Ayelet Kaminitz

Mechanisms of T regulatory cell function.

Regulatory T cells (Treg) play a pivotal role in tolerance to self-antigens and tissue grafts, and suppression of autoimmune reactions. These cells modulate the intensity and quality of immune reactions through attenuation of the cytolytic activities of reactive immune cells. Treg cells operate primarily at the site of inflammation where they modulate the immune reaction through three major mechanisms: a) direct killing of cytotoxic cells through cell-to-cell contact, b) inhibition of cytokine production by cytotoxic cells, in particular interleukin-2, c) direct secretion of immunomodulatory cytokines, in particular TGF-beta and interleukin-10. In addition to differential contributions of these mechanisms under variable inflammatory conditions, mechanistic complexity and diversity evolves from the diverse tasks performed by various Treg cell subsets in different stages of the immune reaction. Here we attempt to integrate the current experimental evidence to delineate the major suppressive pathways of Treg cells.

Apoptosis as a mechanism of T-regulatory cell homeostasis and suppression

Activation‐induced cell death is a general mechanism of immune homeostasis through negative regulation of clonal expansion of activated immune cells. This mechanism is involved in the maintenance of self‐ and transplant tolerance through polarization of the immune responses. The Fas/Fas‐ligand interaction is a major common executioner of apoptosis in lymphocytes, with a dual role in regulatory T cell (Treg) function: Treg cell homeostasis and Treg cell‐mediated suppression. Sensitivity to apoptosis and the patterns of Treg‐cell death are of outmost importance in immune homeostasis that affects the equilibrium between cytolytic and suppressor forces in activation and termination of immune activity. Naive innate (naturally occurring) Treg cells present variable sensitivities to apoptosis, related to their turnover rates in tissue under steady state conditions. Following activation, Treg cells are less sensitive to apoptosis than cytotoxic effector subsets. Their susceptibility to apoptosis is influenced by cytokines within the inflammatory environment (primarily interleukin‐2), the mode of antigenic stimulation and the proliferation rates. Here, we attempt to resolve some controversies surrounding the sensitivity of Treg cells to apoptosis under various experimental conditions, to delineate the function of cell death in regulation of immunity.

Pancreatic islets under attack : Cellular and molecular effectors

Abundant information is available on the involvement of various cellular and molecular mechanisms in beta cell apoptosis. The experimental evidence is controversial and difficult to reconcile, and the mechanisms of evasion of the autoreactive clones from immune surveillance are poorly understood. Multiple apoptotic pathways play a role in destructive insulitis, including perforin/granzyme, Fas/Fas-ligand (FasL), and other members of the necrosis factor superfamily. These pathways present redundant behaviors in both the initial and late stages of beta cell injury, and at the same time, each molecular mechanism is dispensable in the evolution of autoimmune diabetes. There may be a preferential use of perforin/granzyme in CD8(+) T cell-mediated lysis, which participates in onset of autoimmunity, and a predominance of FasL in CD4(+) T cell-mediated insulitis. Several cytokines released in the inflammatory infiltrate induce Fas expression in beta cells, priming them to FasL-mediated apoptosis. In this review, we focus on the possible participation of multiple cell subsets and molecular mechanisms in the pathogenesis of diabetes to the point where inflammation incites an irreversible vicious cycle that perpetuates beta cell death.

The vicious cycle of apoptotic β -cell death in type 1 diabetes

Autoimmune insulitis, the cause of type 1 diabetes, evolves through several discrete stages that culminate in β‐cell death. In the first stage, antigenic epitopes of B‐cell‐specific peptides are processed by antigen presenting cells in local lymph nodes, and auto‐reactive lymphocyte clones are propagated. Subsequently, cell‐mediated and direct cytokine‐mediated reactions are generated against the β‐cells, and the β‐cells are sensitized to apoptosis. Ironically, the β‐cells themselves contribute some of the cytokines and chemokines that provoke the immune reaction within the islets. Once this vicious cycle of autoimmunity is fully developed, the fate of the β‐cells in the islets is sealed, and clinical diabetes inevitably ensues. Differences in various aspects of these concurrent events appear to underlie the significant discrepancies in experimental data observed in experimental models that simulate autoimmune insulitis.

Involvement of IL-2 in homeostasis of regulatory T cells: the IL-2 cycle

A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause-result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.

Low levels of allogeneic but not syngeneic hematopoietic chimerism reverse autoimmune insulitis in prediabetic NOD mice

The relative efficiencies of allogeneic and syngeneic bone marrow transplantation and the threshold levels of donor chimerism required to control autoimmune insulitis were evaluated in prediabetic NOD mice. Male and female NOD mice were conditioned by radiation and grafted with bone marrow cells from allogeneic and syngeneic sex-mismatched donors. Establishment of full allogeneic chimerism in peripheral blood reversed insulitis and restored glucose tolerance despite persistence of residual host immune cells. By contrast, sublethal total body irradiation (with or without syngeneic transplant) reduced the incidence and delayed the onset of diabetes. The latter pattern was also seen in mice that rejected the bone marrow allografts. Low levels of stable allogeneic hematopoietic chimerism (>1%) were sufficient to prevent the evolution of diabetes following allogeneic transplantation. The data indicate that immunomodulation attained at low levels of allogeneic, but not syngeneic, hematopoietic chimerism is effective in resolution of islet inflammation at even relatively late stages in the evolution of the prediabetic state in a preclinical model. However, our data question the efficacy and rationale behind syngeneic (autologous-like) immuno-hematopoietic reconstitution in type 1 diabetes.

