The convergence of bioengineering innovations with advances in immunology has substantially expanded the landscape of cellular therapies (see Glossary) [

]. Cellular therapies aim to treat or manage a disease by introducing living cells that will integrate within the host to restore or eliminate dysfunctional tissues. They typically encompass stem cells (SCs) or non-SCs derived from autologous, allo- or xenogeneic sources, either unaltered or genetically engineered. Currently, hematopoietic SC and chimeric antigen receptor T (CAR-T) cell therapies for hematologic disorders and cancers are the predominant clinically approved products. As of August 2022, there are more than 3000 active clinical trials of cellular therapies. These trials primarily use SCs and blood cells (leukocytes, red blood cells, and platelets) for a spectrum of therapeutic indications such as cancer, hematologic disorders as well as autoimmune, cardiovascular, degenerative, and infectious diseases. A breakdown of the current landscape of active clinical trials of cellular therapies is provided elsewhere [

].

Despite this expanding pipeline, host immune response to cellular therapies remains a challenge that could fundamentally impede clinical adoption and desirable outcomes [

]. Host immune rejection could occur even for cells of allogeneic origin with human leukocyte antigen (HLA) matching, attributable to mismatched minor alleles [

]. Systemic immunosuppression through immunosuppressant drugs is routinely used to reduce rejection. Immunosuppressive treatments are categorized as ‘induction’ (high-intensity immunosuppression immediately post-therapy), ‘maintenance’ (long-term immunosuppression to prevent chronic rejection), and ‘rejection treatment’ (used to treat acute rejection). These immunosuppressants include calcineurin inhibitors (e.g., tacrolimus and cyclosporine), corticosteroids (prednisone), monoclonal antibodies (e.g., basiliximab, adalimumab, and rituximab), inosine monophosphate dehydrogenase inhibitors (mycophenolate mofetil), mechanistic target of rapamycin (mTOR) inhibitor (rapamycin), and depleting antibodies (anti-thymocyte globulins). In general, immunosuppressive agents are administered to target T and B cells, which are key in immune rejection. However, systemic immunosuppression is undesirable due to increased risks of infections, cancers, and organ damage.

Therefore, the clinical success of cellular therapies demands innovations in facilitating as well as maintaining immune acceptance for favorable long-term therapeutic outcome. This review highlights emerging immunomodulatory strategies to attenuate immune rejection or promote tolerance to cellular therapies, with a discussion on local, site-specific immunomodulation measures (Figure 1, Key figure). We exclude solid organ transplantation and tissue grafts, which are extensively reviewed elsewhere [

]. We provide a perspective on opportunities for accelerating translation of innovative immunomodulation strategies that synergize with cellular therapies toward achieving widespread clinical success.

Genome editing technologies such as the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system have led to the development of ‘off-the-shelf’, or ‘universal’, engineered cell therapies with little to no immunogenicity. The CRISPR-Cas9 system generates targeted double-strand breaks (DSBs) in the genome. which can be repaired by the cell. For repair, cells can use non-homologous end joining (NHEJ), which can effectively knockout the gene of interest. Alternatively, homology-directed repair (HDR) can occur if a donor DNA template is provided, which allows for targeted insertion of exogenous genes. Depending on the application, gene editing to avoid immune recognition has mainly focused on the elimination of genes encoding for immunogenic surface markers, such as HLAs and T cell receptors (TCRs). For regenerative therapies using iPSCs, the focus has been on deletion of the B2M and CIITA genes required for the expression of HLA class I and II genes, respectively, which typically drive the alloimmune response (Figure 2). Although complete HLA knockout helps to avoid recognition by host CD4⁺ and CD8⁺ T cells, HLA-1 deficiency can lead to activation of recipient natural killer (NK) cells and transplant rejection [

]. Allele-specific editing of polymorphic HLA-1 to express common HLA-C alleles which could be matched to >90% of the world’s population, in addition to HLA-II elimination, was used to generate iPSCs which suppressed recognition by both NK and T cells [

]. Alternatively, overexpression of nonpolymorphic HLA-1 molecules such as HLA-E in stem and progenitor cells can also inhibit NK cell lysis activity [

,

]. Finally, iPSCs have been edited to simultaneously disrupt HLA-1 expression and overexpress CD47, a ‘don’t-eat-me’ signal which effectively inhibits phagocytosis and thus prevents macrophage and NK cell-mediated transplant rejection [

]. We further expand on iPSCs in the section ‘Stem cell-derived immunomodulatory therapeutics’.

