Nanomedicine, Volume IIA: Biocompatibility
© 2003 Robert A. Freitas Jr. All Rights Reserved.
Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003
184.108.40.206 Immunosuppression, Tolerization, and Camouflage
Transplantation is the transfer of living cells, tissues, or organs from one person, the donor, to another, the recipient (e.g., a blood transfusion), or from one part of the body to another (e.g., skin grafts) with the goal of restoring a missing function . However, even if the patient’s HLA types (histocompatibility locus antigens; Section 220.127.116.11) are closely matched to those of the donor, transplanted organs are usually rejected (beginning within minutes or hours of surgery ) unless the recipient’s immune system is carefully controlled. Immediately after the graft has been implanted, it is necessary to prevent sensitization of pre-existing mature T cells capable of recognizing the graft. Once the graft has escaped the initial acute phase rejection reactions, a cumulative unresponsiveness to the graft develops as the recipient is continually exposed to donor MHC, a stable state that sometimes depends on the development of antigen-specific T-suppressor cells [371, 5349-5354].
In an era of advanced nanomedicine, it should be possible to restrain or reprogram the immune system directly using genetic engineering (Chapters 19 and 23), or by using other means (e.g., temporary systemic white cell sequestration), to reduce or eliminate immunoresponsiveness during the period of nanomedical treatment. Traditional methods are much less desirable. The most general pre-nanomedical method to suppress immune system acute responsiveness is called antigen nonspecific immunosuppression. Antigen nonspecific methods include the use of cytotoxic drugs that interfere with all cell division in the body . Since the immune response to antigen requires clonal proliferation, agents that block mitosis are effective inhibitors of the immune response. But this immunosuppression is general, not specific, thus the patient is more susceptible to infection. If infection occurs, immunosuppression must be suspended whereupon an implanted graft is usually lost due to rejection . These agents also damage all tissues (e.g., gut epithelium, bone marrow) where rapid cell division is occurring, creating other undesirable side effects, thus often may not be suitable for use in medical nanorobotics.
The fungal metabolite cyclosporin A (cyclosporine) [384, 385] has a greater specificity for lymphoid T cells than other cells. Used in isolation, cyclosporine at 10 mg/kg-day effectively suppresses the entire immune system indefinitely , though at great risk of nephrotoxicity. Other newer calcineurin blockers may have fewer side effects . Other pre-nanomedical nonspecific lymphocytotoxic agents commonly include:
(1) Corticosteroids, Purine Analogs, etc. Prednisone and prednisolone  act powerfully to suppress the inflammation accompanying a rejection crisis, and also appear to reduce the expression of class II histocompatibility antigens, thus reducing the immunogenicity of the transplant. First used as anticancer drugs, purine analogs such as 6-mercaptopurine interfere with DNA synthesis and thus are also powerful antimitotic (hence immunosuppressive) agents. Other well-known agents include azothioprine (Imuran) , tacrolimus (FK506) , sirolimus (rapamycin) , mycophenolic acid (mycophenolate mofetil; CellCept) [385, 387], and leflunomide (and its malononitriloamide analogs) [387, 388].
(2) Antilymphocyte Globulin (ALG). ALG is produced by immunizing a large animal such as a horse with human lymphocytes, then purifying the gamma globulin fraction of the serum. Injections of ALG into a graft recipient have a powerful suppressive effect on graft rejection .
(3) Total Lymphoid Irradiation (TLI). A series of sublethal doses of radiation is directed at the patient’s lymphoid tissue (spleen, thymus, and lymph nodes in the neck, chest, and abdomen), with bone marrow and other vital organs shielded from the exposure . In due course, the stem cells in the bone marrow reconstitute the peripheral lymphoid system but the newly formed T cells seem to accept the graft as self . TLI has enabled some transplant patients to quit using other immunosuppressive agents altogether. Photopheresis  is also used for treatment of recurrent rejection.
However, all of these approaches have severe complications and side effects, so the risk benefits would need to be carefully evaluated and almost certainly would be inappropriate in a mature nanomedical technology environment.
(4) Other. T cell activation could also be blocked by altered peptide ligands [389, 390] or synthetic peptides ; or by antibodies to MHC class I [392-395] or class II [396, 397] molecules, or to the T cell receptor [398-401]; or by the presence of solubilized forms of these molecules . Anti-CD3 monoclonal antibodies (acting against all T lymphocytes) [404, 405] are available commercially, as are various other agents. Other methods for terminating lymphocyte responsiveness have been discussed [2545, 2550], and M. Sprintz suggests considering plasmapheresis to decrease levels of preformed antibody.
