ALBERT

Description: We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF )-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)–induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 μmol/L and plateau effects were reached at 0.3 to 3 μmol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells.

Description: In this study we present two novel leukemia-associated antigens and their specific targeting through TCR-transfer. First, two CD8+ CTL clones were characterized that specifically recognized normal and malignant male B and T cell lymphoblasts, but not fibroblasts, resting lymphocytes or monocytes. One of clones recognized the HLA-B*5201-restricted TIRYPDPVI epitope of the RPS4Y protein, while the other recognized the HLA-B*5201-restricted MQQMRHKEV epitope of the UTY protein. Western blot analysis demonstrated the overexpression of RPS4 proteins in normal and malignant lymphoblasts. However, for UTY, real time RT-PCR failed to reveal differential expression of UTY mRNA in lymphoblasts, indicating that posttranscriptional regulation of UTY protein expression and/or improved processing and presentation of this epitope in lymphoblasts are likely causes of its differential recognition. These findings open up the possibility to specifically eliminate leukemic cells by targeting either RPS4Y or UTY. We evaluated the feasibility of transfer of the RPS4Y- and UTY-specific T cell receptors (TCRRPS4Y and TCRUTY, respectively) to naïve T cells to specifically eliminate RPS4Y- and UTY-expressing lymphoblasts. For each clone, the TCR α- and β-chains were cloned into separate pMX retroviral vectors. T cell transduction with either TCRRPS4Y or TCRUTY and subsequent purification resulted in 57–75% of cells expressing both α- and β-chain. TCRRPS4Y/TCRUTY-transduced T cells efficiently lysed B*5201-positive EBV-LCL (100%/92%), PHA blasts (72%/72%), and B*5201-transduced Raji (71%/56%), YT (21%/53%) and RPMI 8226 (28%/47%) cell lines, in the absence of significant recognition of B*5201-transduced fibroblasts. Killing of B*5201-positive primary leukemias could not be checked due to the paucity of B*5201-positive samples. Interestingly, despite the 1.4–1.7 fold lower expression of the exogenous TCRs on transduced T cells, both TCR-transduced populations had the same requirements for the minimal antigenic peptide concentration as the respective clones. This suggests that the limited peptide availability and not the TCR-affinity determines the selective pattern of recognition of these HY antigens. In addition, the amount of IFN-γ produced by TCRUTY-transduced T cells was significantly higher (5455±1286 pg/ml) than that of the original clone (721±134 pg/ml) (p=0.003). Similarly, IFN-γ production by the original RPS4Y-specific clone and TCRRPS4Y-transduced T cells was 391±35 vs. 1498±431 pg/ml (p=0.01). TCR-transduced T cells also displayed a significant increase in stimulation-induced production of IL-2 and TNF-α in comparison to T cell clones, produced less IL-10 and demonstrated great proliferative potential. This suggests that while preserving the fine specificity and differential pattern of recognition of the original clone, TCR-transduced T cells may have much more pronounced anti-tumor effect in vivo. This is likely due to their ‘freshness’ compared to the original clones, which had been in culture for more than 6 months. In conclusion, this is the first demonstration of the possibility to preserve the specific pattern of a T cell response to a differentially recognized antigen after TCR-transfer and to augment the amplitude of this response concomitantly. Our experiments support the feasibility of the selective targeting of male B and T cell lymphoblasts by T cells transduced with UTY- and RPS4Y-specific TCRs. This approach is currently being investigated in vivo in RAG2−/− γc−/− mice bearing male EBV-LCL.

Description: Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. γ9δ2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred γ9δ2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human γ9δ2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual γ9δ2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic γ9δ2T-cell repertoire and, most likely, limit the protective and therapeutic antitumor efficacy of γ9δ2T cells. Based on these findings, we propose combinatorial-γδTCR-chain exchange as an efficient method for designing high-affinity γ9δ2TCRs that mediate improved antitumor responses when expressed in αβT cells both in vitro and in vivo in a humanized mouse model.

Description: We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF )-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)–induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 μmol/L and plateau effects were reached at 0.3 to 3 μmol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells.

Description: Abstract 4504 Introduction Currently innate immune cells such as γ9δ2T cells are explored in tumor immunotherapy e.g. by adoptive transfer of in vitro expanded bulk γ9δ2T cells. Objective We speculated that γ9δ2T cells are highly variable in function and specificity due to differences in γ9δ2TCR, NKG2D and KIR expression and that efficacy of adoptively transferred γ9δ2T cells can be increased by transfer of defined subpopulations or clones rather than bulk γ9δ2T cells. Methods A variety of γ9δ2T cell clones, derived from a healthy donor, were tested for expression levels of γ9δ2TCR, NKG2D and KIRs by flow cytometry analysis. The sequence of the γ9δ2TCR of different clones was further analyzed. Reactivity of γ9δ2T cell clones to a panel of tumor and normal cell lines was tested and these functional analyses were correlated to receptor expression levels and compared with bulk γ9δ2T cells of the same donor. Results Functional analyses revealed a high interclonal variability in recognizing leukemia or solid tumor cell lines. Consequently, γ9δ2T cell clones with high anti-tumor reactivity were superior in killing tumor cells when compared to bulk γ9δ2T cells. Different variable regions of γ9δ2-chains and different expression levels of NKG2D and KIRs were detected in multiple clones. No correlations could be found between TCR, NKG2D, and KIR expression on γ9δ2T cell clones and their response to different tumor cell lines when clones expressed different γ9δ2TCRs. However, analysis of γ9δ2T-cell clones with identical γ9δ2TCRs revealed that a clone with higher reactivity against cancer cells expresses higher amounts of NKG2D and lower inhibitory KIRs when compared to a clone with lower reactivity. Conclusion γ9δ2TCR, NKG2D and KIR expression in γ9δ2T cells is highly variable and cannot be directly correlated to an effective anti-tumor response. Only, when T-cells express one defined γ9δ2TCR, a modulating activity of NKG2D and KIRs can be observed. Thus, we conclude that anti-tumor reactivity is fine tuned by all three receptors and thereby we speculate that the γ9δ2TCR defines tumor-specificity and activity is further modulated by NKG2D and KIRs. These results support the application of distinct subpopulations or of genetically engineered γδT cells with defined receptors rather than bulk γ9δ2T cells in the context of anti-tumor immunotherapies. Disclosures: No relevant conflicts of interest to declare.