T-Cell manipulation strategies to prevent graft-versus-host disease in haploidentical stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (HSCT) from an human leukocyte antigen (HLA)-identical donor can be curative for eligible patients with non-malignant and malignant haematological disorders. HSCT from alternative donor sources, such as HLA-mismatched haploidentical donors, is increasingly considered as a viable therapeutic option for patients lacking HLA-matched donors. Initial attempts at haploidentical HSCT were associated with vigorous bidirectional alloreactivity, leading to unacceptably high rates of graft rejection and graft-versus-host disease (GVHD). More recently, new approaches for mitigating harmful T-cell alloreactivity that mediates GVHD, while preserving the function of tumour-reactive natural killer (NK) cells and γδ T cells, have led to markedly improved clinical outcomes, and are successfully being implemented in the clinic. This article will provide an update on in vitro strategies and in vivo approaches aimed at preventing GVHD by selectively manipulating key components of the adaptive immune response, such as T-cell receptor (TCR)- αβ T cells and CD45RA-expressing naive T cells

Targeting the hematopoietic stem cell antigen FLT3 by high-affinity T cell receptor for the treatment of high-risk acute myeloid leukemia

Acute myeloid leukemia (AML) is a disease with poor prognosis. Fsm-like tyrosine kinase 3 (FLT3) is a promising target because of its overexpression in AML cells. Efforts have been put to develop new therapeutics targeting FLT3 by small molecule inhibitors and most recently with chimeric antigen receptor (CAR) modified T cells. We generated HLA-A2-restricted, FLT3-specific T cell receptors (TCR) to target FLT3-positive AML and hematopoietic stem cells (HSCs) in an HLA-A2-mismatched allogeneic-HSC transplantation. In our proposed set up, FLT3-specific TCRs would eliminate AML cells as well as HLA-A2-positive HSCs of the patient allowing engraftment of a healthy, HLA-A2-negative hematopoietic system. FLT3 is a self-antigen, therefore, T cells bearing high-affinity TCRs against epitopes derived from it are deleted in the thymus during T cell development. To circumvent the tolerance, we immunized a transgenic mouse model expressing a diverse human TCR repertoire and HLA-A2 molecule (ABabDII). The candidate epitopes for immunizations, FLT3839 and FLT3986, were selected among in silico predicted epitopes based on their binding affinity to HLA-A2 and homology to the mouse FLT3. We identified one TCR against FLT3839 (6546-IMS) and two TCRs against FLT3986 (6780-GLL and 6782-GLL). IFN- release was detected only from 6782-GLL T cells after overnight co-culture with a K562 cell line that was modified to express high levels of FLT3 and HLA-A2 proving FLT3986 epitope is naturally processed and presented. We tested the FLT3986-specific TCRs on three different cell lines that express FLT3 endogenously. We did not detect any CD137 upregulation by FACS or IFN- release by ELISA from neither of the FLT3986-specific TCRs against an AML cell line THP1. On the other hand, co-culture with SEM and MV-4;11 cell lines that express FLT3 endogenously and were modified to express HLA-A2 molecule, and with THP1 cells modified to overexpress FLT3 induced CD137 upregulation only on 6780-GLL T cells, but did not trigger any IFN- secretion suggesting higher FLT3 availability might be required for target cell recognition by the 6780-GLL TCR. This could be due to i) the sub-optimal avidities of the identified TCRs to the pMHC complex ii) low binding affinity of FLT3986 epitope to HLA-A2 molecule resulting in a poor presentation on the cell surface. In addition, recognition of MV-4;11 cells which carry the FLT3-ITD mutation suggested FLT3986 epitope is produced from both the wild type and mutated FLT3. During the in vitro safety testing, we discovered high, intracellular FLT3 expression in the Purkinje cells of the human cerebellum. We have stopped our attempt to identify high-affinity FLT3-specific TCRs due to potential cerebellar toxicity. We believe FLT3 could still be a safe, valuable target for therapies other than TCR-modified T cells

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Targeting the CD19/CD21 complex on B cells in the mixed lymphocyte reaction

