3

Lessons Learned from Immune Tolerance and Graft Acceptance

The first session of the workshop explored the current state of knowledge about immune tolerance mechanisms and the lessons learned from other areas of research, including transplant immunology, cancer immunotherapy, and the microbiome. Sohel Talib, scientific program officer at the California Institute for Regenerative Medicine, moderated the session.

THE MICROBIOME AND IMMUNE TOLERANCE: LESSONS FROM ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION

Robert Jenq, deputy department chair of genomic medicine and associate professor of genomic medicine and stem cell transplantation at the University of Texas MD Anderson Cancer Center, stated that allotransplant is a standard treatment for hematological malignancies and is commonly complicated by graft-versus-host disease (GVHD). Approximately 16 percent of mortality in adults who die within 100 days of unrelated donor hematopoietic cell transplantation (HCT) is attributed to GVHD (Phelan et al., 2020).

Pathophysiology of Graft-Versus-Host Disease

The paradigm for how GVHD is believed to occur begins with a patient who undergoes chemotherapy, radiation, or immune-depleting antibody treatment to facilitate engraftment of the hematopoietic cell graft from an unrelated donor (Jenq and van den Brink, 2010). The bacterial products within the patient are thought to translocate and activate host antigen–presenting cells, which in turn activate the effector immune system via donor T cells within the stem cell graft, Jenq explained. These donor T cells then recruit other immune pathways through a variety of mechanisms, such as inflammatory cytokines and cytotoxic ligands. The T cells can also recruit other immune cells, including natural killer (NK) cells and macrophages. Together, the T cells and recruited innate myeloid cells can target a variety of tissues. Classically, these targets are the skin, gastrointestinal tract, liver, and hematopoietic system. A “silver lining” of this process, Jenq said, is an antitumor response that occurs during the inflammatory cascade in which the leukemia, lymphoma, or hematological malignancy becomes better controlled.

Early Studies and Interventions for Graft-Versus-Host Disease

The pathophysiology of GVHD is derived from studies conducted over the past century, said Jenq. One of the earliest germ-free isolators was developed in the 1920s. Housing animal subjects, these sterilizable steel isolators featured ports with attached gloves and windows for handling and viewing

the subjects. While the isolator design and materials have changed in past decades, the basic features remain the same. In the 1970s, two pioneering papers were published—the first detailed a study conducted on germ-free mice in isolators, and the second looked at normal mice treated with gut-decontaminating antibiotics (Jones et al., 1971; van Bekkum et al., 1974). These two studies indicate that in the absence of a microbiome, GVHD is much milder than it otherwise would be. Thus, the microbiome contributes to the pathophysiology of GVHD, he explained.

The microbiome studies on mice were soon followed by clinical application, Jenq said. In the 1980s, Rainer Storb at the Fred Hutchinson Cancer Center in Seattle studied the effects of sterilization on bone marrow transplant patients. A 1983 study reported findings on patients with severe aplastic anemia who were conditioned with cyclophosphamide, followed by a bone marrow transplant from matched siblings (Storb et al., 1983). Thirty-nine of the 130 patients were randomly assigned to a protective environment with laminar airflow. Food was sterilized, utensils were autoclaved, and any items given to patients were sterilized, encased in plastic, and slipped through a slot in the room. Before examining patients, medical staff took the same precautions normally taken prior to performing surgery. Patients remained in this protective environment for 50 days. The study, Jenq said, suggested a benefit from near-total bacterial decontamination, with a markedly reduced incidence of GVHD in the protective isolation conditions that translated into a sizable improvement in overall survival. Patients in the protective environment had an 87 percent probability of survival, compared to 69 percent for patients in settings without laminar airflow (Storb et al., 1983). Therefore, the protective environment became standard practice in the 1980s and early 1990s. However, later studies did not indicate a clear benefit, and this research—coupled with the expense of providing a protective environment—led to the practice gradually falling out of favor (Passweg et al., 1998; Petersen et al., 1987; Russell et al., 2000). Jenq recalled asking Storb why the results did not hold up; Storb attributed it to the absence of cyclosporine in the earlier study, which was conducted prior to the use of calcineurin inhibitors such as cyclosporine, now commonly used to inhibit T cells and prevent GVHD. Once cyclosporine-based GVHD prophylactic treatment was available, the advantage of protective isolation was no longer as apparent.

