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  • Several investigators including ourselves have previously sh

    2021-09-02

    Several investigators including ourselves have previously shown, that allogeneic HLA-DPB1 class II-alleles are powerful leukemia rejection β-Pompilidotoxin for CD4 T-cell therapy following allogeneic hematopoietic stem cell transplantation (Herr et al., 2017, Rutten et al., 2008, Stevanovic et al., 2012) and that allo-HLA-DPB1 reactive cytolytic CD4 T-cell populations can be reliably generated from naive-enriched CD4 T cells by in vitro stimulation methods (Herr et al., 2017). Based on this approach, we tested herein whether CD4 blockade facilitates the targeted generation of CD4 co-receptor independent CD4 effector T cells recognizing allogeneic HLA-DPB1 alleles. We hypothesized that the CD4 blockade preferentially induces the activation of CD4 T-cells with strong TCR/HLA interactions, eventually driving the outgrowth of CD4 T cells acting without CD4 contribution. We show herein that the addition of a CD4 antibody to the T-cell stimulation cultures indeed results in the outgrowth of CD4 co-receptor independent CD4 T cells that are characterized by specific secretion of IFN-γ and cytolytic effector function upon recognition of primary leukemia cells expressing allogeneic HLA-DPB1 alleles.
    Materials and methods
    Results
    Discussion and conclusion We herein developed a reliable and novel protocol to generate CD4 T-cell populations that specifically induce T-cell effector functions without the contribution of the CD4 co-receptor. The method was successfully applied to naive-enriched CD4 T cells from different healthy donors stimulated against a panel of allogeneic HLA-DPB1-alleles. Outgrowing CD4 populations did not only specifically secrete IFN-γ but also induced the lysis of primary AML blasts upon CD4-independent binding to allogeneic HLA-DPB1 antigen. Although the overall number of allo-HLA-DP reactive CD4 T-cell populations was decreased in the presence of CD4 blocking mAb, cytokine secretion and cytotoxicity of outgrowing CD4 T cells was comparable to allo-HLA-DP reactive CD4 T-cell populations generated in the absence of CD4 blockade (Herr et al., 2017), suggesting that activation and functional characteristics of T-cell populations were not markedly hampered by CD4 blocking mAb. Since monocyte derived DCs in situ express high levels of activating Fc receptors for IgG (FcγRs), from which FcγRI (CD64) can bind to monomeric IgG, CD4 mAb may enhance antigen presentation and T-cell responses potentially influencing T-helper cell polarization or promoting activation of autoreactive CD4 T cells (reviewed by Guilliams et al., 2014). However, monocyte-derived DCs cultured in the presence of IL-4 express very low levels of FcγRI mRNA, as IL-4 induce the rapid downregulation of FcγRI by monocyte-derived DCs (Boruchov et al., 2005). Although we have not studied possible side effects on DCs in detail, proportions of autoreactive CD4 T-cell populations generated in the presence or absence of CD4 blocking mAb were comparable (see T-cell population from donor 166 in Fig. 1A). Moreover and in line with our previous data CD4 T-cell populations predominantly secreted the T-helper 1 cytokine IFN-γ and effectively lysed primary leukemia cells (Herr et al., 2017). Since our stimulation approach promotes the outgrowth of CD4-independent T-cell populations it appeared likely that the allo-HLA-DP reactive TCRs from these T cells have a high affinity, which could overcome the need for CD4-costimulation. To allow for detailed characterization of the TCRs, next steps will include the isolation of TCR genes from CD4 T-cell clones and their genetic transfer into CD4 and CD8 T cells. However, T cells redirected with high-affinity TCRs can be extremely sensitive to specific HLA/peptide complexes, which might be coincidented with toxicities due to on-target/off-tumor reactivity when the target antigen is expressed in lower levels in healthy tissues or off-target cross-reactivity to structurally related peptides (reviewed by Stone et al., 2015). In contrast, the lower average affinities of naturally TCRs with specificity for tumor-associated self-peptides may be too low for optimal CD8 and CD4 T-cell activity. To overcome such low affinities, wild-type TCRs can be affinity matured by amino acid modifications within the CDR loops (Robbins et al., 2008) or by directed evolution strategies including yeast (Richman and Kranz, 2007) or phage display (Li et al., 2005). Alternatively, high-affinity TCRs with specificity to human HLA/peptide epitopes can be isolated from the non-tolerant T-cell repertoire of mice engineered to express the human TCR repertoire and/or human MHC antigens (Li et al., 2010, Kuball et al., 2005). However, independent of the TCR source, selection of TCR candidates for TCR gene therapy should include careful specificity and toxicity screening.