Application of Genome-Editing Technologies for Off-the-Shelf T Cell-Based Cancer Immunotherapy

: Genetically, engineered T-cell therapy is a personalized treatment that has demonstrated considerable therapeutic potential for solid tumors and hematopoietic cancers. However, endogenous T-cell receptors (TCRs) on the surface of engineered T cells hamper their application in the allogeneic settings by inducing graft-versus-host disease, where endogenous TCRs on the surface of engineered T cells respond to the recipient’s tissues. Since the cause of this allogeneic response is the TCR complex on the surface of the engineered T cells, preventing the expression of TCR components on the surface of these cells is a promising strategy to address this challenge. This review discusses the production of allogeneic chimeric antigen receptor T cells using genome-editing methods.

Humanized Mouse Model as a Novel Approach in the Assessment of Human Allogeneic Responses in Organ Transplantation

The outcome of organ transplantation is largely dictated by selection of a well-matched donor, which results in less chance of graft rejection. An allogeneic immune response is the main immunological barrier for successful organ transplantation. Donor and recipient human leukocyte antigen (HLA) mismatching diminishes outcomes after solid organ transplantation. The current evaluation of HLA incompatibility does not provide information on the immunogenicity of individual HLA mismatches and impact of non-HLA-related alloantigens, especially in vivo. Here we demonstrate a new method for analysis of alloimmune responsiveness between donor and recipient in vivo by introducing a humanized mouse model. Using molecular, cellular, and genomic analyses, we demonstrated that a recipient’s personalized humanized mouse provided the most sensitive assessment of allogeneic responsiveness to potential donors. In our study, HLA typing provided a better recipient-donor match for one donor among two related donors. In contrast, assessment of an allogeneic response by mixed lymphocyte reaction (MLR) was indistinguishable between these donors. We determined that, in the recipient’s humanized mouse model, the donor selected by HLA typing induced the strongest allogeneic response with markedly increased allograft rejection markers, including activated cytotoxic Granzyme B-expressing CD8+ T cells. Moreover, the same donor induced stronger upregulation of genes involved in the allograft rejection pathway as determined by transcriptome analysis of isolated human CD45+cells. Thus, the humanized mouse model determined the lowest degree of recipient-donor alloimmune response, allowing for better selection of donor and minimized immunological risk of allograft rejection in organ transplantation. In addition, this approach could be used to evaluate the level of alloresponse in allogeneic cell-based therapies that include cell products derived from pluripotent embryonic stem cells or adult stem cells, both undifferentiated and differentiated, all of which will produce allogeneic immune responses.

Characteristics of endothelial corneal transplant rejection following immunisation with SARS-CoV-2 messenger RNA vaccine

AimWe report two cases of endothelial corneal allograft rejection following immunisation with SARS-CoV-2 messenger RNA (mRNA) vaccine BNT162b2 and describe the implications for management of transplant recipients postvaccination for COVID-19.MethodsA 66-year-old woman with Fuchs endothelial corneal dystrophy (FECD) and a unilateral Descemet’s membrane endothelial keratoplasty (DMEK) transplant received COVID-19 mRNA vaccine BNT162b2 14 days post-transplant. Seven days later, she presented with symptoms and signs of endothelial graft rejection. An 83-year-old woman with bilateral DMEK transplants for FECD 3 and 6 years earlier developed simultaneous acute endothelial rejection in both eyes, 3 weeks post second dose of COVID-19 mRNA vaccine BNT162b2. Rejection in both cases was treated successfully with topical corticosteroids.ConclusionsWe believe this is the first report of temporal association between corneal transplant rejection following immunisation against COVID-19 and the first report of DMEK rejection following any immunisation. We hypothesise that the allogeneic response may have been initiated by the host antibody response following vaccination. Clinicians and patients should be aware of the potential of corneal graft rejection associated with vaccine administration and may wish to consider vaccination in advance of planned non-urgent keratoplasties. Patients should be counselled on the symptoms and signs that require urgent review to allow early treatment of any confirmed rejection episode.

Benefit of Belatacept in Cord Blood-Derived Regulatory T Cell-Mediated Suppression of Alloimmune Response

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

The Impact of the Epigenetic Cancer Drug Azacitidine on Host Immunity: The Role of Myelosuppression, Iron Overload and tp53 Mutations in a Zebrafish Model

The unsatisfactory real-world efficacy of the hypomethylating agent azacitidine in treating myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has prompted us to investigate the hematological adverse events and host variables that may compromise the use of this epigenetic drug. Using the zebrafish, we found that azacitidine destroyed their myeloid precursors and impaired myeloid function by inhibiting antigen processing, allogeneic response and phagocytic activity, resulting in increased susceptibility to infection even by the normal flora E. coli. In addition, iron overload, a MDS-associated condition following repeated transfusions, exacerbated bacterial infection especially by V. vulnificus with known iron dependence. Furthermore, we show that the tp53M214K mutant zebrafish survived longer than the wild-type (WT) when challenged with bacteria following azacitidine treatment. This was attributed to the mutant’s hematopoietic cells rather than its general genetic background, since the WT animals reconstituted with the tp53M214K mutant kidney marrow became more resistant to bacterial infection following treatment with azacitidine. The clinical relevance of our findings was indicated by a MDS case with severe azacitidine-induced bone marrow suppression and by the association of hyperferritinemia with bacteremia in azacitidine-treated patients, while tp53M214K-mediated resistance to azacitidine-induced myelosuppression may explain the survival advantage of malignant MDS and AML clones over their normal counterparts under azacitidine treatment. Together, we propose that myelosuppression, iron overload and TP53 mutations may represent the host variables that compromise the azacitidine efficacy.

DOCK8-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. The relative role of dendritic cells, B cells and macrophages in the induction of RBC alloimmunization remain unclear. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused RBCs. A particularly useful transgenic "HOD mouse" has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffyb blood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune cells or receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs. Using these models, we recently discovered that mice lacking the GEF (guanine nucleotide exchange factor) DOCK8 fail to develop alloimmunity to transfused RBCs. Dendritic cells in these knockout mice fail to migrate to T cells due to lack of coordinated actin rearrangement governed by this GEF. Both B cell and T cell activation in the spleen to the transgenic transfused RBCs is abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in DOCK8-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. Our preliminary data now suggest that DOCK8-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The need for dendritic cell migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed; these mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion and the specific role of splenic dendritic cell subsets in CD4+ T cell priming to allogeneic RBCs. Disclosures No relevant conflicts of interest to declare.