Effect of the Microbiome on Allotransplant Mortality

Two decades later, research continues to focus on whether the microbiome can predict mortality in allotransplant patients, Jenq stated. A multicenter study—with researchers from Memorial Sloan Kettering Cancer Center, Duke University, Regensburg University Hospital, and Hokkaido

University—explored fecal microbiome diversity in transplant patients (Peled et al., 2020). Researchers collected 8,767 fecal samples from 1,362 patients across the globe. Profiling the samples revealed that patients have fairly healthy microbiomes with high diversity when they begin their bone marrow transplant hospitalizations. However, during the course of the two- or three-week hospitalizations, most patients rapidly lose much of that diversity. This finding held across all four research centers and indicates that microbiome diversity matters. Researchers stratified the patients by their microbiome diversity to compare the outcomes of those with higher diversity to those with lower diversity. Patients with higher microbiome diversity had improved overall survival rates compared to their counterparts. Mortality related to GVHD appears to be the primary driver in the difference in overall survival rates, Jenq explained.

Further analysis identified that the use of antibiotics could account for some of the differences in loss of microbiome diversity (Peled et al., 2020). When patients lose neutrophils, they often experience high fevers that are treated with empiric, broad-spectrum antibiotics. However, Jenq said, not all antibiotics are equally harmful to the microbiome. For instance, researchers found that cefepime was not highly associated with loss of diversity, whereas meropenem and piperacillin-tazobactam were more highly associated. Some antibiotic associations were only observed in particular centers, likely due to center-specific practices in first-line treatment for neutropenic fever, Jenq noted.

Factors Underlying Loss of Microbiome Diversity

Jenq and his colleagues at MD Anderson Cancer Center have further explored the associations between antibiotics and GVHD. In a study of almost 300 patients with myelodysplastic syndromes or leukemia who received allotransplants, researchers examined the rates of GVHD in patients subgrouped by the antibiotic treatment they received: meropenem, cefepime, both meropenem and cefepime, or no antibiotics. A pattern emerged, with patients that received no antibiotics showing a low incidence of intestinal GVHD (approximately 10 percent). Cefepime, which is not associated with loss of diversity due to its fairly narrow spectrum of activity, showed a similar incidence of GVHD to the group that received no antibiotics. However, patients who received meropenem—either on its own or in combination with cefepime—had a much higher incidence of GVHD, at almost 25 percent.

Retrospective clinical studies may indicate associations, but they do not demonstrate causality, Jenq explained. Therefore, he and his colleagues turned to a mouse model of experimentally induced GVHD (Hayase et al., 2021). Some mice were given transplants with a human leukocyte antigen

(HLA)-identical graft, which avoids GVHD. Other mice received HLA-disparate grafts, leading to the development of GVHD approximately one month post-transplant. Meropenem treatment was added to the drinking water of a third set of mice receiving HLA-disparate grafts, resulting in aggravation of GVHD and providing evidence for causality. Jenq noted that an additional advantage of preclinical animal studies is the opportunity to investigate potential mechanisms. Jenq described the work of Eiko Hayase, postdoctoral fellow at MD Anderson Cancer Center, who explored whether the meropenem was depleting beneficial bacteria or contributing to the expansion of harmful bacteria. Hayase tested for this by adding oral decontamination, and the results were similar to the previously mentioned 1970s decontamination studies in that the mice receiving decontamination and meropenem showed reduced GVHD mortality compared to mice without decontamination. The result indicates that one of the mechanisms of meropenem is selection for a harmful, pro-inflammatory bacterial population, Jenq added.

Categories of Intestinal Bacteria

Jenq explained that intestinal bacteria can be either gram positive or negative and are categorized by type of anaerobe, either facultative or obligate. Bacteria that are tolerant of oxygen are facultative anaerobes, and oxygen-intolerant bacteria are obligate anaerobes. Clostridia are gram positive, obligate anaerobes. Generally thought to be friendly commensals, clostridia help digest food, regulate the immune system, and produce short-chain fatty acids that can recruit regulatory T cells and produce nutrients for the epithelium. Another class of bacteria—bacteroidia—are also obligate anaerobes, but they are gram negative. Typically, they are also thought to be friendly commensals, and under normal conditions, bacteroidia help people digest fiber and starches; however, in the absence of fiber and starches, this subpopulation can alter their gene expression and consume mucus as a source of carbohydrates. Jenq explained that Hayase and her colleagues used 16S sequencing to profile the microbiome of mice and compared mice without GVHD, with GVHD, and with GVHD plus meropenem treatment. Clostridia were sensitive to meropenem, shown by loss of bacteria within that class, while Bacteroides—a genus within the bacteroidia class—exhibited significant enrichment (Hayase et al., 2021).

