Application of advanced molecular techniques and novel animal models has provided new insights into basic mechanisms underlying clinical transplantation. Investigations in diverse areas, including graft rejection and tolerance, autoimmunity and infectious diseases, have revealed increasing complexity of the mechanisms controlling immune function, notably at the interface of the innate and adaptive immune systems and within secondary lymphoid organs. New roles have been identified for NK and dendritic cells, B-lymphocytes and for Th17 and regulatory T cells, notably in novel animal models of costimulatory blockade and tolerance. Confirmation of these observations will be needed in normal animals and in humans undergoing organ and cell transplantation. The impact of the microbiome, of vaccines, and of antimicrobial therapies on immune memory and reconstitution after lymphocyte depletion is beginning to be defined.
antibodymediated rejection Activator of Transcription 3
donor specific transfusions
latent membrane protein-1
myeloid differentiation primary response gene-88
pathogen-associated molecular patterns
posttransplant lymphoproliferative disorder
segmented filamentous bacteria
Signal Transducer and
transforming growth factor
The breadth of scientific investigation in transplantation has been fueled by progressive application of new technologies and animal models. Studies in diverse areas of investigation including immunology (graft rejection, tolerance and autoimmunity), microbiology (the microbiome and infectious disease), tumor biology and tissue injury and repair have unearthed new roles for the cellular components of the immune system. These rapid advances have eroded some of the clarity of previously accepted models of immune function, e.g. as B cells, NK cells and macrophages take on some of the functions previously reserved for T cells. The roles of the anatomy of cellular interactions in lymphoid tissues, of Th17 cells, and the importance of the microbiome are continuing to be unraveled.
MiRNAs and Gene Expression Profiling
Advanced molecular techniques have provided new data on intracellular mechanisms of disease and allowed the development of new clinical biomarkers. In 1978, the Nobel Prize in Physiology or Medicine was awarded for the discovery of restriction enzymes and their application to problems of molecular genetics. However, the availability of full genomic sequences for humans and other species has yet to provide the promised insights into disease prediction, notably for complex traits. At the same time, molecular approaches are beginning to unravel some of the control mechanisms underlying biological processes in transplantation.
MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs of 21–22 nucleotides in length that target complementary messenger RNAs to control (repress) posttranscriptional gene expression. MiRNAs regulate multiple cellular functions including differentiation, proliferation, apoptosis, transformation and gene expression. Altered patterns of miRNAs (arrays) suggest that cellular physiology has been altered, as in infection or cellular injury. Posttransplant lymphoproliferative disorder (PTLD) is a spectrum of diseases often associated with Epstein–Barr virus (EBV) infection. A major oncogene of EBV is latent membrane protein-1 (LMP-1) which protects infected B-lymphocytes from apoptosis and promotes proliferation of EBV-infected cells. This process requires cellular production of interleukin-10 (IL-10) as an autocrine growth factor (Figure 1). B cells infected with a laboratory strain of EBV have altered expression of over 300 miRNAs while PTLD cells have alteration in an overlapping but distinct group of nearly 400 miRNAs . Of the miRNAs produced in an LMP-1 inducible B cell lymphoma cell line, some increased IL-10 production while others suppressed IL-10, ostensibly by binding at the 3′ untranslated negative regulatory element for the IL-10 gene. Thus, miRNAs are potential therapeutic targets for EBV-associated PTLD.
Further evidence of the roles of miRNAs in diverse processes can be derived from pattern analysis (expression profiling) in renal ischemia-reperfusion injury . Principal component analysis determines whether variance of a given data set (e.g. miRNAs) over time has direction. This allows miRNA arrays to be used as biomarkers for processes such as ischemia-reperfusion injury and repair. The miRNA profile following ischemia-reperfusion injury reflects shifts in a surprisingly small number of miRNAs from host cells and reflects lymphocyte-independent changes in kidney cells. Thus, miRNAs are emerging as therapeutic targets in cellular injury and repair, malignancy and immune function.
Th17 cells are a relatively recently described subset of T-lymphocytes that produce the inflammatory cytokine interleukin-17 (IL-17) . These cells produce IL-17A and IL-17F, important cytokines in neutrophil activation, and also IL-21 and IL-22. Functional differences in Th17 cells exist between mice and humans. Th17 cells play central roles in autoimmunity, antitumor effects and antibacterial and antifungal activities notably at epithelial-mucosal barriers. Th17 cells represent promising therapeutic targets in diverse immune disorders.