Interestingly, a few viruses and protozoa can also cause antigen nonspecific immunosuppression, and suppressed immune responses are observed rarely during chronic bacterial infections such as tuberculosis and leprosy . In leprosy (caused by Mycobacterium leprae), the response both to leprosy antigens and to unrelated antigens is poor. Immunological reactivity reappears after successful treatment, an observation that implicates the microbe as the likely cause of the general immunosuppression. At present, little is known of the mechanisms by which pathogens initiate generalized immunosuppression, though it is probably due to interference with the normal immune functions of B cells, T cells, or macrophages . However, the strategy appears to be rare among bacteria because general immunosuppression is not particularly useful for the invader if it merely promotes infection by competing unrelated microorganisms . Many viruses also include genes that can modulate the immune response – for instance, Epstein-Barr virus encodes a gene which produces a protein that is a homolog of IL-10 that downregulates the immune response – and the fungus Cryptococcus neoformans sheds large amounts of capsular polysaccharide that interfere with the formation of inflammatory responses in tissue .
Antigen-specific immunosuppressive agents disable specific targets within the immune system. For example, after specific antigen activation, the responding T cells expand and express IL-2 receptors on their surface. Lymphokine toxin coupled to IL-2 binds and specifically removes this population. Monoclonal antibodies are also available that are specific for the IL-2 receptor. Their presence prevents T cells from proliferating in response to IL-2 . Agents that block CD28-mediated T-cell costimulatory signals inhibit T cell activation and induce a state of antigen-specific unresponsiveness in both in vitro and in vivo experiments [406, 407]. Dendritic cells (ordinarily highly potent activators of naive T cells) that are transfected with CD95 ligand cDNA, called “killer DCs,” deliver death signals, not activation signals, to T cells after antigen-specific interaction . Direct inhibition of complement-mediated responses using modified C3 has been reported . Additionally, in mild cases of leprosy the bacterium can induce an antigen-specific immunosuppression against M. leprae antigens only. This is perhaps due to a lack of costimulatory signals (interference with cytokine secretion), activation of suppressor T cells, or disturbances in TH1/TH2 cell activities .
In traditional organ transplantation work, immunological tolerance  to the histocompatibility antigens on the transplant can be induced by the use of tolerogenic antibodies  or other agents, called tolerogens  or antigen-specific tolerization therapy [413, 414]. For example, several small donor blood transfusions to the recipient prior to transplantation are observed to improve graft retention [415-417], and pretransplant implantation of donor bone marrow has induced donor-specific tolerance . Mitomycin-C-treated spleen cells from a donor rat, when injected preoperatively into a recipient rat, induce immune unresponsiveness when the recipient subsequently receives a cardiac allograft from the donor . Anergic antigen-specific CD4 T cells can inhibit T cells restricted by a different MHC class II molecule. The anergic T cells act as suppressor cells by competing for the membrane of the antigen-presenting cell and the locally-produced IL-2. Induction of tolerance to a single alloantigen could serve to regulate the immune response to an allograft carrying several MHC (and minor antigen) differences . The body can also learn to accept foreign material as “self” by placing the material to be tolerated into the thymus , where cells that recognize it will be inactivated or killed, or by using Starzl’s trick  of transplanting the graft along with immune cells that have the specificity of the graft (in his case, bone marrow from the organ donor), such that, again, cells attacking the graft are themselves attacked. The liver is also known to have a certain degree of “intrinsic tolerogenicity” .
By the late 1990s, strategies were being sought to induce specific tolerance to allogeneic biological transplants without affecting other immune functions. The “veto effect” [372-378] permits one such technique , wherein, for example, CD8 T cells suppress responses of MHC-restricted T-lymphocyte precursors to antigens expressed by those CD8 veto cells. Veto inhibition normally cannot provide complete tolerance to allogeneic grafts since it only operates on CD8-expressing cell populations. But Staerz, Qi and colleagues  have produced a hybrid antibody (Hab) combining a monoclonal antibody for a class I MHC molecule with a soluble CD8 molecule, which can specifically and effectively transfer veto inhibition to different stimulator cell populations, thus promising to selectively and completely tolerize graft-specific cytotoxic T lymphocytes without affecting normal immune responses. Another Hab combines CD4 and an anti-MHC class II antibody, which binds to class II molecules bringing CD4 accessory molecules to the surface of class II-bearing stimulator cells. CD4 T cells with specificity to Hab-coated stimulator cells cannot engage their CD4 molecules and are no longer activated . There is also evidence that retrovirus-infected cells possibly may employ a veto-like mechanism to evade immune T-cell recognition .