Role of Bacteroides thetaiotaomicron in Graft-Versus-Host Disease

Hayase utilized this information, Jenq said, to add another component to the decontamination model by reintroducing Bacteroides thetaiotaomicron (B. theta), the predominant bacteroidia species in mice. The

introduction of B. theta resulted in aggravated GVHD, indicating that B. theta is a pro-inflammatory, potentially harmful bacteria, said Jenq. Having broad capability to digest dietary fiber polysaccharides and glycans, B. theta is believed to be versatile in its carbohydrate utilization. Cultivating B. theta with four different carbohydrates it is capable of consuming has shown that it will not consume all four carbohydrates simultaneously. Instead, B. theta will target one carbohydrate until it is depleted, at which point it will target the next carbohydrate. A hierarchy of carbohydrate preference has been found for this organism, Jenq noted. The mice treated with meropenem showed loss of the mucus layer in the colon, leading to increased translocation of bacteria and compromised barrier function. Research on gene expression profiled how Bacteroides behave under different conditions (Hayase et al., 2021). Jenq and his colleagues found that (1) the carbohydrate xylose is lost with meropenem treatment and (2) xylose is beneficial for reverting Bacteroides back to friendly commensals.

Applications to Other Fields

Jenq outlined lessons learned from this research that might apply to other fields (see Figure 3-1). When the microbiome is healthy and diverse, it does not contribute to inflammation or to rejection. The removal or depletion of the microbiome—as was done in early studies—also lowers the inflammatory risk. However, a microbiome that is in an unstable state between the two extremes of healthy or depleted poses the largest risk for GVHD, translocation, or alloimmune rejection of stem cells. When asked how an individual at high risk of GVHD can be moved toward a lower-risk microbiome state, Jenq replied that for decades, Leiden University in the Netherlands has used an approach to avoid the high-risk microbiome state. It involves decontamination until the greatest risk for GVHD has passed. Once the patient’s neutrophils have recovered and cytokine levels

have settled, microbiome diversity and colonization resistance are rapidly restored through a fecal transplant.

IMMUNE TOLERANCE AND GRAFT ACCEPTANCE: LESSONS FROM TRANSPLANT IMMUNOLOGY

Megan Sykes, Michael J. Friedlander Professor of Medicine, professor of microbiology and immunology and surgical sciences (in surgery) and director of the Columbia Center for Translational Immunology at Columbia University, provided an overview of lessons learned from transplant immunology with application to immune tolerance and graft acceptance. The success of organ transplantation depends on immunosuppressant drugs that must be taken for life and are broadly immunosuppressive, Sykes said. Complications associated with immunosuppression include viral reactivation, susceptibility to cancer, and side effects such as diabetes and kidney toxicity, among others. These drug treatment–related complications are a major limitation to the success of organ transplantation. Furthermore, late graft rejection due to a chronic immune response is an ongoing problem. The holy grail in the field of organ transplantation, Sykes said, is to induce immune tolerance, thus avoiding the need for immunosuppressive drugs. Immune tolerance can be defined as “long-term graft acceptance with normal immunocompetence without requiring immunosuppressive therapy,” she said. Tolerance retains normal immune function, preserving the ability to resist infection and cancer.

Cell Engineering to Avoid Graft Rejection

An alternative strategy to tolerance, Sykes highlighted, is cell engineering to avoid the rejection of tissues, and possibly organs, derived from pluripotent hematopoietic stem cells. Current research is exploring the removal of major histocompatibility complex (MHC) molecules—the HLA—from such stem cells to avoid recognition by T lymphocytes, which are the major drivers of immune rejection. Although the removal of HLA enables cells to evade T-cell immunity, the cells are more susceptible to NK cell–mediated rejection. Evading T-cell immunity would also result in the loss of normal tumor surveillance on the transplanted cells and the inability to protect the graft from infections. Sykes noted that other approaches, such as expressing immunosuppressive molecules like programmed death-ligand 1 (PD-L1), do not involve HLA removal. However, concerns about the susceptibility of organs or tissues to infection persist with these approaches. Immune tolerance is not associated with these concerns, Sykes said, and is therefore a viable alternative approach to achieve acceptance of pluripotent stem-cell-derived grafts.

