Effect of Different Therapeutic Strategies on Regulatory T Cells in Kidney Transplantation
Condition: Kidney Transplantation
Study type: Interventional

Summary: 
      The objective of the study will be to evaluate the effect of different therapeutic
      immunosuppressive strategies currently employed in common clinical practice on regulatory T
      lymphocytes and to verify the hypothesis that the association of thymoglobulins - mTOR
      inhibitors - small doses of Tacrolimus not only represents a safe anti-rejection therapy but
      it can also lead to mid-term formation of a high amount of regulatory T cells and,
      consequently, a high grade of tolerance.
    

Detailed description: 
      Immune response is mediated by the interaction between antigen-presenting cells (APC), CD4+
      helper T cells (Th) and regulatory T cells (Treg), a subpopulation of CD4+ T cells which
      intensively expresses IL-2 receptor (CD25) and FoxP3 transcription factor. Treg cells
      contribute maintaining tolerance by suppressing immune response to normal or tumour
      self-antigens. Treg cells originate in the thymus during ontogenesis and represent
      approximately 10% of peripheral CD4+ cells. All effector T lymphocytes generate in the thymus
      at the early stages of life and evolve through the production of new T lymphocytes as well as
      through antigen-induced expansion of virgin (naive) peripheral T lymphocytes which convert to
      "memory" T lymphocytes and lie in peripheral lymphoid organs (17). Mature T lymphocytes
      constitute 70-80% of normal peripheral blood lymphocytes, 30-40% of lymph nodes cells and
      20-30% of splenic lymphoid cells.

      T lymphocytes are primary effectors of cell-mediated immunity and differentiate into a
      subpopulation of CD8+ cytotoxic T lymphocytes which are able to lyse foreign cells or virus
      infected host-cells and a subpopulation of CD4+ T lymphocytes with a regulating activity on T
      and B lymphocytes and monocytes, through the production of cytokines and cell-to-cell
      contact.

      Treg lymphocytes play a central role among CD4+ cells in balancing tolerance and immunity, as
      they are responsible for maintaining peripheral tolerance through the control of
      self-reactive T cells which escaped thymic deletion (19). Studies have in fact demonstrated
      that whether on one side a defect in their development or activity can lead to serious
      autoimmune diseases, an excessive immunosuppression mediated by these cells stimulates, on
      the other side, an immunodeficient condition also towards antigens produced by neoplastic
      cells favouring, as a consequence, tumour growth.

      Treg lymphocytes represent approximately 10% of all CD4+ T cells present in the thymus,
      peripheral blood and lymphoid tissues; they consist of various populations which differ in
      terms of particular cell-surface molecules expression and the production of diverse
      cytokines, but share a common scarce response to antigenic stimulation and an
      immunosuppressive activity.

      The best characterized Treg lymphocytes are the so called "natural occurring Tregs" (nTregs),
      a sub-group of CD4+ T cells which originates and develops in the thymus during T-cellular
      maturation process and is afterwards normally present in peripheral blood with the function
      of controlling self-antigens and preventing autoimmune diseases. These lymphocytes are
      characterized by the constitutive expression of CD-25 (interleukin-2 receptor α chain), Foxp3
      transcription factor (specific to these cells and implied in the development control within
      the thymus), CTLA-4, GITR and LAG-3 surface molecules as well as TGF-β cytokine, which is
      present in great quantity on cellular surface and is fundamental to their functioning.

      So called "induced Tregs" (iTregs) build the other Treg lymphocytes category: these are CD4+
      T lymphocytes which lack in intrinsic regulatory potential but acquire their suppressive
      activity thanks to a specific and cytokine-mediated activation (26). They are produced in
      periphery and allow the development of peripheral tolerance to self-antigens, not or scarcely
      expressed in the thymus. iTregs consist themselves of two different lymphocytic
      subpopulations: Tr1, present in intestinal mucosa and characterized by a high production of
      IL-10 and TGF-β but Foxp3 negative; and T helper 3 (Th3), deriving from the induction of
      native CD4+ T cells due to the ingestion of alimentary antigens, responsible for oral
      tolerance. Th3 are Foxp3 positive and release high levels of TGF-β (27).