A more valuable approach from the standpoint of nanomedicine is to reduce the immunogenicity of the implant itself, before it is implanted. Traditionally, much of this work involves the “camouflaging” of graft cells. For example, Scott and Murad  used coatings of nonimmunogenic long-chain polymers such as methoxypoly(ethylene glycol) (mPEG) to globally camouflage the surface of foreign cells. This effectively attenuated antibody to surface epitopes and decreased the inherent immunogenicity of foreign, even xenogeneic, cells. Pegylated red blood cells (RBCs) lost ABO blood group reactivity, anti-blood group antibody binding was profoundly decreased, and pegylated sheep RBCs were ineffectively phagocytosed by human monocytes, unlike untreated sheep RBCs  – with no significant detrimental effects on RBC structure or function . Pegylation of antigen presenting cells and T lymphocytes prevented recognition of foreign class II MHC molecules and prevented T cell proliferation in response to foreign MHC molecules . Loss of peripheral blood mononuclear cell (PBMC) proliferation was not due either to mPEG-induced cytotoxicity, since viability was normal, or to cellular anergy, because phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, which suggested that the mPEG-modified T cells were not antigen primed . Similar experiments by Stuhlmeier and Lin  on pegylated endothelial cells showed that mPEG inhibits binding of several antibodies, LPS, and the cytokine TNF-alpha to the cells.
Some natural human cell types stimulate a stronger immune response than others if foreign members are put into the body. Strong immune response comes from leukocytes and endothelial cells . A weak or no immune response comes from keratinocytes, smooth muscle cells, and fibroblasts . Fetal cells may exhibit immune tolerance because of the expression of nonclassical HLA-G molecules at their surface .
Nanorobot architects must take care to avoid designs that might inadvertently trigger or facilitate an autoimmune response. Autoimmune disease (Chapter 23) is the consequence of an immune response against self-antigens that results in the damage and eventual dysfunction of organs that become targeted by the immune system. In most cases the triggering event is unknown, although for decades an infectious cause has been postulated to explain the development of autoimmunity. According to the “molecular mimicry” hypothesis [1153-1156], infectious agents (or other exogenous substances) may trigger an immune response against autoantigens when a susceptible host acquires an infection with a pathogen that has antigens that are immunologically similar to certain host antigens but differ sufficiently to induce an immune response when presented to T cells. The tolerance to autoantigens breaks down, and the otherwise pathogen-specific immune response that is generated cross-reacts with host tissues to cause tissue damage and disease. If a medical nanomachine is designed with organic components that are epitopically similar to components of the natural human body, then an autoimmune attack against those natural human components could also be directed against the nanorobots. Other medical nanorobots that present both human and viral  (or bacterial) components on their exterior blood-contacting surfaces could facilitate autoimmune sensitization of a human patient by providing a previously nonexistent immunological bridge between pathogenic and human epitopes.
Alternatively, resemblance between bacterial antigens and host (or even nanorobot-surface-displayed) epitopes, also called molecular mimicry, could weaken the immune response to that bacterium by inducing a certain degree of immune system tolerance to the pathogen. This is a potential negative “side effect” of nanorobotic treatment that may be avoidable using good design.
Antigenic disguise is another simple camouflage tactic found in nature . Pathogens may hide their unique antigens from opsonizing antibodies or complement by coating themselves with host proteins such as fibrin, fibronectin, or immunoglobulins. For example, S. aureus produces cell-bound coagulase , which binds to fibrinogen [1724-1726] and prothrombin [1725-1727] and activates it to form staphylothrombin, causing fibrin to clot and to deposit on the cell surface . This may immunologically disguise the bacterium so that its natural immunogenicity is not recognized as a target for an immune response . Protein A produced by S. aureus , and the analogous Protein G produced by Streptococcus pyogenes [1729, 1730], bind the Fc- or Fab-regions of immunoglobulins, thus coating the bacterium with antibodies and canceling their opsonizing ability. As yet another example, the fibronectin coat of Treponema pallidum  may provide an immunological disguise for these bacteria . Microbes can also simulate mammalian complex carbohydrates at cell surfaces to use as immune masks – for example, N-acetyl heparosan , colominic acid, and fructosyl chondroitin analog in E. coli , LeX in H. pylori , and hyaluronic acid in some bacteria .
Last updated on 30 April 2004