Mechanisms of Immune Tolerance and Allograft Tolerance Induction

There are three major mechanisms of tolerance: clonal deletion, anergy, and suppression. In clonal deletion, the cells that recognize the donor antigens, such as T cells, are not present, Sykes explained. With anergy, T cells persist but no longer respond to the antigen through their T-cell receptors. In suppression, T cells persist but are actively suppressed, for example, through regulatory T cells (Tregs). Allograft tolerance induction has been explored in the transplantation field for many years, resulting in thousands of reports of successful tolerance induction, said Sykes, though most of these results involved rodents and vascularized allografts, which are highly tolerogenic. A T-cell response involves both a destructive response and a suppressive, regulatory response, and it appears that rodent vascularized allografts have a strong ability to induce suppressive Treg responses. Partial or temporary immunosuppression allows this regulatory response to dominate, leading to graft acceptance. However, these approaches have rarely been successfully applied in large animal models and humans, Sykes added.

Regulatory Cell Therapies in Clinical Trials of Organ Transplantation

Several regulatory cell therapies are currently being explored in clinical trials. One approach involves cell therapy of various cell types that have demonstrated a regulatory function in animal models, said Sykes. Multiple clinical trials in HCT and organ transplantation are examining Tregs. These trials include three types of Tregs: (1) polyclonal cells that are nonspecifically expanded ex vivo, (2) cells that are expanded ex vivo in response to donor antigens, and (3) cells into which a chimeric antigen receptor has been introduced. Other studies explore approaches that involve regulatory dendritic cells, mesenchymal stem cells, and tolerogenic monocytes. None of the trials of these various approaches have an endpoint of complete immunosuppression withdrawal, Sykes said; therefore, they do not assess allograft tolerance. The only way to test tolerance would be to completely remove immunosuppression, which denies the patient the standard of care. This approach would not be attempted unless allograft tolerance were achieved in large animal models, and thus far these approaches generally have not been shown to achieve tolerance.

Efficacy data in the most stringent rodent models—such as skin grafts mismatched for MHC—as well as efficacy and safety data in large animal preclinical models might be needed before clinical trials in tolerance induction and immunosuppression removal should begin, Sykes suggested. A good model of transplant rejection of all kinds has been established in nonhuman primates, she noted. Furthermore, safety data for the proposed drugs or biological agents in humans are required. HCT has been explored

in large animal models and is the approach that comes the closest to meeting these criteria, and it has been tested in immunosuppression withdrawal trials in humans, Sykes added.

Non-Myeloablative Mixed Chimerism Protocol Studies

Hematopoietic stem cell transplantation (HSCT) can cause GVHD and other toxic side effects, and thus cannot be used in its traditional form in a person who needs an organ transplant but who does not have a hematological malignancy, Sykes explained. Meeting the requirements of HCT for tolerance induction is a challenge in the field; it involves developing a minimally toxic, non-myeloablative conditioning regimen that allows hematopoietic cell engraftments across HLA barriers while completely avoiding GVHD. This has been achieved in mice through non-myeloablative mixed chimerism protocols. An early model specifically targeted recipient T cells in the periphery with monoclonal antibodies and in the thymus with local irradiation to the thymus, combined with a low dose of total body irradiation (Sharabi and Sachs, 1989). This model achieved mixed hematopoietic chimerism in which donor and host cells coexisted for life, and the recipient animals were tolerant to the donor.

Pure Deletional Tolerance through Durable Mixed Chimerism

Mechanisms of this and other regimens have since been shown to involve depletion of alloreactive T cells in both the peripheral and thymic compartments, said Sykes. Donor hematopoietic stem cells are grafted in the bone marrow, and these hematopoietic stem cells then send progeny and coexisting recipient cells to the recipient thymus. This leads to the production of dendritic cells that mediate clonal deletion of newly developing T cells, allowing the tolerant T cells to fill the depleted peripheral T-cell compartment. The emerging T cells are tolerant of the donor and recipient, creating a centrally tolerized T-cell compartment. The procedure ultimately yields lifelong mixed chimerism and donor-specific tolerance, Sykes explained.

