ALBERT

Description: Background: Although T cell immunotherapy is considered a promising therapeutic approach in B cell malignancies, autologous T cell based therapy proved to be far less effective in CLL than in more aggressive B cell malignancies. This has been attributed to an acquired state of T cell dysfunction. Disturbances in conventional (αβ-)T cells include expansion of CD4+ and CD8+ T cells, increased expression of exhaustion markers and impaired cytotoxicity and cytokine production. Vγ9Vδ2-T cells are a conserved subset of cytotoxic T lymphocytes with potent antitumor activity, due to recognition of phosphoantigen-induced changes in CD277 in tumor cells. Aminobisphosphonate (ABP) treatment leads to intracellular accumulation of phosphoantigens and increased Vγ9Vδ2 antitumor responses. Vγ9Vδ2-T cells have been shown to effectively kill malignant B cell lines in vitro. Moreover, in clinical trials Vγ9Vδ2-T cells have been shown to recognize and kill B cell lymphomas. Whether Vγ9Vδ2-T cells could be exploited for CLL immunotherapy has not yet been explored. The aim of this study is to investigate the phenotype and function of Vγ9Vδ2-T cells in CLL patients, in order to determine whether Vγ9Vδ2-T cells can effectively kill CLL cells. Results: Frequencies of Vγ9Vδ2-T cells do not differ between untreated CLL patients (n=46) and age-matched healthy controls (HC) (n=20) as assessed by flow cytometry. Vγ9Vδ2-T cell subpopulations are skewed towards effector type (CD27- CD45RA-) in CLL patients, while numbers of naïve (CD27+ CD45RA+) Vγ9Vδ2-T cells are decreased. Expression of exhaustion markers PD-1 and BTLA is comparable between CLL and HC, as is expression of CD16, mediating antibody-dependent cellular cytotoxicity. Next, we compared the functionality of Vγ9Vδ2-T cells from CLL patients and HC. We first examined cytokine production and CD107a expression, a marker of degranulation. Production of TNFα and IFNγ upon PMA/ionomycin stimulation was significantly diminished in CLL Vγ9Vδ2-T cells as compared to HC Vγ9Vδ2-T cells. Similarly, CD107a expression was significantly reduced. Overnight coculture with primary CLL cells or the Vγ9Vδ2-T cell sensitive Daudi lymphoma cell line also induced expression of TNFα, IFNγ and CD107a. However, upon co-culture, HC Vγ9Vδ2-T cells expressed significantly more TNFα, IFNγ and CD107 than CLL Vγ9Vδ2-T cells. Subsequently, we compared cytotoxicity of Vγ9Vδ2-T cells towards Daudi cells. HC-derived Vγ9Vδ2-T cells killed Daudi cells 3-4 times more effectively at 1:5 and 1:2.5 effector:target ratios. Although ABP pretreatment of Daudi cells increased both CLL-derived and HC-derived Vγ9Vδ2-mediated killing, differences between CLL and HC could not be overcome. We then looked at Vγ9Vδ2-T cell cytotoxicity towards CLL cells. Vγ9Vδ2-T cells from HCs effectively recognized and killed primary CLL cells, irrespective of ABP pretreatment. CLL-derived Vγ9Vδ2-T cells killed allogeneic CLL cells significantly less efficiently. Finally, we investigated whether the Vγ9Vδ2-T cell dysfunction in CLL patients was reversible upon ex vivo activation without the presence of leukemic B cells. Purified Vγ9Vδ2-T cells were cocultured with mature monocytic-derived dendritic cells in the presence of ABP for 8 days. Following these culture conditions, no difference was observed in production of TNFα, IFNγ and IL-4 upon PMA/ionomycin stimulation between HC- and CLL-derived activated Vγ9Vδ2-T cells. Likewise, there was no difference in CD107a expression. The activated Vγ9Vδ2-T cells of HCs and CLL patients were equally effective at killing Daudi cells. Conclusion: Vγ9Vδ2-T cells are capable of recognizing and killing CLL cells. Yet, CLL-derived Vγ9Vδ2-T cells are functionally impaired in terms of cytokine production and cytotoxic capacity in comparison to age-matched HCs. Functional impairments of Vγ9Vδ2-T cells are reversible upon ex vivo activation. If dysfunction can be overcome effectively, the antileukemic properties of autologous Vγ9Vδ2-T cells can be efficiently employed. Disclosures No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to increased susceptibility to infections and a decreased immunosurveillance (Görgün et al. JCI, 2005). Furthermore, T-cell dysfunction impairs novel treatment strategies that rely on T-cell mediated effects. The dysfunction of T-cells in CLL is characterized by an inability to form immune synapses, increased expression of exhaustion markers and impaired cytotoxicity and proliferative capacity (Ramsay et al. JCI 2008; Ramsay et al. Blood 2012; Riches et al. Blood 2013). However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact with respect to cytokine production, cytotoxicity and immune synapse formation when compared to age-matched healthy controls (HC)(te Raa et al. Blood 2014). The finding that specific subsets of T-cells in CLL patients are functionally intact challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Aim To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection. Methods EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 42 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation based on surface markers CD45RA, CCR7, CD27 and CD28 and 2 master regulators of T-cell differentiation, the transcription factors T-bet and Eomes. We also measured expression of exhaustion markers (PD-1, CD244 and CD160), functional markers (such as KLRG1, CD127, granzyme B, granzyme K and Ki-67) and homing markers (CXCR3 and CX3CR1). To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Results Using a comprehensive T-cell differentiation staining we found that when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of "early" effector memory cells (also called EM1, CD45RA- CCR7- CD27+ CD28+; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression patterns of the transcription factors T-bet and Eomes; 25.79% EBV-specific T-cells of CLL patients display a T-bethigh Eomeshigh phenotype vs 17.44% in HC. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160 (MFI 4896.42 vs 3130.56 and 2320.09 vs 1097.38, respectively), but not PD-1. However, there were no significant differences in granzyme B and K expression in EBV-specific T-cells, suggesting an unaltered cytotoxic potential. On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β of EBV-specific T-cells after peptide stimulation. Also, degranulation (measured as CD107a+ cells) was similar between CLL patients and healthy controls after peptide stimulation. Finally, polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. We are currently determining cytotoxicity and immune synapse formation. Conclusion So far, although the phenotype may suggest an increased exhaustive state, we have not observed signs of dysfunction of EBV-specific T-cells in CLL patients when compared to HC. We are currently performing experiments to test cytotoxicity and ability to produce immune synapses of EBV-specific T-cells (which we will be able to present during the ASH meeting). Based on these results, we will be able to conclude if EBV-specific CD8+ T-cells are also functionally intact in CLL patients, and whether this population joins CMV-specific T-cells as a subset that eludes CLL induced T-cell dysfunction. T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects. T-cell populations that escape suppression may be good targets for future therapies to build around. Disclosures No relevant conflicts of interest to declare.

Description: Introduction T-cell exhaustion is a state of acquired T-cell dysfunction that arises during chronic infections, but also in the presence of cancer. Chronic lymphocytic leukemia (CLL)-derived CD8+ T cells were recently found to exhibit features of exhaustion as shown by increased expression of inhibitory receptors (including PD-1), impaired proliferative and cytotoxic capacity and disturbed immunological synapse (IS) formation (Riches et al, Blood ’13). In contrast, we have reported a decreased expression of PD-1 on effector-type CD45RA+/-CD27- CD8+ T cells in CLL patients (Tonino et al., Leuk&Lymph ‘12). As this population is enriched for CMV-specific T cells, this implies that PD-1 expression might also be decreased on CMV-specific T cells. Importantly, CMV viral loads are generally undetectable in untreated CLL patients. Taken together these data suggest that the phenotypical and functional changes found in the total CD8+ T-cell population in CLL may not be applicable to CMV-specific CD8+ T cells. In this study, we have thoroughly analyzed phenotype and function of CMV-specific CD8+T cells in CLL. Methods and Results In agreement with Riches et al. we found an increased expression of the exhaustion markers PD-1, CD160 and CD244 (assessed by flow cytometry) on CLL-derived total CD8+ T cells (n=38) as compared to CD8+ T cells from age-matched healthy controls (HC; n=13). In sharp contrast, we found a significantly lower expression of these markers on CMV-tetramer+CD8+ T cells in CLL patients (n=18) as compared to HC (n=7). Next we analyzed the functionality of CMV-specific CD8+ T cells derived from CLL patients and HC. First, the sensitivity of the functional assays used was verified by treating CD8+ T cells with cyclosporine, which mimics intrinsic T-cell defects in vitro. Cytokine production was measured upon stimulation with PMA/Ionomycin. We found no differences between CLL patients and age-matched HC as to IFNγ, TNFα and IL-2 production by CMV-tetramer+CD8+ cells. Interestingly, the fraction of CMV-tetramer+CD8+ T cells producing cytokines after stimulation with CMV-pp65-peptide loaded EBV-transformed B-lymphoblastoid cell lines (EBV-LCLs) derived from normal B lymphocytes also did no differ between CLL patients and HC. Next, the cytotoxic potential of these cells was examined. CMV-specific CD8+ T cells from CLL patients were as effective as those from HC in killing CMV-peptide-loaded EBV-LCLs at varying effector:target ratios. Finally, CMV-specific IS formation was assessed by imaging flow cytometry analysis. This technique combines quantitative and qualitative data and has advantages over the current gold standard in the study of IS, i.e. confocal laser-scanning microscopy, since it is able to measure large cell populations in an automated fashion. On average 0.6x105 total cells per condition were acquired containing approximately 550 doublets. Again, percentages of CD8+T cells forming specific proper IS with CMV-peptide loaded EBV-LCLs were comparable in CLL and HC T cells (around 45%). Conclusion In contrast to evidence for a global disturbed T-cell functionality in CLL, characterized by T cells exhibiting phenotypic and functional signs of exhaustion, we now show that CMV-specific CD8+ T cells derived from CLL patients have a lower expression of exhaustion markers and are functionally intact. These data shed new light on the complexity of interactions between T-lymphocytes and CLL cells and indicate that changes found in the total CD8+ T-cell population in CLL cannot be extrapolated to CMV-specific CD8+ T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Background: At border sites, and in internal organs, tissue resident memory T cells (TRM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. Methods: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). Results: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFNγ than circulating cells and were frequently polyfunctional. Conclusion: Functional T cells with the characteristic traits of TRM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6.