from Harvard University in Boston, Mass, described the exciting discovery of a new member of the T-box family of transcription factors that governs the development of T-helper cells along the Th1 lineage.^[1] This transcription factor, designated T-bet, is also expressed in natural killer (NK) cells. Since T-bet controls the Th1 lineage, it can convert Th1 to Th 2 cells.

The activation of T cells begins with the recognition of antigen (ie, transplanted organ). Antigen is presented to the T cells by antigen-presenting cells (APC), such as dendritic cells. This is the first of two signals necessary for T-cell activation. The second signal involves proteins on the surface of the APC that interact with cell surface molecules on the T cell, which provide a costimulatory signal. Once this occurs, the T cell is fully activated. Upon activation, the T cells produce cytokines that have various effects on both T cells and other inflammatory cells within the vicinity.

Naive helper T cells (Th0) exposed to different stimuli can differentiate into either Th1 or Th2 cells. The predominant Th cells vary according to the disease process. For example, a predominance of Th2 cells is seen in allergic conditions, while Th1 cells drive the response in allo- and autoimmune mechanisms. Each type of helper cell has a characteristic pattern of cytokine production. Th1 predominantly produces interleukin (IL)-2 and IFN-gamma, and Th2 predominantly produces IL-4 and IL-10. In general, Th1 cytokines are considered to be pro-inflammatory, while Th2 cytokines are anti-inflammatory.

Many other factors influence which path a Th0 takes, including the amount and form of antigen, the presence or absence of costimulation, and the cytokines themselves. It is known that IL-12 drives Th1 differentiation, while IL-4 results in Th2 differentiation. The lack of IL-4 or STAT 6 results in failure of Th2 development. Likewise, if IL-12 and IFN-gamma are absent, Th1 cells do not generate.

Alteration of the immune response by cytokines varies with respect to disease process and may be potentially disrupted at several levels. Th1 and Th2 cytokine signaling may be prevented by targeting the signaling proteins STAT 4 and STAT 6, respectively, or the transcription of cytokine genes themselves may be altered. By understanding the tissue-specific nature of cytokine production, it may be possible to alter the expression of either Th1 or Th2 cytokines.

TH1 REGULATION

T-bet is a recently identified tissue-specific regulator of Th1 cytokines whose expression is increased by T-cell receptor (TCR) signaling. Immunohistologic studies show a similar pattern for both T-bet and IFN-gamma.

In assays using a luciferase reporter gene, no IFN-gamma is produced when the IFN-gamma gene is expressed alone. However, coexpression of T-bet results in IFN-gamma production. Not only does T-bet transactivate IFN-gamma, but it also represses IL-2 and turns off Th2 cytokines. Retroviral transfection of CD4⁺ cells results in 70% of cells producing IFN-gamma in non-Th1 skewed conditions.

Of great interest, transfected Th2 cells can change their pattern of cytokine production to produce IFN-gamma, which stops Th2 cytokine production and thus reprograms the cells. This mechanism can also be demonstrated in cells committed to Th2 long-term cytokine production and Th2 clones.

TH2 REGULATION

A previously described proto-oncogene, c-MAf, was identified as a tissue-specific regulator of IL-4 several years ago by the Glimcher group. It is only expressed in Th2 cells and is induced by TCR signaling. Ectopic expression of c-MAf is capable of transactivating the IL-4 promoter in Th1 cells, B cells, and nonlymphoid cells.

Studies of c-MAf transgenic mice by Dr. Glimcher and associates demonstrated IL-4 production in all cells in the body. In the study population, IL-4 was produced in quantities comparable to cells that had been skewed to Th2 phenotype, and the mice had increased amounts of IgE and IgG1. Deletion of c-MAf resulted in the absence of IL-4 production. The absence of c-MAf did not affect the production of other Th2 cytokines. Regulators of c-MAf are currently being investigated as potential therapeutic modalities for autoimmune diseases and transplantation. In addition, the effect of retroviral expression in islets on graft survival is under investigation.

It has been recognized that expression of T-bet may be induced by IL-12. Studies are currently underway to search for signal pathways involved in the regulation of T-bet and for agents that can alter its activity.