Although achieving this process in large animals and humans has been elusive thus far, Sykes said, other animal models, primarily rodents, have shown the complete deletion of donor-reactive cells. Pure deletional tolerance, with no long-term role for regulatory mechanisms, has been observed when durable mixed chimerism is achieved with complete, global T-cell ablation in the periphery and thymus (Khan et al., 1996; Nikolic et al., 2001; Sharabi et al., 1990; Tomita et al., 1994). It has also been accomplished with other models that specifically remove preexisting donor-reactive T cells in the periphery and thymus—without global depletion—by

combining costimulatory blockade with the bone marrow transplant (Fehr et al., 2008; Fehr et al., 2010; Fehr et al., 2005; Haspot et al., 2008; Kurtz et al., 2004; Lucas et al., 2011; Takeuchi et al., 2004). In other models, complete deletion of donor-reactive cells was not achieved; instead, Tregs were expanded in response to the donor, resulting in a combination of Treg mediation and ongoing central deletional tolerance (Bemelman et al., 1998; Bigenzahn et al., 2005; Domenig et al., 2005; Yamazaki et al., 2007). The combination of regulation and deletion can be a powerful method for inducing tolerance, Sykes emphasized.

Hematopoietic Stem Cell Transplantation for Kidney Allograft Tolerance

Three clinical trials of HSCT for kidney allograft tolerance have been conducted. While working at Massachusetts General Hospital (MGH), Sykes and her colleagues carried out a study involving non-myeloablative conditioning and succeeded in achieving tolerance across HLA barriers, which she noted is the most challenging immunological barrier. Northwestern University also achieved tolerance in HLA-mismatched full chimeras; however, the regimen was associated with GVHD and infectious complications. This trial is now in phase III. Stanford University tested another regimen that achieved mixed chimerism, but has thus far only worked in HLA-identical transplants and has not yet been successfully achieved in HLA-mismatched trials. Samsung Medical Center in South Korea is currently testing a protocol similar to that of MGH.

Translational Studies between Hematopoietic Cell Transplantation and Organ Transplantation

Translational work between HCT and organ transplantation led to the MGH protocol, Sykes said. While testing a monkey model, she and her colleagues learned that transient mixed chimerism associated with HCT transplantation at the time of kidney transplant from the same donor could lead to tolerance. This discovery led to successful tolerance protocols supported by the Immune Tolerance Network (Kawai et al., 2008; Kawai et al., 2013). The chimerism lasted only a few weeks, and therefore the long-term central deletional mechanism of tolerance referenced earlier could not be applied to these patients (LoCascio et al., 2010). Sykes and her colleagues studied the mechanisms of tolerance in these patients and developed a method of identifying the alloreactive repertoire based on T-cell receptor sequencing (Morris et al., 2015). In tracking these data, researchers found deletion of preexisting donor-reactive T cells in long-term patients (Morris et al., 2015). Furthermore, a specific expansion of donor-specific Treg clones was observed during the early period (Savage et al., 2018). This transient

chimerism leverages two mechanisms of tolerance, combining the early role of expanded donor-specific Tregs with the long-term deletion of preexisting donor-reactive T cells, Sykes explained.

Overcoming Limitations of Transient Chimerism

While this approach has worked in monkey models of kidney transplantation, it has not worked for other organs that are not tolerogenic, such as lung, liver, heart, or islets of Langerhans in the pancreas. Sykes outlined several limitations of transient chimerism: (1) the kidney plays an important role in promoting tolerance and must be grafted with the bone marrow transplant, (2) not all organs are tolerogenic like the kidney, and (3) no success has yet been achieved with a regimen that would be relevant for cadaveric donation. To help overcome these limitations, she and her colleagues at Columbia University have established a nonhuman primate transplant program aimed at creating a more durable mixed chimerism that will succeed for all types of organs. They use a non-myeloablative model that builds on previous transient chimerism studies and adds autologous polyclonal Tregs. The kidney graft is delayed to four months after the bone marrow transplant in order to conduct a stringent test of bone marrow–induced tolerance. The kidney is known to promote tolerance, she noted, and therefore if tolerance persists to 120 days without the kidney graft, then the model does not rely on the kidney itself for tolerance induction. They found that when a donor kidney was transplanted into the animal four months after the bone marrow transplant, the kidney was accepted with no immunosuppression and showed no infiltrates (Duran-Struuck et al., 2017). The control group did not receive Tregs, and these animals rejected the delayed kidney transplants. The results demonstrate that the combination of Tregs and a low-intensity conditioning regimen can achieve more robust tolerance than has previously been seen, Sykes explained. This tolerance does not depend upon the presence of the donor kidney, and therefore it should be applicable to other types of transplants.

Applications of Immune Tolerance and Graft Acceptance to Regenerative Medicine

Sykes outlined the relevance of immune tolerance and graft acceptance to regenerative medicine. The transplant community is working to develop gentler, non-myeloablative regimens for mixed chimerism induction that foster systemic tolerance to the donor. Several groups are advancing the development of pluripotent hematopoietic stem cells from human pluripotent stem cells. Advances will continue in the achievement of durable mixed chimerism with relatively nontoxic conditioning regimens, she said.

Success in these two areas could result in an off-the-shelf, pluripotent stem cell-derived donor HSCT that could be used to achieve tolerance for organs and tissues derived from those same pluripotent stem cells. This approach is optimal because it does not require the graft to be immunosuppressive; rather, the entire recipient immune system remains functional and becomes donor-tolerant, Sykes remarked.

DISCUSSION

Microbiome Manipulation

Immune system cells play a role in the suppression of GVHD, said Talib, and those cells have their own metabolism that influences GVHD suppression. He asked whether genetic engineering can be used to manipulate the microbiome to aid in engraftment, allograft acceptance, or the suppression of GVHD. Jenq replied that the microbiome was traditionally believed to be limited to epithelial surfaces such as the gastrointestinal tract, the skin, and perhaps the genital urinary tract. However, studies indicate the possibility of a circulating microbiome that can be detected in the blood. Research is also exploring a tumor microbiome, with studies examining viable bacteria within tumor cells in the context of pancreatic and colon cancer. Thus, the belief that the microbiome is only on epithelial surfaces is shifting, he added. Engineering the microbiome is an area of active study, as groups work toward genetic manipulation of bacteria. The tools to genetically manipulate commensal bacteria are limited, and each tool developed only works for a particular species or closely related strains of bacteria. Lactobacilli, Escherichia coli, and Bacteroides are easy to genetically manipulate, while clostridia are substantially more challenging, said Jenq. Therefore, the target for genetic manipulation should be carefully considered. Once the modality is developed, a researcher can be creative in terms of the bacteria that can potentially be made, he added.

Effects of Systemic Homeostasis and Inflammation on Immune Response

Given that most work on immune response and regeneration focuses on the local niche yet is also influenced by systemic immune homeostasis, Talib asked how systems-level immune function dysregulation can be understood and manipulated to aid local regeneration. Ruslan Medzhitov, Sterling Professor of Immunobiology at Yale University School of Medicine, replied that most knowledge about the effects of systemic homeostasis on local immune function is in regard to systemic metabolic homeostasis—that is, the availability of different types of metabolic fuels such as glucose, fatty

acids, and ketones. Other aspects of systemic homeostasis are less clearly understood. Systemic control of core body temperature has long generated interest because it changes with fever during infection, but the influence of increased body temperature on immune function remains unclear after many years of research. Some systemic homeostatic circuits, such as calcium concentration, are so tightly controlled that little variation is allowed. Generally, systemic homeostasis creates indirect effects as it translates to tissue-level homeostasis. Aside from metabolic homeostasis, these effects and the quantitative degree to which they are meaningful are not yet known, said Medzhitov.

Talib asked whether the role of inflammation in immune response could be reappropriated, and if so, what threshold of inflammation would be necessary to initiate a protective immune response. Traditionally, inflammation was viewed as a response to tissue injury or infection, Medzhitov said, whereas current thinking places that type of inflammatory response at one end of a spectrum. Homeostasis is at the other end of the spectrum, and between the endpoints are deviations from homeostasis that are less extreme than the inflammatory response to injury or infection. The question of how much inflammation is necessary to initiate the immune response is complex, because not all immune responses are the same, he said. Measuring a quantitative aspect—such as the amount of cytokines produced—will not fully capture the immune response. Generally, immune responses have been primarily considered from the perspective of defense from microbes, he added. This perspective is shifting as the idea that immune responses might have other functions unrelated to antimicrobial defense garners more attention. The role of inflammation for such immune responses likely differs from that for responses associated with antimicrobial need. This area of research is emerging, as most knowledge of the immune system is based on antimicrobial functions, Medzhitov commented.

Mechanisms of Tolerance in HLA-mismatched Transplants

Given that the MGH protocol can generate tolerance in HLA-identical or mismatched transplants, whereas the protocol from Stanford University has only been able to generate tolerance in HLA-matched transplants, Talib asked how these protocols differ and what lessons from the differences can be applied to tolerance induction in HLA-mismatched cases. Overall, the two protocols are quite different, Sykes said. The MGH protocol involves pre-transplant non-myeloablative doses of cyclophosphamide, a monoclonal antibody against cluster of differentiation 2 (CD2) to exhaustively deplete peripheral T cells, thymic irradiation to deplete existing alloreactive thymocytes, and a short course of calcineurin inhibitor post-transplant.