Differentiation of CD4+ helper T cells into Th17 cells occurs in the presence of transforming growth factor (TGF)-β and IL-6. Studies of the control of IL-17 gene expression established the regulatory role of genomic, intrageneic, noncoding elements and of DNA methylation at CpG dinucleotides . The methylation target is located within a consensus element (CCpGTCA) for Signal Transducer and Activator of Transcription 3 (STAT3). Multiple transcription factors (STAT3, Runx1, Ratf and IRF4) and nuclear receptors (RORγt, RORa and Ahr) are required for IL-17 induction during Th17 differentiation. The roles for the multiple regulatory factors were clarified using a 120 kb genomic region construct containing the il17a and il17f genes and six conserved, intragenic, noncoding sequences [4, 5]. A genomic element has been identified that synergizes with both STAT3 and RORγt to drive transcription from the il17a and il17f promoters. Demethylation of the promoter region CCpGTCA consensus element and of cellular histone proteins is required for differentiation of Th17 cells. Transcription factor RORγt-positive Th17 cells also play a role in hepatic ischemia-reperfusion injury . However, much of the IL17A produced in hepatic ischemia-reperfusion injury appears to be produced by NK cells rather than by Th17 cells . Given the breadth of processes in which Th17 cells participate, the inhibition of Th17 functions in graft rejection may provoke adverse effects in host defenses or in autoimmunity .
NK Cells and Rejection
In translational trials, the power of microarray profiling has been harnessed to clarify the roles of various immune cells in clinical syndromes. Late renal transplant glomerulopathy (TG) has been ascribed largely to antibody-mediated injury. In studies of a cohort of 51 presensitized DSA+ kidney recipients presenting with early (type 1) antibody-mediated rejection (ABMR) and intense microvascular inflammation, the occurrence of graft fibrosis and TG was evaluated using clinical parameters, graft biopsies for histology and C4d-immunostaining and using microarrays focusing on the NK cell burden, IFN-γ effects and markers of endothelial activation . At the time humoral rejection was diagnosed many renal biopsies (41%) remained C4d negative. NK cell burden and production of endothelial activation transcripts correlated with chronic ABMR and graft loss. These data are consistent with the growing evidence that NK cells play a previously underestimated role in ABMR [10-12]. Effective treatment of chronic ABMR may require therapies targeting NK cells and other components of the innate immune system.
NK cells appear to play a significant role in the rejection of cellular grafts as well as in ABMR. Embryonic stem cell-derived hematopoietic progenitor cells (HPCs) are deleted by natural killer (NK) cells in vivo. Proteomics from transplanted HPCs purified from bone marrow cells identified increased expression of 29 distinct proteins including one unique protein, Ym1 . Ym1 is a chitinase-like protein of ambiguous function. Recombinant Ym1 strongly enhanced NK cell cytotoxicity toward HPCs in vitro and augmented the killing of tumor cell lines in vivo. Ym1 activated NK cells with increased production of IFN-γ, perforin and granzyme B. Thus, organ rejection may depend not only on antigenic disparity between donor and recipient, but on the induction of new bioactive proteins posttransplantation that may enhance NK cytotoxicity and accelerate organ rejection.
New roles for the innate immune system can also be demonstrated in Rag knockout mice deficient for T and B cells. In this model, NK cells are the primary effector cells in rejection of allogeneic cells in naive mice. However, in these mice and in other models with donor antigen priming and help from CD4+ T cells, macrophages can be alloreactive . The importance of this role for macrophages in the human alloimmune response is unknown; however, the spectrum of macrophage functions is widening .
Costimulatory Blockade, Tregs, Dendritic Cells and Lymphoid anatomy
The role of Th17 cells has been examined in the context of the possibly higher-than-expected rate of graft rejection (and of PTLD) in kidney transplant recipients treated in clinical trials with belatacept despite improved long-term renal allograft function. Th17 cells have reduced costimulatory requirements and may mediate relative resistance to costimulatory blockade. In MHC-mismatched murine skin graft and heterotopic cardiac transplant systems, use of αIL12/23 antibody to block cytokines required for Th1 and Th17 T cell development when combined with costimulatory blockade substantially prolonged graft survival compared to either costimulatory blockade or αIL12/23 alone . It appeared that IL-23 blockade was the main factor in the improved effect of costimulatory blockade, highlighting the role of Th17 cells in graft rejection. Interestingly, IL-17 production was increased in recipients treated with costimulatory blockade alone compared to untreated recipients, suggesting that costimulatory blockade may amplify Th17 alloresponses. This effect might reflect the suppression of Th1 cytokines (e.g. IFNγ) by costimulatory blockade with increased Th17 development.
STAT 3 has been implicated in the control of FoxP3 expression in regulatory T cells (Tregs) in the context of alloimmune responses. Multiple laboratories are investigating uses for Tregs in protection of allografts. However, preloading of CD4+CD25+ Tregs with pancreatic islets before transplantation failed to induce long-term allograft survival in normal or wild-type animals . Blocking of STAT3 expression in islets in advance of transplantation improved islet survival.
