ALBERT

Description: Although current allo-transplantation therapy can induce considerable graft-versus-tumor (GvT) effects in RCC patients, it is also accompanied by severe, even life-threatening side effects, mainly due to graft-versus-host disease (GvHD). Efforts aiming to improve the specificity and efficiency of allogeneic cell therapy in this disease (e.g. specific donor lymphocyte infusion or vaccination) will certainly benefit from the identification of potential anti-tumor effector mechanisms and their corresponding target structures. We recently demonstrated that RCC-reactive cytotoxic T-lymphocyte (CTL) clones can be isolated from peripheral blood of healthy donors matched with the RCC stimulators for HLA-class I. These CTL were found to recognize a broad panel of RCC antigens with restricted or ubiquitous tissue expression (Doerrschuck A, et al., Blood July 1, 2004, Epub). We now extended our analyses on peripheral blood mononuclear cells (PBMC) of further HLA-matched healthy sibling (1) and unrelated individuals (4) and compared these results with available autologous patient PBMC. While mixed lymphocyte/tumor-cell culture (MLTC) responders derived from allogeneic donors showed a robust antigen-dependent proliferation over several weeks, a weak if any proliferative response was seen with autologous MLTC populations. By analysing the fine specificity of MLTC-derived clonal CTL the majority of allogeneic effectors recognized exclusively their RCC stimulators, but not corresponding lymphoblastoid-cell lines or natural killer target K562. These CTL were restricted by various HLA-A, -B or -C molecules. We further isolated CTL clones that exhibit an extraordinary strong recognition of RCC and various epithelial tumor-cell lines. Antibody blocking experiments provided clear evidence that these CTL are restricted by a not yet defined HLA-Ib molecule and, simultaneously, by a NKG2D-dependent mechanism. Other rapidly proliferating CD3+ CD8+ CTL clones were obtained that showed a non-HLA-restricted reactivity against RCC and a minor but consistent reactivity against targets with low or absent HLA-class I expression (e.g. K562). In conclusion, our results demonstrate that a heterogeneous panel of RCC-reactive HLA-Ia/Ib-restricted CTL can be isolated from PBMC of HLA-class I-matched healthy individuals. Alternatively, CTL recognizing RCC in a non-HLA restricted manner can be obtained. Our observations might reflect the superior ability to activate and expand RCC-directed T cells from PBMC of allogeneic healthy donors compared to the autologous setting. At this point, we cannot conclude whether these various CTL populations contribute to effective anti-RCC immune responses occurring in vivo. Answering this question will certainly require to identify further CTL-defined target structures at the molecular level. This would allow us to analyse the expression of candidate antigens and the frequency of specific CTL in RCC patients after allogeneic blood stem-cell transplantation, and to correlate these findings with clinical GvT and GvH events.

Description: Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. After 2 weeks, IL-12 was replaced by IL-2. Mini-MLLCs were performed in seven different healthy donor-AML pairs that were matched for HLA class I according to high-resolution molecular typing. Following 2 weekly re-stimulations with primary AML blasts, mini-MLLC responder populations were analyzed for reactivity on day 19 of culture using split-well IFN-gamma ELISPOT assays. AML-reactive CD8+ T-cell responders were obtained from all 7 donor-AML pairs. The majority of reactive cultures originated from the CD62L(high)+ subfractions. In 4 out of 7 pairs MLLC responder populations mainly recognized AML blasts, but not Epstein-Barr virus transformed B-lymphoblastoid cell lines of donor and patient origin. The AML-reactive CD8+ T cells were restricted by single HLA class I alleles as determined by blocking experiments using a panel of HLA allele-specific monoclonal antibodies. Representative mini-MLLC responders demonstrated strong cytotoxicity against primary AML blasts in 51Chromium-release assay. Cross-reactivity testing identified an HLA-A*0201-restricted CTL population that recognized AML blasts much stronger than non-malignant monocytes of the same patient. This CTL neither recognized recipient-derived primary fibroblasts nor other hematopoietic cells suggesting a leukemia-associated rather than a minor histocompatibility antigen as the target structure. Several MLLC-derived CTL populations expressed unique T cell receptor Vbeta chains consistent with clonal origin from AML-reactive precursors. Multiple CTL responders reached a cell yield exceeding 108 after 6 to 10 weekly re-stimulations with AML blasts. Our results suggest that in healthy individuals most AML-reactive CD8+ CTLs originate from the CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. This mini-MLLC approach allows the rapid expansion of AML-reactive CD8+ CTLs from HLA-matched healthy donors to cell numbers sufficient for antigen identification strategies or adoptive immunotherapy trials.

