**3. Transplantation tolerance**

The alloimmune response can be divided into central and peripheral tolerance, according to the mechanisms that induce a tolerance state. These are related and not exclusive [27] (Figure 2).

#### **3.1. Central tolerance**

Central tolerance is the most important means by which T and B autoreactive lymphocytes are eliminated in a process termed clonal deletion. T and B cells mature and are educated in the thymus and the bone marrow, respectively (Figure 2B).

Immature T lineage cells emerge from hematopoietic progenitors in the bone marrow and enter the thymus without expressing either the TCR or coreceptors. Since they lack CD4 and CD8 antigens, these cells are called double-negative (DN) cells or thymocytes. T cell selection begins after DN cells have undergone a TCR-mediated rearrangement process and up-regulated both CD4 and CD8 antigens, thus becoming double-positive (DP) cells [28]. From here, the thymo‐ cyte's fate is determined by the nature of its interaction with self-peptides that are presented on the self-HLA molecules of thymic stromal cells. This process is called "the affinity-avidity model". If a T cell reacts too strongly with self-antigens presented on bone marrow–derived APCs, it is eliminated by apoptosis or negative selection in the thymus [29]. Thymocytes with TCRs that interact with self HLA/peptides with lesser avidity, are positively selected and evolve into mature T cells that express either the CD4 or CD8 receptor (single positive T cells). The cells with very low avidity interactions fail to induce survival signals and die within the thymus. At the end of the process, only 3% of the total number of CD4+ CD8+ DP cells are exported from the thymus, having developed into single positive CD4+ or CD8+ cells [30].

Currently, it is not completely understood how many peripheral tissue-specific antigens are expressed and presented in the thymus to ensure central T-cell tolerance to antigens that will be encountered in the periphery eventually. The expression of peripheral pro‐ teins in the thymus (such as insulin, thyroglobulin, and renal autoantigens) is driven in part by a gene called AIRE (autoimmune regulator). Mutations in the AIRE gene result in a disease known as autoimmune polyglandular syndrome type I. Interestingly, only cer‐ tain organs and systems are involved, and within these, only particular parts of the organ tend to be affected, confirming that additional mechanisms must be involved to maintain systemic tolerance [31].

B cells undergo a similar process, as they are tested for reactivity to self-antigens before they enter the periphery. Immature B cells, developing in the bone marrow, test antigen through their antigen receptor, a surface IgM called the B cell receptor (BCR). If signaling through the BCR is sufficiently weak, immature B cells can be rendered permanently unresponsive or anergic. However, if immature B cells are strongly self-reactive, there are two possible scenarios to ensure tolerance. The first is deletion of these self-reactive B cells. The second is receptor editing, a process by which a new receptor with altered specificity is generated through another sequence of B cell receptor gene rearrangements [32].

#### **3.2. Peripheral tolerance**

including HLA class I and IL-2 [26]. There are other important events implicated in the activation of T cells, including leukocyte migration and the interaction of chemokines with

The alloimmune response can be divided into central and peripheral tolerance, according to the mechanisms that induce a tolerance state. These are related and not exclusive [27] (Figure 2).

Central tolerance is the most important means by which T and B autoreactive lymphocytes are eliminated in a process termed clonal deletion. T and B cells mature and are educated in the

Immature T lineage cells emerge from hematopoietic progenitors in the bone marrow and enter the thymus without expressing either the TCR or coreceptors. Since they lack CD4 and CD8 antigens, these cells are called double-negative (DN) cells or thymocytes. T cell selection begins after DN cells have undergone a TCR-mediated rearrangement process and up-regulated both CD4 and CD8 antigens, thus becoming double-positive (DP) cells [28]. From here, the thymo‐ cyte's fate is determined by the nature of its interaction with self-peptides that are presented on the self-HLA molecules of thymic stromal cells. This process is called "the affinity-avidity model". If a T cell reacts too strongly with self-antigens presented on bone marrow–derived APCs, it is eliminated by apoptosis or negative selection in the thymus [29]. Thymocytes with TCRs that interact with self HLA/peptides with lesser avidity, are positively selected and evolve into mature T cells that express either the CD4 or CD8 receptor (single positive T cells). The cells with very low avidity interactions fail to induce survival signals and die within the