The Stanford protocol is based on total lymphoid irradiation and antithymocyte globulin, followed by an association with the kidney transplant, and then a delayed donor HCT.

Sykes noted that Dixon Kaufman, of University of Wisconsin Health, is currently evaluating the Stanford protocol in a nonhuman primate model in an effort to achieve tolerance across MHC barriers. Mechanistic studies are needed to explain how the tolerance is achieved and why it fails when HLA barriers are crossed. The Kaufman study has found that when immunosuppression—in the form of two post-transplant drugs—is stopped, the chimerism disappears and rejection occurs. In the MGH protocol, the chimerism is more transient, yet the graft is not rejected despite the loss of chimerism, she explained. Research on the mechanisms involved indicates expansion of donor-specific Tregs in the early period. This may partly depend on the way in which anti-CD2 selectively spares Tregs while depleting effector memory cells. Each method entails a different combination of mechanisms involving regulation and, to some extent, deletion. Modeling these protocols in nonhuman primates first allows researchers to better understand what to expect for results in humans, she said.

Tolerance Induction in Xenotransplantation

Given the advancement of genetic engineering, a participant asked Sykes about her thoughts on xenotransplantation. Sykes and her colleagues are currently exploring tolerance induction to xenografts. Immune responses against xenografts are more formidable and involve more mechanisms than responses to allografts, she said. In the allograft response, for instance, when the T-cell response in the naïve recipient is targeted, the response can be overcome, and no other significant activity takes place. In xenotransplantation, natural antibodies independent of T cells, in addition to NK cells, increase innate recognition of the xenograft, creating a much more powerful indirect T-cell response. Thus, the xenograft barrier is stronger, and studying xenograft tolerance is therefore important, Sykes said.

Genetic engineering is a key aspect of preventing xenograft rejection by natural antibodies, which has already been demonstrated with the development of Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout pigs, she added. The combination of genetic engineering and tolerance mechanisms will likely lead to advances. Sykes and her team are specifically targeting their genetic engineering approach to make the bone marrow of pigs better able to survive in a human marrow microenvironment and to resist immediate rejection by human macrophages. Although genetic engineering is a useful tool that helped researchers move past hyperacute rejection, removing additional natural antibody carbohydrate targets of the xenograft, beyond

that accomplished with Gal, will not necessarily be the best solution to non-Gal natural antibody-mediated rejection, said Sykes. Further study in nonhuman primates could help make that determination. Some data suggest that removing too many carbohydrate terminal sugars can reveal other natural antibody targets. For this reason, Sykes prefers the mixed chimerism approach because it tolerizes both the T cells and the natural antibody-producing B cells. She and her colleagues are working toward a goal of establishing permanent mixed chimerism in the nonhuman primate model.

Regulatory T-Cell Expansion

Belumosudil, which inhibits ROCK2,¹ has been approved by the U.S. Food and Drug Administration for chronic GVHD, Talib noted. Given that ROCK2 regulates T helper 17 cells (Th17), he asked whether a ROCK2 inhibitor might increase endogenous Tregs and be used in place of anti-thymocyte globulin to help suppress GVHD for solid organ transplants. Sykes replied that Treg instability is a major limitation in the use of exogenous Tregs and has been a barrier in extending studies to additional animals. The inflammatory environment produced by conditioning and lymphopenia contains substantial interleukin 6 (IL6). Current understanding is that Tregs in that environment may deviate toward the Th17 phenotype. Determining how to control this tendency is a challenge for researchers. Expanding endogenous Tregs is a research area of interest, Sykes noted. Approaches to supporting the survival and stability of infused Tregs are being explored, and clinical trials are conducting safety tests on methods to expand endogenous Tregs via drugs or cytokines and on several cell-based mechanisms. Tolerogenic dendritic cells and mesenchymal stem cells have been shown to be associated with expanded donor-specific Tregs. Engineering the population of antigen-presenting cells is one approach that will likely be an important component of a robust tolerance regimen, Sykes said.

Blocking the Initiation of Immune Response

A participant asked how the immune system’s potential can be harnessed to improve patient responses to regenerative therapies. Opportunities for transplantation depend on the type of tissue involved, Medzhitov said, as well as the cell type and its role within the tissue. Transplanted cells require appropriate growth factors in the extracellular matrix in order to survive, flourish, and differentiate. They also need to be protected from

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¹ ROCK2 is the abbreviated name for rho-associated coiled-coil-containing protein kinase. It is involved in a signaling pathway that regulates the balance between regulatory T cells and T helper 17 cells (Jagasia et al., 2021).

destructive aspects of the immune response. Two strategies for protecting the transplanted cells include (1) suppressing the response, which involves unwanted side effects, or (2) blocking signals that initiate the immune response. This second strategy could lead to a more desirable outcome because the immune system is not suppressed indiscriminately and remains functional, yet it is prevented from attacking transplanted cells. Achieving this outcome will require understanding the signals involved in recognition beyond matching molecular signatures. The immune system may be put on alert both because of a foreign signal from a mismatched MHC or microbial foreign antigen and because of a loss of normal homeostasis within the tissue. These functions of the immune system are noncanonical since they do not involve antimicrobial defense. Better understanding of such immune system inputs is necessary to pursue the preferred strategy of preventing activation of the immune system rather than suppressing an ongoing immune response, he said.

Organoid Models

Given that tissues have specific immune environments with tissue-resident immune cells playing an important role, Talib asked how organoid models with induced pluripotent stem cells can be used to understand the local immune system environment and improve methods for tissue and cell transplantation. Medzhitov replied that these technologies are proving to be incredibly powerful, but their full power will be realized when organoids incorporate more cell types and when they become self-sufficient such that they no longer require exogenous provision of growth factors. This development could provide a platform for interrogating all the parameters of the system that affect acceptance or rejection of different cell types, which could lead to an understanding of the rules that govern those interactions. Personalizing organoid models allows analysis of the role of particular genetic variance on organ function and immune susceptibility, Sykes added. This type of analysis has benefited the ability to generate insulin-producing beta cells from stem cells, she noted. Various types of organoids and chromatin immunoprecipitation assays can be used in high-throughput screening, Jenq said. Since organoid systems can directly study human cells, they circumvent concerns about whether findings in animal studies will hold up in human biology. Although there are challenges associated, some researchers have begun introducing bacteria or a combination of epithelial cells, immune cells, and bacteria to organoid systems, Jenq noted.

Applying Lessons from COVID-19 to Regenerative Medicine

The COVID-19 pandemic has increased the pace of science, Talib said. He asked whether any lessons from COVID-19 could be applied to move the field of regenerative medicine forward. Sykes remarked that although transplant patients are susceptible to COVID-19, the disease has not been particularly worse for them than for other patient groups. She added, it is not clear that immunosuppression increases the severity of COVID-19 and researchers need to learn more about the pathogenesis of the virus and how the timing and context of the immune system’s components affect its role in combating COVID-19. Furthermore, transplant patients and other people with compromised immune systems have not had optimal responses to COVID-19 vaccination, said Sykes. Messenger RNA (mRNA) vaccines have potential applications beyond infectious disease, and researchers are exploring their use in tolerance induction. Enabled by an infusion of funding, the development of COVID-19 vaccines is an “incredible tour de force” that demonstrates how increases in collaboration and energy dedicated to an issue can result in great strides made in a shorter time period, she said. Since the simple, underlying method of manipulating biological systems can be a tool for study to encode anything of interest, mRNA vaccines likely have uses beyond disease treatment and immunity, Medzhitov said. He noted that the COVID-19 pandemic has demonstrated the critical nature of reducing the time from discovery to dissemination of information. The current lengthy process in making research findings broadly available to scientists and the public is unsustainable and should be addressed, Medzhitov remarked.

Applying Lessons from Transplantation Immunology to Regenerative Medicine

Talib asked the panelists for their final thoughts on how lessons learned from transplantation immunology can be applied to regenerative medicine. History indicates that the most impressive advances in science are not spurred by funding, but by a research area that attracts interest in exploration, Medzhitov stated. Development of technology and tools can increase the feasibility of answering interesting questions. He remarked that making regenerative medicine more attractive through tool development could be a tipping point for the field, given that interesting questions beyond practical implications are already being asked. Sykes emphasized the potential benefits of engineering or directly differentiating pluripotent stem cells into hematopoietic stem cells. Greater investment in this effort could increase progress to benefit tolerance induction and the field of regenerative medicine

as a whole. Jenq advocated for the approach of reverse translation, involving a heterogeneous patient population. The approach is fertile ground for generating hypotheses and experimental testing, he continued. Talib added that the California Institute of Regenerative Medicine utilizes this type of approach in “translation going from bench to bedside and from bedside back to the bench” to better understand basic biology or immunology to advance the field.