Götz Ulrich Grigoleit

Lowest numbers of primary CD8 + T cells can reconstitute protective immunity upon adoptive immunotherapy

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of todays virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Dendritic Cell Vaccination in Allogeneic Stem Cell Recipients: Induction of Human Cytomegalovirus (HCMV)—Specific Cytotoxic T Lymphocyte Responses Even in Patients Receiving a Transplant from an HCMV-Seronegative Donor

Vaccination with peptide-loaded dendritic cells (DCs) has been shown to be potent immunostimulatory therapy for the management of serious infections. After allogeneic stem cell transplant (SCT), a prolonged and severe immune deficiency often leads to infectious complications. Human cytomegalovirus (HCMV) infection is one such life-threatening complication after allogeneic SCT. A phase 1/2 study including 24 allogeneic SCT recipients at high risk for HCMV disease was performed to analyze the feasibility and efficacy of vaccination with HCMV peptide-loaded DCs. No acute adverse effects were observed, and a significant clinical benefit could be demonstrated in comparison to our historical control group. An induction or expansion of HCMV-specific cytotoxic T lymphocytes was observed in 5 patients after DC vaccination.

SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells.

Background Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. Principal Findings BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. Significance The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.

Adoptive immunotherapy with virus-specific T cells

Viral infections are still common causes of morbidity and mortality in immunosuppressed patients after allogeneic hematopoietic stem cell transplantation. Infections caused by virus such as cytomegalovirus, adenovirus and Epstein-Barr virus are well-known. In addition, several other viruses such as polyomavirus and human herpesvirus 6 have been recently reported to be causes of significant complications. As the delay in recovery of virus-specific cellular immune response after transplant is associated with viral reactivation and viral disease, adoptive immunotherapy to restore virus-specific cellular immunity is an attractive option. Recent clinical trials showed the safety and effectiveness of adoptive immunotherapy against viral diseases. In this review, we summarize the current status of adoptive immunotherapy against several viral diseases including cytomegalovirus, adenovirus, Epstein-Barr virus and polyomavirus.

Adoptive immunotherapy of HCMV infection

Human cytomegalovirus (HCMV) infection or reactivation is a frequent cause of morbidity and mortality in immunocompromised individuals such as transplant recipients. Primary HCMV infection or reactivation of HCMV from latency is mostly asymptomatic in immunocompetent individuals and is controlled by the hosts cell-mediated immune response. Healthy HCMV seropositive individuals develop high frequencies of HCMV-specific cytotoxic T lymphocytes (CTL) in the peripheral blood. Furthermore, a direct correlation between the recovery of HCMV-specific CTL responses and an improved outcome of HCMV disease could be demonstrated in immunocompromised patients. Deriving from these observations, the strategy of an adoptive transfer of HCMV-specific T cells has been developed. Protective immunity can be transferred successfully by the infusion of donor-derived HCMV-specific CD8+ cytotoxic T-cell clones or cell lines. In addition, several studies have supported the importance of antiviral effector functions of Th cells in maintaining CTL responses after adoptive transfer and their capacity to produce antiviral cytokines. Until today, a broad variety of clinical protocols for HCMV-specific immunotherapy has been published. These protocols vary regarding the isolation procedure, composition of cellular product, number of transferred cells and thus treatment efficacy. In this review, we aim to provide a comprehensive synopsis of the current standard of knowledge concerning cellular HCMV-specific immunotherapeutic approaches.

Life-Threatening Infection Caused by Daptomycin-Resistant Corynebacterium jeikeium in a Neutropenic Patient

ABSTRACT Daptomycin is a novel lipopeptide antibiotic agent approved for the treatment of gram-positive life-threatening infections. Here we report, for the first time, the isolation of a highly daptomycin-resistant strain of Corynebacterium jeikeium causing a life-threatening infection in a neutropenic patient undergoing cord blood transplantation for secondary acute myeloid leukemia.

Dendritic cell vaccination in an allogeneic stem cell recipient receiving a transplant from a human cytomegalovirus (HCMV)-seronegative donor: induction of a HCMV-specific Thelper cell response

BACKGROUND AIMS In the absence of a protective immune response, human cytomegalovirus (HCMV) infection remains a life-threatening complication after allogeneic stem cell transplantation (SCT), especially in recipients of grafts from HCMV-seronegative donors. After allogeneic SCT from a seronegative donor, prolonged and severe immune deficiency often leads to infectious complications. Vaccination with antigen-loaded dendritic cells (DC) has been shown to be a potent approach for the induction of antigen-specific cytotoxic T-cell responses in vivo. For protection from subsequent HCMV reactivation, a sustained immune response is necessary, including antigen-specific CD4(+) T cells. METHODS We report the case of an 18-year-old girl with high-risk acute lymphoblastic leukemia that received an allogeneic SCT in CR2. After an HCMV infection, the graft was rejected and she received a second transplant from an HLA-mismatched, HCMV-seronegative family donor. She was treated with pp65-pulsed monocyte-derived DC at day 200 post-SCT, using a recombinant pp65 protein. Until day 200 post-SCT, HCMV reactivated six times with emerging viral resistance to antiviral chemotherapy. RESULTS After vaccination with protein-pulsed DC, an induction and expansion of HCMV-specific T(helper) cells and cytotoxic T lymphocytes was observed, associated with a sustained clearance of the HCMV viremia. Antiviral treatment could be tapered without recurrence of viremia within the first year post-SCT. CONCLUSIONS pp65-pulsed DC could induce antigen-specific T-cell responses even after a SCT from an HCMV-seronegative donor. After vaccination with pp65-pulsed DC, a sustained antigen-specific T-cell response prevented concurrent HCMV viremia. Emergence of antigen-specific T(helper) cells may be essential for a sustained, functional T-cell response post-SCT.