Currently, it is not completely understood how many peripheral tissue-specific antigens are expressed and presented in the thymus to ensure central T-cell tolerance to antigens that will be encountered in the periphery eventually. The expression of peripheral pro‐ teins in the thymus (such as insulin, thyroglobulin, and renal autoantigens) is driven in part by a gene called AIRE (autoimmune regulator). Mutations in the AIRE gene result in a disease known as autoimmune polyglandular syndrome type I. Interestingly, only cer‐ tain organs and systems are involved, and within these, only particular parts of the organ tend to be affected, confirming that additional mechanisms must be involved to maintain

B cells undergo a similar process, as they are tested for reactivity to self-antigens before they enter the periphery. Immature B cells, developing in the bone marrow, test antigen through their antigen receptor, a surface IgM called the B cell receptor (BCR). If signaling through the BCR is sufficiently weak, immature B cells can be rendered permanently unresponsive or

thymus. At the end of the process, only 3% of the total number of CD4+

exported from the thymus, having developed into single positive CD4+

their receptors.

**3.1. Central tolerance**

systemic tolerance [31].

**3. Transplantation tolerance**

468 Current Issues and Future Direction in Kidney Transplantation

thymus and the bone marrow, respectively (Figure 2B).

Besides the deletion process of autoreactive cells occurring during central tolerance, some T or B cells with self-reactivity may escape from the thymus or bone marrow, making the loss of self-tolerance easier. However, several mechanisms, collectively named peripheral toler‐ ance, can control or eliminate such cells. Peripheral tolerance involves deletion and apoptosis, anergy, and regulation or suppression (Figure 2C).

### *3.2.1. Deletion and apoptosis*

This mechanism is used to eliminate activated T cells specific for self-antigen. The programmed cell death, or apoptosis, is also termed activation-induced cell death (AICD). This process is mediated by the interaction of Fas (CD95) with its ligand (Fas-L or CD95L) on T cells, and can occur in developing thymocytes as well as mature T cells [33]. IL-2 can activate the STAT 5 sig‐ naling pathway through the IL-2 receptor (IL-2R), which in turn potentiates the up-regulation of Fas-L and the down-regulation of Bcl2 expression on T cells, thus promoting AICD. Con‐ versely, IL-15 acts as a growth and survival factor for T cells [34, 35]. Since augmented AICD can induce tolerance through elimination of populations of reactive lymphocytes [36], certain tol‐ erogenic models which use IL-15 antagonists and IL-2 agonists during transplantation havere‐ sulted in donor-specific tolerance [37]. Further research on this topic is needed before considering this peripheral mechanism as a therapeutic approach.

#### *3.2.2. Anergy*

CD8+ DP cells are

cells [30].

or CD8+

The hyporesponsiveness of T or B cells to further antigenic stimulation, also called anergy, is a process that can result from antigenic stimulation in the absence of costimulation. In the case of T cells, complete activation requires the presentation of peptide on the HLA molecule to the TCR (first signal), and costimulatory signals, such as the B7-CD28 and CD40-CD154 interac‐ tions (second signal). The second signal is required to induce the multiple pathways that will lead to the activation of IL-2 gene transcription, ultimately inducing T cell activation and proliferation. However, it has been shown that IL-2 production and subsequent signaling through its receptor, IL-2R, is necessary for T cells to escape anergy, since blocking IL-2/IL-2R engagement even after stimulation through the TCR and CD28 still results in induction of T cell anergy [38].

As with T cell activation, B cell activation requires two signals. In this context, naïve B cells can be anergized if their surface immunoglobulins bind to self-antigens (first signal) in the absence of the additional necessary T cell signals (second or costimulatory signal) [39].

#### *3.2.3. Regulation or suppression*

A third mechanism of peripheral tolerance is regulation or suppression of immune responses to self or foreign antigens. Perhaps, the regulatory T cells (Treg cells) are the most important and well documented effectors of this mechanism to date. These cells control the type and magni‐ tude of the immune response to foreign antigen to ensure that the host remains undamaged. Treg cells are also integral to maintaining a lack of response to self-antigens or tolerance [40].

were not compliant with their immunosuppressive regimens or in individuals who had previously received a bone marrow transplant for hematological disorders [52]. At the present time, in looking for tolerance in renal transplantation, physicians in clinical prac‐ tice have implemented protocols and surgical procedures in which tolerance was the