Production of adequate numbers of functional Tregs for clinical trials has been a hurdle to some studies. Nonhuman primate regulatory T cells were expanded using CD3/CD28-coated beads; artificial antigen producing cells substantially increased the efficiency of expansion while maintaining high FoxP3 expression . The combination of these cells with CTLA4-Ig produced enhanced inhibition of alloproliferation in vivo. Treatment of rhesus macaque Tregs with sirolimus caused decreased proliferation in vitro but significantly enhanced the suppression of alloimmune responses by these cells. Dendritic cells (DC) exposed to rapamycin (RAPA-DC) enriched for CD4+FoxP3+ T cells and induced IFN-γ-dependent alloreactive T cell apoptosis . Thus, RAPA-DC also promote experimental allograft survival, yet paradoxically secrete increased IL-12, which is central for the generation of IFN-γ+CD4+ T cells. Thus, increased IL-12 may underlie the capacity of RAPA-DC to prolong experimental allograft survival and prevent GVHD. Among studies of cofactors for costimulatory blockade, among the most surprising was the demonstration of a critical role of B cell-derived IL-10 in transplantation tolerance induced by costimulatory blockade with donor specific transfusions (DST) [20, 21]. Multiple studies have demonstrated a B cell signature associated with tolerance in vivo. In BALB/c cardiac allografts into C57BL/6 mice following DST with anti-CD40 ligand (CD40L) antibody, B cell depletion by anti-CD20 antibody (day +1) prevents tolerance induction and induces acute cellular rejection. The mechanism for this abrogation of tolerance appears to include inhibition of IL-10 production in marginal zone B cells by anti-CD20. Deficiency of IL-10 in CD19+ B cells in a murine knock-out model also prevents tolerance induction. Direct B cell depletion perturbed cytokine expression in follicular helper T cells (Tfh), increased IL-21 expression and pushed differentiation toward inflammatory Th17 cells . Decreased expression of CCR7 in Tfh cells in the absence of B cells produce a shift of Th17 cells from the germinal center to the allograft. These observations suggest that persistent cross-talk of B cells and Tfh cells keeps Tfh cells in the proper location and prevent differentiation into Th17 cells.
The role of anatomic colocalization of cells within the lymph node to achieve tolerance induced by DST and costimulatory blockade was supported by studies of lymphotoxin-αβ (LTbR) on activated lymphocytes. LTbR signaling regulates communication among lymphocytes, myeloid cells and stromal cells required for tolerance. LTbR engagement in lymph node stromal cells is required for DST with costimulatory blockade-induced tolerance .
Studies combining costimulatory and integrin (LFA-1) blockade identified further requirements for cell-to-cell communication in lymph nodes in costimulatory blockade-induced tolerance. Prolonged allograft survival was produced in the face of heterologous memory responses by a mechanism that appears to favor relative Treg accumulation in draining lymph nodes . Thus, increased evidence is accumulating in costimulatory blockade for an important role for B cells, cytokines, and tightly regulated cellular colocalization in lymph nodes.
Links Between Innate and Adaptive Immune Systems
Links between the innate and adaptive immune systems and the increasing importance of Th17, dendritic cells (DC) and NK cells have been identified in multiple models of graft rejection. Toll-like receptors (TLRs) are key members of the innate microbial defense system as well as regulators of adaptive immunity . TLRs are pattern recognition receptors that bind molecules shared by pathogens based on common molecular patterns (pathogen-associated molecular patterns or PAMPs). Myeloid differentiation primary response gene-88 (MYD88) is a universal adapter protein used by all TLRs (except TLR3) to activate transcription of NF-KB. NF-KB is a transcription factor present in a preformed, inactive state and is activated by a wide variety of cellular stimuli including tumor necrosis factor-α, interleukin 1-β, bacterial lipopolysaccharides and other bacterial products. MYD88 knockout (MYD88−/−) mice are more susceptible to bacterial infections but are protected against acute and chronic renal graft rejection in a fully MHC mismatched (BALB/c → B6) model of kidney allograft rejection . MyD88−/− allograft recipients were protected from both acute and chronic kidney allograft rejection with development of donor antigen-specific skin graft tolerance . Depletion of CD4+CD25+FoxP3+ cells abrogated kidney allograft acceptance. Tolerance could be transferred by adoptive transfer of CD4+CD25+ cells from long-term MyD88−/− acceptors into naive RAG−/− mice with donor matched skin allografts. The mechanism of tolerance includes impaired Th17 development in MyD88−/− allograft recipients. This provides evidence that targeting signaling in the innate immune system may facilitate transplantation tolerance. Similarly, a blocking peptide for TLR4 improved the survival and function of murine allogeneneic islet transplants. Inhibition of membrane expression of TLR4 resulted in less TNF-α production and protected islets from cytokine-induced apoptosis . C57BL/6 recipients receiving islets preincubated with the blocking peptide remained normoglycemic for >20 days after transplantation. In a murine heart transplant model, recipient mice treated with diphtheria toxin to deplete host dendritic cells demonstrated significantly delayed rejection kinetics when compared with untreated controls . Recipient dendritic cells acquire intact MHC class I from donor graft parenchymal cells for presentation to CD8 T cells leading to activation.