T-cell therapy for cytomegalovirus infection.

Purpose of reviewHuman cytomegalovirus (CMV) reactivation and disease remains one of the major complications after allogeneic haemopoietic stem cell transplantation. Cell-mediated immunity is essential in counteracting CMV infection as evident by detection of high frequencies of CMV-specific CD8+ and CD4+ lymphocytes among the healthy CMV-seropositive individuals. Adoptive transfer of CMV-specific T cells to speed up reconstitution of CMV-specific immunity potentially offers clinical protection and reduces drug toxicities as well as outgrowth of drug-resistant strains from prolonged antiviral therapy. Recent findingsDifferent strategies to generate CMV-specific T cell have been explored. Similarly, vast diversities in term of cell dose and composition of the cellular product have been infused into small cohorts of patients. To date, a number of phase I/II clinical trials have demonstrated the feasibility of adoptive transferred CMV-specific T cells as prophylaxis, pre-emptive or therapeutic measure. In general, all these strategies showed variable degrees of efficacy without obvious adverse event particularly with regard to the induction of graft-versus-host disease. SummaryIn this review, we would like to give a comprehensive synopsis regarding therapeutic application of CMV-specific T cells in fighting CMV infection.

Targeted delivery of CD40L promotes restricted activation of antigen-presenting cells and induction of cancer cell death

BackgroundStimulation of CD40 can augment anti-cancer T cell immune responses by triggering effective activation and maturation of antigen-presenting cells (APCs). Although CD40 agonists have clinical activity in humans, the associated systemic activation of the immune system triggers dose-limiting side-effects.MethodsTo increase the tumor selectivity of CD40 agonist-based therapies, we developed an approach in which soluble trimeric CD40L (sCD40L) is genetically fused to tumor targeting antibody fragments, yielding scFv:CD40L fusion proteins. We hypothesized that scFv:CD40L fusion proteins would have reduced CD40 agonist activity similar to sCD40L but will be converted to a highly agonistic membrane CD40L-like form of CD40L upon anchoring to cell surface exposed antigen via the scFv domain.ResultsTargeted delivery of CD40L to the carcinoma marker EpCAM on carcinoma cells induced dose-dependent paracrine maturation of DCs ~20-fold more effective than a non-targeted control scFv:CD40L fusion protein. Similarly, targeted delivery of CD40L to the B cell leukemia marker CD20 induced effective paracrine maturation of DCs. Of note, the CD20-selective delivery of CD40L also triggered loss of cell viability in certain B cell leukemic cell lines as a result of CD20-induced apoptosis.ConclusionsTargeted delivery of CD40L to cancer cells is a promising strategy that may help to trigger cancer-localized activation of CD40 and can be modified to exert additional anti-cancer activity via the targeting domain.

Clofarabine-based salvage chemotherapy for relapsed or refractory acute leukemia before allogeneic stem cell transplantation: results from a case series

Treatment options are limited for patients that are refractory to or relapse after standard induction chemotherapy for acute lymphoblastic (ALL) or myeloid leukemia (AML). Complete remissions (CR) are attained in less than 30% and disease-free survival (DFS) at 1 year is observed in less than 10% [1–3]. The last years have seen a number of reports indicating that allogeneic stem cell transplantation (SCT) offers a curative treatment option for these patients and precise subgroup analyses revealed that minimal residual disease (MRD) before allogeneic stem cell transfer rather than age or previous chemotherapy is prerequisite for long term benefit, relapse free, and overall survival [4–6]. Clofarabine is a new nucleoside analogue with high activity against leukemic blasts. Because of a favorable toxicity profile and lack of substantial neurotoxicity it offers various advantages over its precursor substances fludarabine and cladribine. Potent antileukemic activity is well documented in preclinical studies and in clinical trials including pediatric ALL and AML in elderly [7]. Furthermore, a combination of clofarabine with cytarabine or cyclophosphamide elicits a higher response rate than clofarabine alone [8,9]. The efficient reduction of immature leukemic blasts, however, is accompanied by a significant reduction of hematopoietic stem cells that in many cases translates in prolonged periods of neutropenia [10] for upto several weeks which is associated with a high risk for infectious complications. On the basis of this information we set out to design a salvage module for leukemia patients eligible for allogeneic stem cell transplantation but not achieving complete remission by conventional chemotherapy. We opted for a sequential therapy approach to (i) assess and control clofarabine induced tumor cell reduction by bone marrow biopsy before transplantation, to (ii) minimize possible additive toxic effects of Clofarabine, and to (iii) allow the application of a conditioning therapy with well established cytoreductive and immunosuppressive qualities. Between April 2006 and January 2008, six patients were scheduled for allogeneic stem cell transplantation but presented with refractory or relapsed leukemia immediately before conditioning start. In an attempt to enable allogeneic transplantation in the situation of low tumor burden, clofarabine in combination with cytarabine or cyclophosphamide followed by different reduced intensity conditioning regimens was applied as detailed in Table I and supplementary Table I. All patients were informed on the experimental character of the treatment schedule applied and bone marrow biopsies were taken before and after Clofarabine-based salvage therapy to assess best responses. Patients treated with