Killer Treg restore immune homeostasis and suppress autoimmune diabetes in prediabetic NOD mice.

We hypothesized that regulatory T cells (Treg) effectively target diabetogenic cells, and reinforcing their killing capacity will attenuate the course of disease. For proof of concept, Fas-ligand (FasL) protein was conjugated to CD25(+) Treg (killer Treg) to simulate the physiological mechanism of activation-induced cell death. Cytotoxic and suppressive activity of killer Treg was superior to naïve Treg in vitro. Administration of 3-4 × 10(6) Treg prevented hyperglycemia in 65% prediabetic NOD females, however only killer Treg postponed disease onset by 14 weeks. CD25(+) Treg homed to the pancreas and regional lymph nodes of prediabetic NOD females, proliferated and ectopic FasL protein induced apoptosis in CD25(-) T cells in situ. This mechanism of pathogenic cell debulking is specific to killer Treg, as FasL-coated splenocytes have no immunomodulatory effect, and only killer Treg prevent the disease in 80% of NOD.SCID recipients of effector:suppressor T cells (10:1 ratio). All immunomodulated mice displayed increased fractional expression of FoxP3 in the pancreas and draining lymph nodes, which was accompanied by CD25 only in recipients of killer Treg. A therapeutic intervention that uses the affinity of Treg to reduce the pathogenic load has long-term consequences: arrest of destructive insulitis in mice with established disease prior to β-cell extinction.

Participation of adult bone marrow-derived stem cells in pancreatic regeneration: neogenesis versus endogenesis.

Regenerative medicine opens new avenues and promises towards more effective therapies for autoimmune disorders. Current therapeutic strategies for type I diabetes focus on three major directions, with distinct advantages and disadvantages: arrest of autoimmunity, islet transplantation and generation of neoislets. There is mounting evidence that candidate stem cells residing in the hematopoietic compartments participate in regeneration of pancreatic islets following chemical and autoimmune injury in vivo. The apparent major mechanisms include immunomodulation, revascularization, support of endogenous beta-cell regeneration and differentiation into insulin-producing cells. Review of the current evidence suggests that some divergent observations depend primarily on the experimental design, which both limits and accentuates developmental events. The flood of publications reporting negative results appears to reflect primarily suboptimal experimental conditions for differentiation of putative stem cells, rather than limited developmental plasticity. Stem cells modulate the course of autoimmune diabetes through multiple mechanisms, including de novo generation of units capable to sense, produce and secrete insulin. Therefore, the charged debate over controversies surrounding developmental plasticity should not impede attempts to design curative therapies for this disease.

Immunosuppressive therapy exacerbates autoimmunity in NOD mice and diminishes the protective activity of regulatory T cells

Mounting evidence indicates that immunosuppressive therapy and autologous bone marrow transplantation are relatively inefficient approaches to treat autoimmune diabetes. In this study we assessed the impact of immunosuppression on inflammatory insulitis in NOD mice, and the effect of radiation on immunomodulation mediated by adoptive transfer of various cell subsets. Sublethal radiation of NOD females at the age of 14 weeks (onset of hyperglycemia) delayed the onset of hyperglycemia, however two thirds of the mice became diabetic. Adoptive transfer of splenocytes into irradiated NON and NOD mice precipitated disease onset despite increased contents of CD25(+)FoxP3(+) T cells in the pancreas and regional lymphatics. Similar phenotypic changes were observed when CD25(+) T cells were infused after radiation, which also delayed disease onset without affecting its incidence. Importantly, irradiation increased the susceptibility to diabetes in NOD and NON mice (71-84%) as compared to immunomodulation with splenocytes and CD25(+) T cells in naïve recipients (44-50%). Although irradiation had significant and durable influence on pancreatic infiltrates and the fractions of functional CD25(+)FoxP3(+) Treg cells were elevated by adoptive cell transfer, this approach conferred no protection from disease progression. Irradiation was ineffective both in debulking of pathogenic clones and in restoring immune homeostasis, and the consequent homeostatic expansion evolves as an unfavorable factor in attempts to restore self-tolerance and might even provoke uncontrolled proliferation of pathogenic clones. The obstacles imposed by immunosuppression on abrogation of autoimmune insulitis require replacement of non-specific immunosuppressive therapy by selective immunomodulation that does not cause lymphopenia.

Apoptosis of purified CD4+ T cell subsets is dominated by cytokine deprivation and absence of other cells in new onset diabetic NOD mice.

BACKGROUND Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures. PRINCIPAL FINDINGS Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25(+) T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25(-) T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25(+) T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays. CONCLUSIONS The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression.