ALBERT

Description: Key Points Anti–third-party Tcm kill malignant B cells in a T-cell receptor–independent mechanism while sparing naive B cells.

Description: Donor derived CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but actively suppress GVHD induced by CD25− conventional T (Tconv) cells when co-transplanted at a 1:1 ratio in murine model systems. Purified human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as their murine counterparts, i.e. they constitutively express high levels of intracellular CTLA-4, preferentially express the forkhead/winged helix transcription factor Foxp3, they are anergic to T cell receptor-mediated stimulation, and suppress the proliferation of Tconv cells after polyclonal or allogeneic activation in vitro. Thus, their adoptive transfer is a promising strategy for the prevention of GVHD in patients after allogeneic BMT. For potential clinical applications, efficient protocols for the isolation of CD4+CD25+ Treg cells from peripheral blood are required. In laboratory-scale experiments we determined that depletion of B cells (that partially express CD25), followed by repetitive positive selection of the remaining CD25+ cells with paramagnetic beads, yields a more than 90% pure population of CD4+CD25+ T cells. We now applied this protocol for the large-scale isolation of human CD4+CD25+ T cells from leukapheresis products of healthy volunteers under GMP conditions using the CliniMACS® device. B cell depletion with CD19-beads, followed by three cycles of CD25-enrichment with CD25-coupled microbeads resulted in an average 90% purity of CD4+CD25+ T cells (range 86% to 94%). Positively selected cells constituted on average 1.56% of MNC (range 0.84% to 2.0%), resulting in absolute numbers of 63 to 261 x 106 cells, depending on the initial size of the leukapheresis product. Since T cells with regulatory capacity predominantly reside within the CD4+CD25high subpopulation, we further determined the relative proportion of CD25high to CD25intermediate CD4+ T cells within the cell product. CD4+CD25high T cells represented on average 42% of the purified CD4+CD25+ T cell population (range 26% to 60%) as compared to 10.6% (range 8.5% to 13%) of the CD4+CD25+ T cells within the starting population. CD4+CD25+ T cells isolated according to this GMP protocol expressed higher levels of Foxp3 mRNA and Foxp3 protein as well as higher levels of intracellular CTLA-4 as compared to CD4+CD25− T cells. In addition, they showed functional characteristics of Treg cells as they were hypoproliferative to polyclonal stimulation in vitro and inhibited the proliferation of co-cultured CD4+CD25− T cells in a standard suppression assay. The ability to isolate highly enriched Treg cell populations in sufficient numbers under GMP conditions will accelerate their clinical application in allogeneic BMT.

Description: CD4+CD25+ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4+CD25+ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4+CD25- T cells. Here we examined the differential effect of CD62L+ and CD62L- subsets of CD4+CD25+ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4+CD25+ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4+CD25- T cells, only the CD62L+ subset of CD4+CD25+ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4+CD25+CD62L+ Treg cells were transferred compared with the CD62L- subset. Subsequently, CD4+CD25+CD62L+ Treg cells showed a significantly higher capacity than their CD62L- counterpart to inhibit the expansion of donor CD4+CD25- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.

Description: The adoptive transfer of donor CD4+CD25high regulatory T (Treg) cells has been suggested for the prevention of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). In preparation of such trials we previously described protocols for the efficient in vitro expansion of human Treg cells (Hoffmann et al. 2004, Blood104:895). Strong costimulation provided by immobilized anti-CD3 and anti-CD28 antibodies together with high-dose IL-2 resulted in a more than 3-log polyclonal expansion of Treg cells with strong suppressive activity. In contrast to CD4+CD25− T cells, the majority of the CD4+CD25high Treg cells maintained expression of the lymph node homing receptors CD62L and CCR7 during in vitro culture. Detailed examination of sorted subpopulations from Treg cell lines now revealed that only CD62L+CCR7+ cells combined all phenotypic and functional characteristics of natural Treg cells, such as FOXP3 expression, lack of cytokine secretion and potent suppression of responder T (Tresp) cell proliferation. To elucidate the origin of this cell population, we initiated cultures from CD45RA+ naive as well as CD45RA− effector/memory-type CD4+CD25high Treg cells. The CD45RA+ population initially contained 95 ± 2.5% CD62L+CCR7+ cells and maintained this phenotype with still over 90% CD62L+CCR7+ cells after 2 weeks and appox. 70% after 3 weeks in culture (n=10). In contrast, CD62L and CCR7 expression in CD45RA− Treg cells was less stable and decreased from 58 ± 8% CD62L+CCR7+ cells after isolation to 32 ± 20% after culture for 3 weeks. Similar differences were observed with respect to cytokine production, as determined by intracellular staining: Whereas less than 5% (n=8) of expanded CD45RA+ Treg cells expressed IL-2 and/or IFN-γ, almost 40% of expanded CD45RA− Treg cells produced one or both of these pro-inflammatory cytokines upon stimulation with PMA/ionomycin. Interestingly, expanded CD45RA− Treg cells also contained a defined subpopulation of IL-10-producing cells (6.9 ± 5.6%; n=8), which was absent in expanded CD45RA+ Treg cells. Both subpopulations showed suppressive activity after polyclonal restimulation, however, suppression of Tresp cell proliferation by expanded CD45RA+ Treg cells was much more profound with only 13.6 ± 4% proliferating cells as compared to 35.4 ± 7.7% in the presence of expanded CD45RA− Treg cells and 79.6 ± 18.7% in the absence of Treg cells, as measured in a CFSE-dilution assay at a 1:4 ratio of Treg and Tresp cells (n=7). Most importantly, when determined on a single cell level by intracellular staining, 93.6 ± 1 % (n=3) of expanded CD45RA+ Treg cells still expressed FOXP3 after 3 weeks in culture, whereas only 10.8 ± 6.8% of expanded CD45RA− Treg cells remained FOXP3+. No re-expression could be induced in FOXP3− expanded CD45RA− Treg cells by short-term restimulation via CD3/CD28, whereas expanded FOXP3+ CD45RA+ Treg cells transiently upregulated FOXP3, with peak expression levels as early as 24h after stimulation and return to baseline levels by 48 to 72h. Based on these unexpected findings that only naive CD45RA+, but not memory-type CD45RA− CD4+CD25high T cells give rise to homogeneous Treg cell lines, we suggest that isolation and expansion of CD45RA+ CD4+CD25high T cells is the best strategy for adoptive Treg cell therapies.

Description: Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation caused by donor T cells in the stem cell graft. Prophylactic treatment of GVHD usually comprises immunosuppressive medication including cyclosporin A (CsA) that blocks calcineurin phosphatase activity and thereby inhibits nuclear factor of activated T cells (NFAT)-mediated cytokine production. CD4+CD25+ natural regulatory T (Treg) cells have been shown to suppress the proliferation and cytokine secretion of conventional CD4+ and CD8+ T cells in vitro and to protect from GVHD lethality in murine models of allogeneic BMT. As the adoptive transfer of human CD4+CD25+ Treg cells as a means of GVHD prophylaxis is currently under investigation, we aimed to explore the influence of CsA on survival, cytokine production and suppressive activity of this T cell subpopulation. To this end, in vitro expanded human CD4+CD25high and CD4+CD25− T cells were stimulated with anti-CD3 and anti-CD28 antibodies and simultaneously exposed to various concentrations of CsA. While T cell death occurred in both in vitro expanded subpopulations at high CsA concentrations of 1000 ng/ml, with only 10 to 20% viable cells after 72h exposure, CD4+CD25high T cells showed significantly enhanced resistance to lower CsA concentrations of 100 ng/ml with still approx. 60% viable cells, as compared to only 20% viability in expanded CD4+CD25− T cells. At this lower concentration, CsA also blocked the proliferation of the two subpopulations in response to anti-CD3/CD28 stimulation, as determined by CFSE-dilution, and inhibited the IL-2 and IFN-γ production of CD4+CD25− T cells as well as the IL-10 production by expanded CD4+CD25high Treg cells, as shown by intracellular staining after stimulation with PMA/ionomycin. Addition of exogenous IL-2 protected both subpopulations from CsA-induced cell death and restored their proliferative capacity but was unable to abrogate the block in cytokine production. Surprisingly, the suppressive activity of in vitro expanded CD4+CD25high Treg cells was neither affected by a pre-incubation of the cells with 100 ng/ml CsA, nor by its presence during the suppression assay. These results indicate that in vitro expanded CD4+CD25+ Treg cells are less sensitive to CsA-induced apoptosis and that their yet unknown mechanism of suppression does not involve the calcineurin pathway.

Description: Graft-versus-host disease (GvHD) is a frequent and life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT) and is therefore one of the main factors that limits the broad application of HSCT. Over the last decades, several studies have reported a clinical association between GvHD and reactivation of cytomegalovirus (CMV). Using a lethal murine GvHD model with major MHC mismatch (C57BL/6 -〉 Balb/c), we were able to demonstrate that recipients latently infected with murine CMV (MCMV) before transplantation showed recurrence of CMV infection concomitant with the manifestation of GvHD. Moreover, these preinfected recipients showed an accelerated mortality compared to recipients that were not preinfected. The therapeutic co-infusion of CD4+CD25+ regulatory T cells (Tregs) with conventional T cells (Tcons) prevented lethal GvHD in preinfected mice and, markedly reduced the recurrence of MCMV infection. Remarkably, these mice showed clearance of MCMV 5 weeks post transplantation in contrast to mice receiving only Tcons in which massive virus infection persisted. Enhanced reconstitution of T lymphocytes and establishment of an anti-MCMV antibody titer from donor B cells in these animals suggest that CD4+CD25+ Tregs do not interfere with an anti-viral response while suppressing Tcon-mediated GvHD. Therefore, our study revealed that the suppressive function of CD4+CD25+ Tregs is not affected by CMV reactivation and more importantly, that Tregs do not adversely affect the anti-viral immunity in the recipient. In sum, these results provide important information on the correlation of GvHD and CMV reactivation and underline the possible clinical benefit of Treg application in GvHD patients. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Under the auspices of the EC-Horizon 2020 project "TREGeneration", we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs. Methods For repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative "DeepImmune" technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample. Results As first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product. Conclusion By applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.