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.

The clinical application of T-cells for adoptive immunotherapy often requires the ex vivo stimulation and expansion of antigen-specific T cells and the re-transfer of the expanded T cells into patients. One strategy to generate such T-cell products includes the use of autologous antigen-presenting cells (APCs) which vary essentially in quality and quantity. Furthermore, other strategies for the generation of APCs such as dendritic cells (DCs) are labor intensive and expensive. To circumvent these disadvantages, many investigators have focussed their research on the development of artificial APCs (aAPCs). Recent studies have demonstrated that microbeads loaded with MHC-peptide molecules in monomeric or Ig-coupled dimeric forms and co-stimulatory antibodies such as anti-CD28 and anti-4-1BB are reproducibly capable to induce high numbers of antigen-specific T-cells. In our study, we wanted to address the question if the use of further stimulatory signals in addition to CD28/4-1BB could induce higher stimulatory capacities in aAPCs. We loaded epoxy surface activated magnetic beads (DynaBeads Epoxy M-450) with HLA-B*0702/CMV_pp65 (TPRVTGGGAM) monomer and activating antibodies (AB) and/or ligands (L) against co-stimulatory molecules of the CD28 and/or the TNF-receptor (TNFR) family: namely CD28 (AB), ICOS (AB), 4-1BB (AB+L), CD27 (L), OX40 (L). The molar ratio between MHC and the co-stimulatory molecules was between 1:1 and 1:5 with a total amount of MHC-molecules of approximately 2×105 molecules/aAPC. Cell cultures were maintained as described previously 1 and analyzed weekly by staining with HLA-B*0702/CMV_pp65 (TPRVTGGGAM) tetramers and surface antibodies against the surface markers CD3, CD8, CD45RA, CCR7, CD57 and CD25. Our results demonstrate that CD28 triggering is pivotal on aAPCs besides the antigen-specific signal for an optimal expansion of CMV-specific CTL. This signal can not be replaced by ICOS or 4-1BB either alone or in combination. The addition of ICOS and/or 4-1BB to CD28 did not significantly improve the percentage of specific T-cells after 3 weeks of culture although a trend to higher cell numbers and higher specificity when combining CD28, ICOS (AB) and 4-1BB (AB) was observed. Here, longer time periods may be required to evaluate the capacity of this combination to maintain long term cultures of antigen-specific CTL. Therefore, we always applied HLA-B*0702/CMV_pp65 monomer and CD28 (AB) on the aAPCs in further experiments and supplemented these molecules by the other co-stimulatory signals as described above. Our data suggests that the phenotype (and thereby potential effector functions/in vivo survival) varies when triggering different costimulatory pathways via aAPCs. Interestingly, we could detect a higher amount of central memory T-cells (CCR7+/CD45RA−) in samples co-stimulated with the 4-1BB antibody, compared to samples treated with other co-stimulations [e.g. MHC/CD28: 6.2 % of CD8+/Tet+ vs. MHC/CD28/4-1BB (AB): 23.5 % of CD8+/Tet+]. Another difference was seen in OX40 ligand co-stimulated samples when analyzing CD57 and CD25 expression: Here, CD8+/tetramer+ T-cells had a higher content of non-activated (CD25−) T-cells with less replicative history (CD57−) than samples co-stimulated with other co-stimulations [e.g. MHC/CD28: 23 % of CD8+/Tet+ vs. MHC/CD28/OX40 (L): 45.2 % of CD8+/Tet+]. These preliminary findings suggest that the CD28 signal on aAPCs is indispensable for expansion of CMV-specific T-cells. But the phenotype and thereby in vivo functionality may be controlled by the use of defined other co-stimulatory signals like members of the CD28 and/or TNFR family. Further tests will have to define optimal amounts of MHC-molecules on aAPCs and their ratio to co-stimulatory molecules.