**9. Immune suppression and tolerance in xenotransplantation**

Advancements in understanding of immune mechanisms in immune rejection have elucidated a number of targets and pathways for intervention, and discovered a variety of small molecule and protein therapeutics for the suppression and manipulation of the immune system. However, the restraint of the immune system required to prevent xeno-organ rejection places the patient at significant risk of infections, tumors and other diseases which are preventable by an intact immune system. Therefore, there is a growing interest in the application of immune tolerance mechanisms in the transplant setting.

Immune tolerance is the natural unresponsiveness of the immune system to targets which may otherwise create an immune response. As mentioned previously, there are many mechanisms used by the immune system to identify non-self-antigens to prevent autoimmune diseases. As the body of literature regarding the molecular basis of immune tolerance has grown, interest in testing tolerance mechanisms in xenotransplantation has also increased [50].

Mixed chimerism is one route that has shown significant promise in both allo- and xenotransplant settings. This approach combines the transfer to the recipient of both the organ and hematopoietic cells from the donor. Typically, the patient is pre-treated with radiation or drugs to allow hematopoietic cell engraftment prior to the organ transplant. The combination of hematopoietic cells from host and donor allows cross-tolerance of host immune cells to donor tissue as well as donor immune cells to host tissue. Therefore, the resulting immune system is a combination of the donor and host, or a "mixed chimera," which recognizes the donor organ and host tissue as "self" despite the differences in genetic origin [51].

of genetically-modified pigs. In addition, in the normal situation human cells display human peptides in the HLA, the overwhelming majority of which will be conserved between a human donor and recipient, and thus much less likely to induce a response. If pigs were engineered to express human HLA which is perfectly matched to the patient, the donor porcine cells could now be significantly more efficient at displaying porcine peptides to the human immune system and more rapidly induce T cell activation. Therefore, introduction of human HLA in place of porcine SLA may not provide a benefit without additional engineering.

Humans possess a number of pre-existing antibodies specific for porcine antigens which can contribute to the xeno-organ damage during HAR. As the donor tissue is damaged, the antigens are released and presented to T cells as described above, causing the activation of helper T cells. These T cells interact with B cells in lymphoid organs, inducing the activation of any B cells which express antibodies specific for the xeno-antigens. This initiates the germinal center reaction, in which antigen-specific B cells rapidly proliferate and mutate their antibody sequences and are then progressively selected for improved antibody function. The resulting B cells expressing the affinity-matured antibodies exit the germinal center and can differentiate further to plasma cells, which act as factories that can produce extraordinarily high levels of serum antibody [48]. These induced antibodies, like natural antibodies, further amplify

The *de novo* production of antibodies can be quite rapid and are a risk for the lifetime of the transplant whether for allo- or xenotransplantation. There are a number of drugs available for the control of B cell reactions. One of the most effective approaches is the depletion of B cells using antibody therapeutics such as Rituxan, specific for the CD20 surface molecule [49]. However, the constitutive ablation of host B cells will create long term immunosuppression and could be prohibitively expensive. Although highly related to CR in allotransplant, the B cell responses in xenotransplant are stronger and more challenging and likely to require more

Advancements in understanding of immune mechanisms in immune rejection have elucidated a number of targets and pathways for intervention, and discovered a variety of small molecule and protein therapeutics for the suppression and manipulation of the immune system. However, the restraint of the immune system required to prevent xeno-organ rejection places the patient at significant risk of infections, tumors and other diseases which are preventable by an intact immune system. Therefore, there is a growing interest in the application

Immune tolerance is the natural unresponsiveness of the immune system to targets which may otherwise create an immune response. As mentioned previously, there are many mechanisms used by the immune system to identify non-self-antigens to prevent autoimmune diseases. As the body of literature regarding the molecular basis of immune tolerance has grown, interest in testing tolerance mechanisms in xenotransplantation has also increased [50].

AVR/AHXR and contribute to the destruction of the xeno-organ.

342 Organ Donation and Transplantation - Current Status and Future Challenges

**9. Immune suppression and tolerance in xenotransplantation**

of immune tolerance mechanisms in the transplant setting.

stringent therapeutic control.

A further refinement of mixed chimerism includes transplant of donor thymus into the recipient, allowing selection of host T cells via donor antigen presentation [52], suggesting that tolerance is T cell dependent. A large body of evidence points to the role of regulatory T cells (Treg) as a driver of immune tolerance. Treg cells are antigen-specific but upon binding of the specific HLA-peptide complex on antigen-presenting cells will produce a variety of immune inhibiting and tolerogenic factors. The Treg cells may be derived from either thymus selection (central tolerance) or selection in tissues (peripheral tolerance), with central tolerance believed to be more durable, and the conceptual basis for donor thymus transplantation in mixed chimerism [53].

A critical factor in the maintenance of tolerance is the balance between Treg and effector T cells over time. Any imbalance that increases the number of effector T cells can rapidly lead to immune rejection. If indeed the Treg population is the main active component of immune tolerance, then it may be desirable to specifically bolster the numbers of Treg cells transferred to the recipient to more greatly ensure that the balance is biased firmly toward tolerance. A number of groups have established protocols for the generation of Treg cells that are specific for xeno-organs and tissues through *in vitro* selections and expansions [54]. While this has been shown to have positive effects in allograft tolerance, the durability is variable and, worse, some studies have described conversion of Treg to effector T cells which then contribute to rejection [55]. Despite these concerns, mixed chimerism, with or without Treg supplementation, remains a potentially valuable approach to immune tolerance.