      While iTregs seem to function only by the production of immunosuppressive cytokines such as
      IL-10 and TGF-β, nTregs can exert their activity also through various mechanisms, such as
      cytokine-dependent, cell contact-dependent or both, according to the nature and intensity of
      the inflammatory response and of the target tissue to which the latter is directed. This
      context-dependent regulatory mechanism leads to the elaboration of the model of adaptability
      and diversification within the function of these cells: in other words, Tregs would
      constitute a functionally homogeneous cellular subgroup in which every single cell can exert
      a different mechanism according to the characteristics of inflammatory response. Some authors
      believe instead that Tregs consist of several subpopulations, each of these presenting its
      own inhibitory mechanisms (27-28).

      Phenotypic characterization of Tregs among circulating human lymphocytes has been complicated
      by the fact that CD25 is not exclusively expressed by Tregs but also by non-regulatory
      lymphocytes after activation, so at present only CD4+ cells expressing the highest CD25
      levels (CD4+CD25+bright) can be considered authentic Tregs.

      In 2003 the Forkhead Transcription Factor (Foxp3) was identified as major regulator in Tregs
      development; its expression is predominantly restricted to CD4+CD25+ T lymphocytes and Foxp3
      expression in naive T cells supports their conversion into a regulatory T phenotype
      functionally similar to nTregs. Foxp3 is therefore considered an extremely specific marker of
      Treg cells and is fundamental to the control of their development und functioning.
      Particularly, some studies have demonstrated that the lack of Foxp3 at birth in humans and
      rats leads to a massive T-cellular hyperproliferation, resulting in multi-organ autoimmunity
      and premature death (24), whereas its deletion in adult rats causes hyperproliferation and
      expansion of dendritic cells as well as death within two weeks. Based on these results Foxp3
      seems to be the ideal marker for tolerance study.

      Tregs production is strictly controlled by signals emanated by various types of cells, such
      as epithelial cells, T lymphocytes and APCs. Although the presence of the latter is
      fundamental to TCR-mediated activation of Tregs, the real suppressive phase in vitro induced
      by nTregs is actually APC-independent. It implies in fact a mechanism of contact between
      CD4+CD25- effector T lymphocytes and CD4+CD25+ nTregs.

      At present we dispose of little knowledge regarding functional consequences of suppression
      carried out by Tregs on CD4+ and CD8+ lymphocytes which are sensitive to them, beside that
      they inhibit IL-2 transcription and, consequently, induce cell cycle interruption. Further
      immune suppression mechanisms reported by other studies, but not yet confirmed, are
      following: anergy induction in responsive T cells, refractory reaction to the mitogenic
      effects of IL-2, release of new iTregs which are likely to suppress Th1s and Th2s by
      producing TGFβ1 and/or IL-10.

      NTregs require particular cytokines for their development and homeostasis. Moreover, Tregs
      result to be hypoproliferative or anergic to TCR activation, probably due to their
      incapability of transcribing and actively secreting IL-2. This cytokine is principally
      released by activated T lymphocytes but not by nTregs and it exerts its biologic activities
      by binding to its receptor, a membrane protein complex formed by three subunits: α (CD25), β
      and γ chain.

      When IL-2 binds to its receptor in T cells it activates Janus kinases (JAKs) which
      consequently phosphorylate and release STAT5 proteins (signal transducers and transcription
      activators). IL-2 exerts in fact its effects on T cells by regulating several target genes
      primarily at transcriptional level; it does not only increase the expression of IL-2R α and β
      chains but it also modulates the expression of genes involved in cell cycle regulation, by
      inducing for instance the up-regulation of proto-oncogenes such as c-myc, c-fos and c-jun, as
      well as anti-apoptotic genes such as bcl-2 or pro-apoptotic genes like Fas ligand. IL-2 is
      essential for nTreg development and maintenance, as some studies have demonstrated: induced
      IL-2 depletion in rats causes autoimmune diseases which can be associated to a reduced number
      of Foxp3+ nTreg lymphocytes in peripheral compartment.