Description: Current methods for the detection and isolation of antigen-specific CD4+ and CD8+ T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We have recently reported that de novo cell surface expression of the TNF receptor family member CD137 (4-1BB) identifies currently activated, but not resting, human alloreactive CD8+ T cells. This observation allowed us to develop a CD137-based technology for the depletion of alloreactive CD8+ T cells in vitro (Wehler et al. Blood2007;109:365–373). More recently, a similar approach has been described that uses activation-induced CD137 expression for the detection and enrichment of antigen-specific CD8+ T cells (Wolfl et al. Blood2007;110:201–210). In the current study we complement this work and demonstrate the transient up-regulation of CD137 directly on activated cytomegalovirus (CMV) or Epstein-Barr virus specific CD8+ T cells using peptide/HLA tetramer staining of PBMC from seropositive healthy individuals. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. Most importantly, we also observed a similar CD137 expression kinetics (i.e. low baseline, maximum at 24h) on virus-specific CD4+ T cells upon activation with CMVpp65 peptides. The median frequencies of CMVpp65-reactive CD137+ cells measured ex vivo in 4 different CMV+ healthy donors after 24h of stimulation were 3.6% (range, 1.1–6.7) in CD8+ T cells and 2.7% (range, 0.8–6.4) in CD4+ T cells, respectively. We also analyzed PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors (n=3) stimulated with CMV peptides. None of these samples contained more than 0.3% CD137+ cells per total CD4+ and CD8+ T cells, thereby confirming the specificity of antigen-induced CD137 expression. We next established a two-step in vitro approach allowing the activation and subsequent CD137-based immunomagnetic cell sorting of virus-reactive CD4+ and CD8+ T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65-reactive CD4+ and CD8+ T cells from PBMC of 6 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 69.4% (range 12.7–94.1) among CD4+ T cells and 70.6% (range, 28.5–93.4) among CD8+ T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4+ and CD8+ T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-γ secretion assay to isolate CMVpp65-specific CD4+ and CD8+ T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-γ+ cells (n=5), suggesting that CD137 might detect a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid detection and isolation of viable virus-reactive CD4+ and CD8+ T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials.

Description: Recent reports have demonstrated the feasibility of using the anti-CD52 antibody Campath-1H for in vivo T-cell depletion (TCD) in the context of a fludarabine/melphalan-based reduced-intensity conditioning regimen (Kottaridis et al. Blood 2000, 96:2419). Major disadvantage of this protocol is the severe immunosuppression which results in an increased rate of opportunistic infections and disease relapses. Donor lymphocyte infusions (DLI) are frequently used to overcome this limitation. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvHD (Nimer et al. Transplantation 1994, 57:82; Giralt et al. Blood 1995, 86:4337). We set up a phase-I allotransplantation study which combines the Campath-1H (100mg)/fludarabine (150mg/m2)/melphalan (140mg/m2) preparative regimen with the preemptive administration of CD8-depleted DLI in patients with hematological malignancies not eligible for myeloablative conditioning. After early withdrawal of CsA immunosuppression, CD8-depleted DLI are scheduled in a dose-escalating regimen starting on day +60 after HLA-identical sibling transplantation and on day +120 after unrelated donor transplantation, respectively. For CD8-depletion of donor leukaphereses a new CliniMACS-based protocol using clinical grade CD8 Microbeads is used. Our data show that this procedure is efficient in reducing the content of CD8+ T-cells by at least 2.5 to 3.5 logs, while CD4+ cells remain largely unchanged. In this ongoing study, 8 patients (4 myeloma, 2 CMPD, 1 ALL, 1 NHL) were included and received peripheral blood stem cells (PBSC) from HLA-identical sibling (1), HLA-matched (5) or DRB1-mismatched unrelated donors (2), according to high-resolution HLA typing. Chimerism analyses on total bone marrow-derived cells as well as on purified CD4, CD8, CD19, CD14 and CD15-positive peripheral blood cells showed durable complete donor chimerism in all but one patient (see below). After successful withdrawal of CsA, 2 patients already received CD8-depleted DLI without any acute toxicity. In the first patient (UPN 3), who was transplanted from an HLA-identical sibling, DLI were given on days 60 and 120 (1 and 5 x 106 CD4+ T cells, respectively). The second patient (UPN 4), who was transplanted from a matched unrelated donor, received the first dose of 1 x106 CD8-depleted DLI on day 120. DLI were regularly followed by a more than 2-fold increase of CD4+ cells during the first two weeks after application, while CD8+ cells remained unchanged. After the second DLI, patient UPN 3 developed a grade 2 GvHD of the skin (d+22 after DLI). Both patients were at high risk for CMV reactivation (donor: CMV negative, host: CMV positive) and repeatedly received preemptive treatment. Immediately prior to the second DLI, the CD8+ cell count in patient UPN 3 increased during an ongoing CMV reactivation. Line-specific chimerism analysis revealed that more than 50% of these CD8+ cells were of host origin. As expected, transfer of CD8-depleted DLI from CMV-negative donors was unable to entirely prevent CMV reactivation in seropositive patients. Nevertheless, our preliminary data suggest that the preemptive transfer of CD8-depleted DLI is able to accelerate immune reconstitution after reduced intensity TCD allogeneic PBSCT without rapid increase of CD8+ donor cells.