For generating ‘off-the-shelf’ CAR-T cells, human T cells have been edited to eliminate both CD7 and TRAC, with the latter’s deletion blocking TCR-mediated signaling which causes graft-versus-host disease (GVHD) [

]. These doubly edited CAR-T cells were effective in killing T cell acute lymphoblastic leukemia (T-ALL) in vivo without evidence of xenogeneic GVHD [

]. Additionally, CAR-T cells edited to lack TCR and HLA-1 reduced alloreactivity and GVHD associated with the cell therapy with a simultaneous third edit to delete PD1 [

]. The PD1 inhibitory pathway can attenuate CAR-T cell-mediated antitumor activity. As such, the abrogation of the PD1 inhibitory pathway improved antitumor efficacy.

Furthermore, Cas9 was used to endow INS-1E, a rat insulin-secreting β-cell line, with immunomodulatory functions. Specifically, INS-1E was precisely engineered to continuously produce interleukin-10 (IL-10) cytokine in a glucose-responsive manner as the knock-in site was located in the c-peptide region [

]. The continuous local secretion of IL-10 can reduce fibrosis and protect β cells from proinflammatory cytokine-induced cell death, with minimal systemic effect on the host immune system.

The generation and use of genetically engineered cell therapies with minimal immunogenicity come with safety concerns which must be considered. Gene editing with CRISPR-Cas systems often involves DSBs in the genome which can cause unintended large deletions, complex genomic rearrangements, or aneuploidy leading to harmful pathologies if not carefully monitored and addressed [

,

]. These risks are especially important to consider in cell therapies engineered to avoid immune recognition because malignant transformation of the engineered cells may escape sensing by host immune cells. Therefore, there is growing interest in using gene editing tools which do not induce DSBs such as base editors and prime editors, or even epigenomic editing tools, to lower immunogenicity of cell therapies [

].

RNA therapeutics, such as RNAi technologies, for targeted silencing of genes or in vitro-transcribed mRNA for transient expression of encoded peptides or proteins, hold significant potential in promoting immune tolerance toward cell therapies. Similar to CRISPR-Cas9, RNAi offers the ability to silence the expression of immunogenic alloantigens through mRNA transcript degradation or translation inhibition. This is achieved through the use of short, double-stranded RNA (dsRNAs) in combination with the endogenous effector RNA-induced silencing complex to facilitate homology-directed gene silencing at the post-transcriptional level. Therefore, the elimination of surface MHC molecules can be achieved through RNAi without the risks associated with gene editing as mentioned in the previous section (Figure 2).

For vascularized cell therapies, host immune responses to graft endothelial cells (ECs) expressing nonmatched HLA can lead to transplant rejection. Pretreatment of donor blood vessels ex vivo with siRNA targeting CIITA eliminated HLA-II expression in ECs and prevented rejection of donor arteries by adoptively transferred allogeneic peripheral blood mononuclear cells (PBMCs) in immunodeficient mice [

]. While the use of siRNA is promising for transient knockdown of HLA, which may be beneficial in promoting initial immune tolerance, permanent elimination may be more desirable to improve the likelihood of the long-term viability of cell therapy. Lentiviral delivery of short hairpin RNA (shRNA) targeting B2M can enable stable expression of the interfering RNA to achieve a more permanent knockdown of HLA-1. This approach has been used to generate HLA-1 knocked down cells, which prevents CD8⁺ T cell response with residual HLA-1 expression preventing NK cell lysis [

]. Stably expressed shRNA targeting B2M has also been used to generate HLA-1 knocked down iPSCs that can be derived into megakaryocytes capable of generating platelets following transfusion into a mouse model for platelet refractoriness [

].

With the approval of two mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many more mRNA therapies in clinical trials, there is growing interest in the use of mRNA to promote immune tolerance toward cell therapies [

]. The focus of mRNA therapies in this area has been on the activation and expansion of regulatory T cells (Tregs) which play an important role in immunosuppression and prevention of GVHD [

]. mRNA encoding for human IL-2 mutein was designed to preferentially bind IL-2 receptor α (IL-2Rα) receptors on Tregs and avoid proinflammatory T cell activation [

]. Delivery of this mRNA led to Treg activation and expansion in mouse and non-human primate models and effectively reduced acute GVHD in mice. However, the dual role of IL-2 in promoting both Tregs and proinflammatory T cells will require careful monitoring of T cell responses to avoid exacerbating immunogenicity toward cell therapies.

Alternatively, tolerogenic mRNA vaccines may be used to induce alloantigen-specific tolerance. Such vaccines are designed with chemically modified mRNA that are carefully purified to remove dsRNA contaminants. The resulting noninflammatory mRNA vaccines can induce tolerance toward an encoded antigen when presented to T cells in the absence of co-stimulatory molecules [

,

,

]. Currently, the use of tolerogenic mRNA vaccines has been limited to the induction of autoantigen-specific Treg responses for the prevention of autoimmune disease onset in a mouse model of multiple sclerosis [

]. We envision that prophylactic tolerogenic mRNA vaccines encoding for donor HLA could induce tolerance toward HLA-mismatched cell therapies mediated by donor HLA-specific Tregs.

