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.

The GVL effect following allo-SCT is one of the most prominent examples showing the ability of the immune system to eliminate malignant hematological diseases. Tumor-associated Ags (TAA), for instance WT1 and proteinase-3, have been proposed as targets for T cells to establish a GVL effect. Here, we examined an additional TAA (MUC1) as a possible T-cell target of GVL-related immune responses. We have defined new peptide epitopes from the MUC1 Ag to broaden patients' screening and to expand the repertoire of immunologic monitoring as well as for therapeutic approaches in the future. Twenty-eight patients after allo-SCT have been screened for T-cell responses toward TAA (proteinase-3, WT1, MUC1). We could detect a significant relationship between relapse and the absence of a TAA-specific T-cell response, whereby only 2/13 (15%) patients with TAA-specific CTL relapsed, in contrast to 9/15 (60%) patients without TAA-specific CTL responses (Po0.05). In conclusion, CD8 þ T-cell responses directed to TAA might contribute to the GVL effect. These observations highlight both the importance and the potential of immunotherapeutic approaches after allo-SCT.

The Graft-versus-Leukemia (GVL) effect following allogeneic hematopoetic stem cell transplantation (HSCT) is one of the most prominent examples showing the ability of the immune system to eliminate malignant diseases. This effect was a strictly clinically described phenomenon, but in the last years T-cell responses against tumor-associated antigens (TAA) could partly be set in correlation with clinical benefit. Previously, TAA such as WT1 and proteinase-3 have been proposed as the targets for T-cells to establish a GVL effect. Now, we examined in addition other TAA (MUC1 and HM1.24) as possible T-cell targets of GVL related immune responses. We have defined new peptide epitopes from the MUC1 and HM1.24 antigens by the reverse immunology approach to increase the number of patients who can be screened and to expand the repertoire of immunologic monitoring as well as therapeutic approaches. A total of 25 patients after allogeneic stem cell transplantation have been screened and we are able to detect T-cell responses to both the MUC1 and HM1.24 antigens on top of the WT1 and the proteinase-3 antigen. Interestingly, we could detect a significant relationship between relapse and the absence of a T-cell response to TAA: Only 1/10 patients (10%) with TAA-specific CTL relapsed in contrast to 8/15 patients (53.3%) without TAA-specific CTL responses (p < 0.05). Furthermore, we demonstrated MUC1 peptides presented by HLA A*6801, B*0702 and B*4402 to be specifically recognized by CD3+/CD8+ T-cells. In conclusion, CD8+ T-cell responses directed to TAA might contribute to the GVL effect and are not limited to WT1 and proteinase-3. These observations clearly highlight both the importance and the potential of immunotherapeutic approaches in allogeneic stem cell recipients. Figure 1: New defined HLA class I epitopes predicted by computer analysis are recognized by specific CTL in patients post allogeneic HSCT. IFN-γ staining of PBMC from, patient No. 17 (AML, CR), 672 days post transplantation (A), patient No. 8 (AML, CR), 1035 days post transplantation (B) Cells were stimulated with 10μg/ml of the indicated peptides. Gates were set on lymphocytes by forward/side scattering (R1) and on CD3+/CD8+ cells (R2). Percentage numbers show peptide-specific CD3+/CD8+ T-cells from all CD3+/CD8+ T-cells. Figure 1:. New defined HLA class I epitopes predicted by computer analysis are recognized by specific CTL in patients post allogeneic HSCT. . / IFN-γ staining of PBMC from, patient No. 17 (AML, CR), 672 days post transplantation (A), patient No. 8 (AML, CR), 1035 days post transplantation (B) Cells were stimulated with 10μg/ml of the indicated peptides. Gates were set on lymphocytes by forward/side scattering (R1) and on CD3+/CD8+ cells (R2). Percentage numbers show peptide-specific CD3+/CD8+ T-cells from all CD3+/CD8+ T-cells.

4737 Flow cytometry has become a routine method in both clinical and basic immunological research. Its ability to differentiate between distinct populations of cells by surface staining of various parameters is a main advantage since we have the possibility to identify antigen-specific T-cells by flow cytometry through the development of soluble multimeric peptide–MHC complexes. Nevertheless, surface staining does not provide information about the functionality of the analyzed cell populations. Hence, further methods have been described to define cells by detection of intracellular epitopes. These assays include the intracellular staining of distinct cytokines or phosporylated signaling molecules (Phosflow). MHC-multimer approaches combined with intracellular cytokine staining are routinely used, whereas the detection of intracellular p-kinases under MHC-multimer staining applying the Phosflow-protocols has not been realized so far. The use of phosphoepitope analysis in antigen-specific T-cells is of high interest in infections or especially during immunosuppressive drug treatment. Therefore, we aimed to establish a dual multimer-phospho-staining protocol to provide a method to get insight into the biochemical signaling processes in antigen-specific T-cells. We chose CTL responses against CMV as model system due to well established epitopes and high frequency in healthy donors. The original Phosflow-protocols did not turn out to be suitable for a combination with MHC-multimer staining. The very harsh fixation and permeabilization procedures largely or completely abrogated the antigen-specific staining. We have been able to stain both the CMV-specific T-cell-receptor and phosphorylated kinases following polyclonal stimuli (e.g. PMA, IL-2 etc.) using different protocols for some p-kinases (ERK, STAT5, NfKB, p38). These protocols allow a combination of specific T-cell-receptor staining with that of intranuclear phosphoepitopes after polyclonal stimulation. In preliminary experiments, we have also been able to show a specific phosphorylation of the ERK molecule after stimulation with CMV-specific artificial antigen-presenting cells or antibody-coated plates. As mentioned above, the use of phosphoepitope analysis in antigen-specific T-cells may offer the possibility to correlate immunological anergy with distinct signaling processes in defined clinical situations, e.g. in immunosuppressed patients post alloSCT. Disclosures: No relevant conflicts of interest to declare.