Description: The combination of fludarabin (150mg/m2) / melphalan (140mg/m2) based reduced-intensity allo-transplantation (RIT) with in vivo T-cell depletion (TCD) by the anti-CD52 antibody alemtuzumab (100mg) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD) (Kottaridis et al., Blood 2000). However, the slow lymphocyte recovery following this protocol is associated with reduced anti-infectious and antitumor immunity. In an attempt to improve immune-reconstitution after TCD / RIT, we investigated the early preemptive use of CD8-depleted donor lymphocyte infusions (DLIs). For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8 microbeads was applied. This procedure was efficient in reducing the content of CD8 T cells by 2.5 to 4 logs, while NK cells, B cells and CD4 T lymphocytes remained largely unchanged. Up to now, 13 high-risk patients with hematological malignancies were treated. In 4 patients, persistent cutaneous GVHD prevented the preemptive DLI application. These patients exhibited a spontaneous increase in T-cell numbers to 〉 200/μl in the peripheral blood before day +120 after transplantation. Neither increasing T-cell numbers nor persistent acute GvHD were seen in the 9 patients who qualified for preemptive DLIs. Animal data suggest that the local persistence of host antigen-presenting cells might have initiated or triggered the early onset cutaneous GVHD. Therefore, Langerhans cells (LCs) were isolated from skin biopsies for prospective chimerism analyses. In the analyzed patients, LCs demonstrated a delayed switch to full donor status beyond day +100 after transplantation. Nine patients without early GVHD received a total of 13 CD8-depleted DLIs starting on day +60 after sibling and day +120 after unrelated donor transplantation, respectively. CD4 T-cell numbers demonstrated a median increase of 2.2-fold within 2 weeks. Transient DLI associated grade-I GVHD occurred in 5 patients after a median of 32 days (range, 21–59). Only one HLA-C-mismatch patient developed temporary grade-III disease that was associated with a detectable level of mismatch-directed CD8 T cells in peripheral blood. Two patients developed limited chronic GvHD. In cases of decreasing donor-chimerism, the transfer of CD8-depleted DLIs were sufficient to reinduce full donor status. In 3 patients suffering from multiple CMV reactivations, anti-cytomegalovirus CD4 and CD8 responses were markedly enhanced following DLIs. We hereby present the transfer of GMP-grade CD8-depleted donor lymphocytes. In contrast to previous studies, DLIs were applied preemptively to overcome the long-lasting immunosuppression after TCD / RIT-based allo-transplantation. Our results suggest that the preemptive use of clinical-grade CD8-depleted DLIs accelerates immune reconstitution after alemtuzumab-based RIT and thereby leads to an increase in virus-specific immunity and maintenance of donor chimerism with a low risk of GVHD.

Description: Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C–mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

Description: In HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch transfected K562 cells or recipient-derived fibroblasts. Selective allodepletion (SAD) was subsequently performed by a newly developed immunomagnetic depletion approach targeting the tumor necrosis factor receptor molecule CD137 (4-1BB). Compared with other activation-induced antigens, CD137 showed a superior performance based on a consistently low baseline expression and a rapid up-regulation following alloantigen stimulation. In 15 different SAD experiments, the frequency of alloreactive CD8 T cells was reduced to a median of 9.5% compared with undepleted control populations. The allodepleted T-cell subsets maintained significant antitumor and antiviral CD8 responses. In vitro expansion of tumor-reactive T cells followed by CD137-mediated SAD might enhance the antitumor efficacy of T-cell allografts with lower risk of inducing GVHD.