The Microbiome and Immune Function
One of the aspects of transplant immunology that has gained significant traction in recent years is the effect of the microbiome on immune function . The microbiome refers to the entire community of microorganisms, including bacteria, parasites, fungi and viruses that populate the body—and potentially the main source of microbial exposure for the immune system. The microbiome includes acute and chronic infections, colonizing organisms, and is altered by vaccination and antimicrobial therapies. The genetic diversity of microbial species of the human colon, for example, includes >1010–11 cfu of commensal bacteria per gram of tissue. While over a thousand different microbial species from over 10 different divisions colonize the GI tract, just two bacterial divisions—the Bacteroidetes and Firmicutes—and one member of the Archaea (including the segmented filamentous bacteria or SFB) appear to dominate, together accounting for >98% of the 16S rRNA sequences obtained from this site. SFB are spore-forming obligate anaerobic bacteria that have not yet been successfully cultured in vitro.
The concept of heterologous immunity includes all immune “memory” responses to previously encountered pathogens which alter subsequent immune responses (Figure 2). Thus, memory T cells specific for microbial antigens may cross-react with donor MHC antigens and provoke graft rejection or block tolerance induction. In addition, T cell memory can be generated during homeostatic proliferation when T cells repopulate the host after depleting therapies. Graft or tissue injury (e.g. from ischemia-reperfusion), inflammation or microbial antigens may provoke upregulation of antigen presentation, the expression of costimulatory molecules or cytokine production with enhanced T cell priming. The sequence of infectious exposures is also critical to control of T cell memory; elevated frequencies of microbe-specific memory T cells may impact the specificities of subsequent immune responses given the distinct hierarchy of T cell responses  These effects may be mediated by NK and macrophages and dendritic cells as well as by T and B cells.
None of these observations explains how the host becomes tolerant of the huge mass of colonizing organisms or the role of these organisms in the development of immune function. Multiple studies presented at the ATC suggested that the colonic microbial repertoire has an important role in the maintenance of the local, colonic Treg population and may play a central role in the pathogenesis of autoimmunity, inflammatory bowel disease and in determination of immunity to self and nonself antigens. It has been observed that SFB induce the expression of Th17 cells in the lamina propria of the colon [30-32]. This is accompanied by a reduction in Tregs in colonic Peyer's patches but not systemically (e.g. in the spleen). In murine models, arthritis develops in conventionally housed mice mice with T cells displaying a transgene-encoded T cell receptor (TCR) that recognizes a self-peptide derived from glucose-6-phosphate isomerase (GPI) on the MHC class II molecule Ag7 . These autoreactive T cells provide help to GPI-specific B cells, resulting in massive production of GPI autoantibodies. Arthritis ensues rapidly with high penetrance (∼100%). This effect can be duplicated by gavage with SFB in germ-free mice. The Treg population in Peyer's patches of affected animals, but not in the spleen, is reduced by over 50% by exposure to SFB via the gut. The development of arthritis and the loss of Tregs are reversed by antibiotic treatment (vancomycin) targeting SFB. Thus, there appears to be a local effect of the gut flora on the development of Tregs; increased production of Th17 cells over Tregs results in systemic arthritis. The mechanism by which Peyer's patches sample the gut lumen is unclear but may involve cellular extensions that sample gut contents, possibly by dendritic cells.
The role of local Tregs in tolerance to commensal bacteria over development of effector cells has been demonstrated in mice . The T cell receptors of colonic Tregs are bacterial-antigen specific and distinct from the T cell receptor repertoire of systemic Tregs, providing further evidence for the peripheral differentiation of inducible Tregs. The reactivity pattern of colonic Tregs was transferrable by cohousing of naive and infected mice, likely by shared microbial flora and was not transferred by food or self-antigens. These observations have potential implications for clinical practice, including the impact of antimicrobial therapies on immune phenomena (e.g. arthritis) and the control of T cell receptor utilization following T cell depletion and repopulation .
The author of this manuscript has no conflicts of interest to disclose as described by the American Journal of Transplantation.