B cells exert antigen presenting functions at the crossroad between immune activation and tolerance induction, a fate notably governed by the B Cell Receptor (BCR) response regulator CD19/CD21 complex. This duality in B cells raises a particular interest in the case of acute graft-versus-host disease (GvHD), a common complication post allogeneic haematopoietic stem cell transplantation (HSCT). In this context, B cells may act either through the disruption of their capacity to present antigensin an attempt to passively hamper GvHD onset, or, conversely, by harnessing their tolerogenic potential to actively protect host tissues from cytotoxic killing by donor T cells. Hence deciphering a way to fine tune B cell functions to fit these purposes is critical. Here, we proposed that the maintenance of B cells in a resting state with the concourse of the anti-CD22 antibody epratuzumab, inducing reduction in surface CD19/CD21 complex, would mitigate their responsiveness to allopeptides and thus, prevent the acquisition of potent antigen presenting functions. Furthermore, the combination with C3d adjunction would direct antigensto CD21-mediated uptake for class II MHC presentation, taking advantage of the incapacity to derive any costimulatory activities as part of this process. The subset of B cells hereby generated would therefore have constituted a large repertoire of antigens towards which a tolerogenic state would be established. The plausibility of this strategy was investigated in a newly designed mixed lymphocyte reaction (MLR) model in which we attempted to integrate critical component of the cytokine network at the backbone of aGvHD pathophysiology, IL-15, stemming as a result of the post-HSCT lymphopenia and initial tissue damage induced by the pre-conditioning regiment. We found a dichotomy in the effect mediated by C3d. It decreased by 1.46-fold granzyme b production when used alone, but failed to sustain such effects when used in conjunction with epratuzumab. The reduction in CD19/CD21 expression induced by epratuzumab could potentially account for this outcome as CD2llow B cells are reported to be anergic. C3d usually has a role in breaking tolerance, however an opposite effect was observed here. The targeting of C3d-coated antigens to CD21 independent of BCR engagement appears to be taking place. This study provides an insight on the potential of fine-tuning the CD19/CD21 complex and sets the foundation for future research to better characterise the events taking place. This is crucial for the better optimisation of putative therapeutic strategies targeting the CD19/CD21 complex in the settings of aGvHD

EXPRESSION PATTERNS AND ROLES OF THE IKAROS FAMILY OF TRANSCRIPTION FACTORS IN HUMAN REGULATORY T CELL DEVELOPMENT AND FUNCTION.

Regulatory T cells (Tregs) are a small subset of immune cells that are responsible for downregulating the immune response and maintaining immune tolerance. Tregs are generally defined by a high expression of the transcription factor FOXP3. Dysfunction of Treg activity or number is the basis of many inflammatory diseases. Thus, there has been much research on understanding Treg development in the thymus and activity in the periphery and Tregs are being studied as a potential cellular therapy. One form of Treg therapy is generating engineered Tregs (eTregs), which involves expressing Treg genes in conventional T cells through retroviral or lentiviral transduction. The work described here investigates the role of the Ikaros family of transcription factors in eTreg function and thymic Treg development. The Ikaros family members, namely Helios and Eos, have been implicated as critical mediators of Treg induction and function. In the first study, we hypothesized that ectopic expression of Helios with FOXP3 is required for optimal engineered Treg immunosuppression. In the second study, we hypothesized that Ikaros family member expression correlates with Treg marker expression and defines points of Treg lineage commitment within CD4+ mature single positive (MSP) Tregs in the thymus. In the first study, we generated eTregs by retrovirally transducing total human T cells with combinations of FOXP3, Helios (Hel-FL) and Δ3B Helios (Hel-Δ3B), a relevant splice variant of Helios. FOXP3+Hel-FL eTregs were the only eTregs able to delay disease in a xenogenic Graft versus Host Disease model. In vitro, FOXP3+Hel-FL CD4+ eTregs suppressed T cell proliferation more effectively than FOXP3 and FOXP3+Hel-Δ3B CD4+ eTregs. However, both FOXP3+Hel-FL CD8+ eTregs and FOXP3+Hel-Δ3B CD8+ eTregs were more effective than FOXP3 alone. RNA Sequencing of the CD4+ and CD8+ eTregs demonstrated that the addition of Hel-FL to FOXP3 in eTregs changed gene expression in cellular pathways and the Treg signature compared to FOXP3 alone or FOXP3+Hel-Δ3B. Thus, overexpression of Hel-FL with FOXP3 in eTregs changed gene expression in Tconvs and mediated immunosuppression in vivo and in vitro. Additionally, there is a functional difference between the endogenous splice variants of Helios in mediating CD4+ and CD8+ T cell immunosuppression. In the second study, we used novel CD4+ mature single positive (MSP) thymocyte populations that our laboratory previously defined to track CD4+ CD25+ FOXP3+ human Treg development from human thymus samples. We then characterized protein expression of Ikaros family members and Treg markers of Tregs from each of these populations. We found that a majority of Tregs can be found in the distinct MSP6 population and these Tregs have heterogenous expression of Helios and CD39 and CD127. Within the MSP1-MSP5 populations expression of Ikaros family members transiently changed and Helios and Eos correlated with the percent of Tregs within each population. Thus, using Ikaros family members and Treg markers within subsets of CD4+ thymocytes, we were able to more precisely determine where thymic Tregs originate from and critical points of Treg development. Overall, the results of this research provide further insight into the role of the Ikaros family members in Treg function and development and can be used to improve current Treg therapy