      Another important cytokine to the functioning of Tregs is TGF-β. Despite its inhibitory
      effects on other T cells, this growth factor is likely to support extra-thymic production of
      Treg cells and the increase of Foxp3 expression: this action is, however, necessary
      exclusively to the initial induction. TGF-β plays therefore a different but complementary
      role to IL-2.

      Finally, a further critical cytokine to the functioning of Tr1 lymphocytes is IL-10. Chronic
      activation in vitro of CD4+ lymphocytes in the presence of IL-10 leads to their
      differentiation in Tr1, high levels of IL-10 and TGF-β, low levels of IL-2 and IL-4 and a
      scarce proliferation after restimulation. IL-10 can moreover induce immunosuppression by
      supporting the formation of anergic T cells capable of inhibiting the activity of other
      activated T lymphocytes through a contact-dependent mechanism, by competing with the latter
      for specific APC (Antigen Presenting Cells) receptors and for locally produced IL-2. These
      anergic T cells are able to suppress immune response in vivo, as their transfer in rats which
      underwent allogeneic skin graft showed a longer transplant survival.

      Beside the above mentioned cytokines, others seem to be involved in different ways in Treg
      generation and induction, such as IL-4 and IL-13, whereas other cytokines have an antagonist
      effect on them, such as IL-6 for instance, which contrasts their immunosuppressive activity
      without interfering with proliferation. Other studies on Treg function have also showed that
      a transfusion of allogeneic Treg lymphocytes and the subsequent skin graft in IL2Rβ-/- rats
      (rapidly prone to lethal autoimmune diseases if not treated) not only controlled and
      suppressed those diseases although Tregs and self-reactive T cells were MHC-incompatible, but
      also consequently allowed to establish a state of tolerance to those transplants presenting
      common MHC molecules to donor Tregs. This experiment has then proved that Tregs suppress also
      self-reactive T cells deriving from non MHC-correlated donors and so, theoretically, that the
      donor pool for Treg-based immune therapy would be potentially very wide.

      Thanks to their essential role in immune tolerance and their immunosuppressive function, Treg
      lymphocytes are therefore gaining more and more importance in transplantology, as
      experimental models of organ transplants have underlined their capability of modulating CD4+
      and CD8+ cell activity, both responsible for rejection, by inhibiting it, and of suppressing
      GVHD (Graft-versus-host disease), mediating in this way allotransplant tolerance. There are,
      however, controversial results regarding Treg phenotype and their action mechanism, depending
      on the experimental model and on the protocol used to induce this tolerance.
    

Intervention name(s): Tacrolimus + Methylprednisolone; Tacrolimus + Everolimus + Methylprednisolone
Intervention type(s): Drug; Drug
Intervention description(s): Induction therapy:
Methylprednisolone (500 mg), thymoglobulins (1.5 mg/Kg/die, beginning 1 hour before transplantation) and 1 mg/Kg/die during the following 3 days.
Maintenance therapy:
Tacrolimus dosage will be 0.2 mg/kg of body weight and will be administered approximately 12 hours after chirurgical intervention twice a day (mornings and evenings). Dosage will be adapted on individual basis in order to gain pre-dose blood dosages according to indicated plasmatic levels; Induction therapy:
Methylprednisolone (500 mg), thymoglobulins (2.5 mg/Kg/die, beginning 1 hour before transplant) and Mycophenolate mofetil (1000 mg) and no immunosuppressive therapy for the following 3 days (WOFIE hypothesis), and for day 4-5-6 thymoglobulins (1.0 mg/Kg/die).
Maintenance therapy:
Tacrolimus dosage will be 0.1 mg/kg of body weight and will be administered approximately 12 hours after chirurgical intervention twice a day (mornings and evenings). Dosage will be adapted on individual basis in order to gain pre-dose blood dosages according to indicated plasmatic levels
Intervention MeSH terms: Methylprednisolone; Methylprednisolone Acetate; Methylprednisolone Hemisuccinate; Prednisolone; Prednisolone acetate; Everolimus; Tacrolimus; Prednisolone hemisuccinate; Prednisolone phosphate
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: Kidney transplantation; Regulatory T cells; Tolerance; Tacrolimus; Rejection