MSCs secrete cytokines, chemokines, and growth factors responsible for regulation of inflammation and immune response [

]. MCSs can inhibit allogeneic T cell responses, promote Tregs, trigger DC differentiation into tolDCs, transform proinflammatory M1 macrophages to anti-inflammatory M2 phenotype, as well as inhibit NK cell proliferation. These immunosuppressive properties of MSCs have rendered them attractive for codelivery in cellular therapies, including for that of islets (NCT02384018).

In a murine model of retinal degenerative disease, cotransplantation of MSCs with fetal retinal pigment epithelial (RPE) cells suppressed host immunoreaction, allowing prolonged graft survival for preserving retina function [

]. The co-encapsulation of hepatocyte nuclear factor-4 alpha (HNF4α) overexpressing MSCs with hepatocytes promoted M2 macrophage polarization and alleviated inflammation in an acute liver failure murine model [

]. Yoshida et al. showed that syngeneic MSCs induced immune tolerance to iPSC-derived cardiomyocytes by promoting Tregs and triggering CD8⁺ T cell apoptosis [

]. The combination of iPSC-derived cardiomyocytes and MSCs yielded improved cardiac function in a murine model of myocardial infarction, compared with single-cell population transplant alone [

]. In the context of diabetes, good manufacturing practice-compliant human umbilical cord perivascular MSCs cotransplantation with islets in diabetic mice achieved T cell suppression and maintenance of tight glycemic control [

]. Furthermore, MSCs engineered to express PD-L1/CTLA4-Ig (eMSCs) can induce local immunosuppression and support allogeneic rat islet engraftment without systemic immunosuppression [

].

Despite their promise, challenges with SCs include challenges with achieving full functional maturation, unclear long-term fate, immunogenicity, and cost and complexity of large-scale manufacturing. On that note, quality control between different SC sources, ease of procurement, and upscaling while maintaining stable phenotype are important criteria for clinical translatability [

]. We refer the readers to the section ‘Concluding remarks and future perspectives, for considerations regarding clinical translation.

The wide breadth of investigations in the field are exemplified by the numerous clinical trials exploring immunomodulatory strategies for cell therapy, some of which are listed in Table 1. The first-in-human and open-label, clinical trial of CAR-Tregs for inducing and maintaining immune tolerance to kidney transplants was initiated in 2021 (NCT04817774). Recently, a clinical trial in Japan suggested that cord blood transplantation combined with intra-BM injection of MSCs could prevent GVHD without engraftment inhibition [

]. Further, some innovative immunomodulatory interventions include clinical investigations of CRISPR-Cas9- and mRNA-based therapeutics to mitigate immune rejection or promote tolerance (NCT05210530).

As with all novel biomedical technologies, safety and ethical concerns must be addressed prior to clinical translation. Further, critical considerations for translation of cellular therapies include reproducibility, large-scale production [

], and standardization and quality control protocols [

,

] (see Outstanding questions). To resolve these challenges, various public and private programs have established fundamental guidelines for good manufacturing processes in the biofabrication industry. A relevant example of these efforts is the BioFabUSA program established at the Advanced Regenerative Manufacturing Institute (ARMI). BioFabUSA is a public–private partnership consisting of industry, academia, government, and nonprofit organizations. This unique partnership focuses on directing science and engineering resources toward enabling scalable, consistent, and cost-effective manufacturing of cellular therapies. Within this, advances in robotics, information technology, computational sciences, and artificial intelligence infrastructures [

] will be fundamental to rendering cellular therapies more personalized, accessible, and affordable. In addition, as cellular therapies are essentially living drugs, mastering the supply chain from appropriate temperature regulated transportation logistics and storage to proper thawing and administration is important for widescale implementation in a reproducible manner.

Another significant challenge is developing safe and effective delivery strategies for cell therapeutics. Transplantation of pancreatic islets or SC-derived β cells provides a clear example of the importance of the delivery approach on the viability and function of the graft. Despite more than 70 years of research and development in the field, the ideal technological solution for the delivery of these cells has yet to be identified. To this end, novel discoveries in biomaterials and nanomedicine will continue to support these efforts in providing new molecular and cell engineering tools [

,

].

Finally, new research opportunities reside in developing effective strategies to track cell therapeutics upon delivery in the body [

], noninvasively monitor their viability and function, as well as modulate immunological response ad hoc. Here, innovative real-time imaging technologies and optogenetic approaches to manipulate cells using light stimuli at specific wavelengths may pave the way for novel discoveries [

].

These translational challenges are massive and require the convergence of multidisciplinary expertise and capabilities. As exemplified by the global response to the SARS-CoV-2 pandemic via the collective actions of academia and industry, which resulted in the ultra-rapid vaccine development and regulatory approval, concerted efforts could enable cellular therapies to reach their full potential.