EMERGING CONCEPTS OF CYTOTOXIC T-CELL MECHANISMS IN GRAFT REJECTION

R. Chris Bleackley, PhD, from the University of Alberta in Alberta, Canada, contrasted traditional and emerging models of cytotoxic T-cell mechanisms in transplant rejection.^[2] Previously, the correlation of perforin expression with acute allograft rejection implied a central role for perforin in the rejection process. However, deletion of perforin in transplant animal models failed to alter the rate of rejection.

To reconcile these two findings, it has been proposed that alternative mechanisms compensate for the lack of perforin. Hypothesized alternate mechanisms include Fas, antibodies, cytokines, and the delayed-type hypersensitivity (DTH) response. But proposing alternate mechanisms implies a fuller understanding of how cytotoxic T-lymphocytes (CTL) work, and to date, this is not fully understood. Research from the University of Alberta group is contributing to further explication of CTL function.

In previous textbook models, perforin monomers were depicted as singularly boring holes in cell walls, resulting in cell lysis. It was proposed that both perforin and granzyme B (GrB) were present in the electron-dense deposits in CTLs and that both undergo directed exocytosis in the direction of the target cell. Upon release of perforin and GrB into the intercellular space, perforin forms pores in the membrane of the target cell allowing the entry of GrB into the cell.

It is believed that the entry of GrB into the cell results in apoptosis, or programmed cell death. If perforin acts alone, cell death occurs through lysis but no DNA fragmentation takes place. GrB, a serine protease that cleaves at arginine residues, causes DNA fragmentation by cleaving caspase 3 into its active form, which in turn initiates apoptosis.

Dr. Bleackley and colleagues demonstrated that when the effects of GrB are examined using different markers for apoptosis in the presence and absence of caspase inhibitors, all but altered membrane potential were caspase-dependent, thus delineating apoptosis from membrane damage. A remaining question is whether perforin actually causes pore formation. New information is helping to elucidate the answer.

Since adenovirus has been used in gene therapy research to introduce DNA and proteins intracellularly, researchers substituted adenovirus for perforin to see if it could facilitate the entry of GrB into a cell. In the presence of adenovirus, investigators found that GrB induced DNA fragmentation and efficient membrane release. This discovery, along with the demonstration that perforin knockout mice were found to behave similarly to granzymes A and B double-knockout mice, suggests that perforin may not be the mediator of the membrane lytic effect. Instead, perforin may merely facilitate the action of GrB.

It was recently recognized that perforin is required for significant apoptosis to occur, since GrB-treated cells undergo apoptosis following treatment with perforin. Subsequent studies by the Bleackley group have shown that perforin facilitates the release of GrB from the CTL instead of facilitating the entry of GrB into the T cell. This led to the hypothesis that a cell surface receptor must exist that facilitates the internalization of GrB after exposure of the cell to either perforin or adenovirus.

A newly identified, and yet unnamed, receptor for GrB has now been identified through this work. In ongoing studies, deficient expression of this factor has resulted in decreased killing while enhanced expression restored the ability of GrB to induce apoptosis.

This new receptor is an important potential target for immunomodulation. The effects of knock-out on this receptor in an islet model are currently under investigation. Initial results showed that graft destruction was prevented in the experimental group when histology was compared with the control group at 12 days posttreatment.

DENDRITIC CELLS IN ANTIGEN PRESENTATION: WHAT’S IMPORTANT?

Dendritic cells, though rare throughout the body, efficiently capture, process, transport, and present antigen (Ag) to Ag-specific T cells in lymphoid tissue. Indeed, one dendritic cell can effect more than 1,000 T cells. Since they are the most effective antigen-presenting cells (APC) at activating naive CD4⁺ cells, dendritic cells can determine the balance between tolerance and immunity.

The review of the research on the antigen-presenting capacity and tolerogenic potential of dendritic cells was provided by Angus Thomson, PhD, DSc, from the Thomas E. Starzl Transplantation Institute at the University of Pittsburgh Medical Center.^[3] Dr. Thomson also described important in vivo evidence supporting the possibility of maximizing the tolerogenic potential of dendritic cells for therapeutic use in allograft rejection and autoimmune disease.

After organ transplant, it is believed that donor dendritic cells migrate via the blood and lymphatics to T-cell-dependent areas of lymphoid tissue and present antigen to T cells with certain TCRs. Although it has been demonstrated that dendritic cells present antigen and initiate T cell activation in vitro, direct in vivo evidence of this process was lacking until Kudo and colleagues observed proliferating T cells in contact with dendritic cells in T-cell areas of rat lymphoid tissue following injection of allogenic dendritic cells.

Methods are not yet available to permit in situ detection in normal individuals. However, confocal microscopy was successfully used by Ingulli and associates to track in vivo location of dye-labeled dendritic cells and naive TCR CD4⁺ T cells specific for a peptide complex after adoptive honing of both cell populations into syngeneic recipients.

Dendritic cells have the capability to both initiate immune response and regulate immune reactivity. During the life cycle of the cell, dendritic cells progress from an immature to mature form. In rat studies, immature dendritic cells from nonlymphoid tissue proved to be poor stimulators of naive allogenic T cells, eliciting a predominant Th2 response when exposed to a major histocompatibility complex (MHC) peptide. When cells were matured, they demonstrated the capacity to stimulate both Th1 and Th2 responses. Findings that immature donor liver dendritic cells secrete IL-10 and/or modulate host responses toward Th2 predominance imply that the dendritic cell may contribute to the tolerogenicity of hepatic allographs.

In mice, two major dendritic cell lineage subsets derived respectively from myeloid or lymphoid precursors appear to differ in expression of CD8-alpha, effect on Th subsets, localization in lymphoid organs, and types of immune responses generated in vivo. At the present time, the function of these two dendritic cell subsets remains controversial. It has been suggested that the myeloid-derived subsets prime naive T cells and generate memory responses, while the lymphoid subset induces tolerance. in vitro studies of human dendritic cells have extended the concepts of differences in subset function. However, further work is needed to establish the similarities between mice and human dendritic cell subsets.

The question of whether dendritic cells can promote and maintain dendritic cell tolerance is integral to transplantation. Based on increased knowledge of dendritic cell immunobiology and in vitro and in vivo established alloimmunity, it is apparent that these cells have the potential to promote tolerance or immunity. Optimizing these possibilities could inhibit or prevent allograft rejection.

Four possible strategies for enhancing tolerance or immunity were outlined. Tolerogenicity was first described in the context of intrathymic self-tolerance. A recent rat study suggested that injection of peptide exposed to host dendritic cells into a recipient thymus promoted donor-specific immature transplant tolerance to cardiac allografts. Systemic injection of allogenic dendritic cells exhibiting tolerogenic properties in vitro has been demonstrated by several groups to prolong allograft survival. Administration of these cells 1 week before transplantation prolonged survival of transplanted cells or organ allografts in previously nonimmunosuppressed MHC-mismatched subjects. These findings were in direct contrast to the acceleration of graft rejection that occurred when mature dendritic cells were administered at the same dose and time in relation to transplantation.

The capacity of immature dendritic cells to promote heart graft survival was potentiated by blockade of the CD40-CD40L costimulatory pathway in several studies. Tolerogenicity of immature dendritic cells was also potentiated by anti-T cell monoclonal antibody (mAb) and total lymphoid irradiation. When combined with anti-CD40LmAb, anti-CD4⁺ anti-CD8mAbs, anti-IL-12, or IL-13, dendritic cells at various stages of maturation were associated with prolonged skin or heart allograft survival.

A currently understudied possibility is that in vitro manipulated host dendritic cells could be used as systemic antirejection therapy. Last, a large number of agents have been found to inhibit dendritic cell maturation in vitro, resulting in increased tolerogenicity. These agents include anti-inflammatory cytokines (IL-10 and TFG beta), prostaglandin E2 (PGE2), the costimulation blocking fusion protein CTLA4Ig, and anti-inflammatory drugs including corticosteroids, cyclosporin A, and deoxyspergualin. Ultraviolet B irradiation has also been shown to promote dendritic cell tolerogenicity. Studies of in vivo effects are ongoing.

Due to their unique biological properties and immunoregulatory potential, dendritic cells will continue to be a focus of study. Maximizing the tolerogenic properties of these APCs may help achieve the goal of long-term and even drug-free allograft survival.

REFERENCES

Glimcher L. Tissue-specific regulation of T helper cell development. Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.
Bleackley RC. Cytoxic T-cell mechanisms in transplantation rejection. Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.
Thomson A. A critical look at the dendritic cell in antigen presentation: What is the in vivo evidence that it is important? Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.