Tolerance in Renal Transplantation http://dx.doi.org/10.5772/54734 471

Protocols in which tolerance in renal transplantation was the planned objective before the transplant may be divided into three subgroups, namely molecule-based, cell-based, and total

The molecule-based group includes all cases in which the induction of tolerance was attempted through administration of presumed tolerogenic drugs. These tolerogenic drugs include polyclonal antithymocyte globulin antibodies and anti-CD25 monoclonal antibodies. Anti-CD25 monoclonal antibodies competitively inhibit IL-2R-dependent T cell activation, while the polyclonal antithymocyte globulin antibodies are directed against lymphocyte antigens. The goal of the induction treatment was the nonspecific removal of clones of immune cells responsible for rejection before contact with foreign donor antigens occured. Once the donor antigens were in place after implantation of the new kidney, repletion of immune cells occured, favored by the homeostatic expansion triggered by leukocyte depletion. In addition, minimi‐ zation of maintenance immunosuppression was implemented to further reduce the anti donor response with just enough treatment to prevent irreversible immune damage to the graft, but not with such heavy treatment that the donor specific clonal exhaustion-deletion process was

In the cell-based group, patients received a donor-cell infusion of highly enriched CD34+ hematopoietic progenitor cells mixed with CD3+ T cells, [54] ie, patients received heavy conditioning regimens in association with the perioperative infusion of immunomodulatory cells, such as transplant-acceptance inducing cells. Afterward, maintenance immunosuppres‐ sion was given for a few months until complete withdrawal, when possible. Overall, although these trials demonstrated that the infusion of transplant-acceptance inducing cells is feasible, major concerns remain regarding the efficacy and safety of such an approach. Whether this approach confers any benefit in the establishment of minimal immunosuppression in renal transplantation patients when compared with the protocols currently in use is unclear. Lastly, the optimal dose and timing of cell infusions, along with the most appropriate concomitant

Patients who received renal transplantation after bone marrow transplantation from the same donor are also included in this group. Bone marrow transplantation, when success‐ ful, generally results in the total replacement of the recipient's bone marrow with the do‐

immunosuppression regimen, remains to be determined [55,56].

planned objective before the transplant.

**4.1. Strategies and protocols**

*4.1.1. Molecule-based protocols*

lymphoid irradiation.

precluded [53].

*4.1.2. Cell-based protocols*

There are two subsets of Treg cells."Natural" Treg cells, are a thymus-derived population that constitute about 10% of the CD4 population. Natural Treg cells express CD4, CD25, CTLA4, and GITR on their surface [41], and express transcription factor Foxp3 intracellularly [42]. The importance of Foxp3 as the orchestrator of the molecular programs involved in mediating Treg function has been highlighted by diseases such as IPEX syndrome (immune dysfunction, polyendocrinopathy, enteropathy and X-linked inheritance), in which a mutation in the Foxp3 gene has been described [43].

The other subset of Treg cells, commonly termed "adaptative" Treg cells, develops in the periphery, in a thymic-independent manner, following antigen encounter under particular circumstances, namely exposure to transforming growth factor-β (TGF-β). This leads to the expression of Foxp3; the hallmark of Treg cells [44]. Data suggesting the role of these cells in immunologic tolerance has been obtained from different studies in which patients with normal graft function reportedly possess a smaller Treg population compared with patients having chronic allograft rejection, suggesting that Treg cells may prevent damage and graft loss [45]. Other groups have shown that certain immunosuppressive protocols are more permissive than others in generating these populations [46].

The mechanisms by which Treg cells exert their effects are not completely understood. There have been two main mechanisms proposed. One mechanism requires cell contact between CD4+CD25+ Treg and responder cells and interaction between CTLA-4 and GITR molecules [47], while the other mechanism involves the induction of suppression or regulation by newly generated suppressor T cells in a cytokine-dependent manner through IL-10 and/or TGF β [48, 49]. Although promising, there is still too much to learn, before using this subset of cells for tolerance induction in renal transplantation.

In addition to Treg cells, there are other cell phenotypes with regulatory properties, such as CD8+ T cells and certain NK populations [50]. CD8+ T cells with regulatory/suppressive properties have been named "veto cells". Such cells maintain peripheral tolerance by attacking alloreactive T cells which are present in bone marrow with increased frequency, and may be responsible in part for the reduction in graft versus host disease and the induction of chimerism seen in some bone marrow transplant models [51].
