Phenotypic characteristics and protective capacities of HLA-C*0702/IE-1-specific T cells

In recent years, it was suggested by several publications that early differentiated pathogen-specific T cells, such as TSCM or TCM cells, might be superior for ACT than transfer of late differentiated TEM or TEMRA cells. The advantage of these early differentiated T cells are the longevity, the differentiation capacities, and the high polyfunctional abilities (Appay et al., 2008; Buchholz et al., 2013; Gattinoni et al., 2011; Gattinoni & Restifo, 2013; Mahnke et al., 2013). In addition, the transfer of selected T cell populations, such as Tnaive-depleted grafts, was associated with a reduction in GvHD in comparison to donor lymphocyte infusions (Bleakley et al., 2015).

In recent studies, especially TSCM cells were promoted and thought to have increased anti-tumor effect but were also shown in the setting of CMV (Gattinoni et al., 2011; Lugli et al., 2013a; Sallusto & Lanzavecchia, 2011). Notably, even after excessive screening of healthy donors, no HLA-C*0702/IE-1-specific CD8⁺ TSCM cells were detectable. However, since these very early differentiated cells are only available in very low numbers in the peripheral blood, we cannot exclude that our used analytic methods were sensitive enough with the available cell numbers.

TCM cells are also early differentiated and are able to establish a long lasting protective immunity in a non-human primate model (Berger et al., 2008). In addition, in several clinical trials using chimeric antigen receptor-transduced TCM cells, these transferred cells were associated with a beneficial anti-tumor effect (Sommermeyer et al., 2016; X. Wang et al., 2016). In addition to anti-tumor effects, TCM cells also comprise CMV-specific T cells in substantial numbers and are able to establish a functional pathogen-specific immunity. In a serial transfer mouse model using Listeria-specific TCM cells, Graef et al. proved that these cells have a long-term survival and protective capacities in case of a lethal bacterial infection (Graef

et al., 2014). This underlines the positive features of TCM cells and therefore, the high proportion of HLA-C*0702/IE-1-specific CD8⁺ T cells within the central memory compartment makes these cells an attractive target for ACT. Even though the proportion of HLA-C*0702/IE-1-specific CD8⁺ T cells was higher than for HLA-B*0702/pp65-specific T cells, this does not imply a superior role of the IE-1-specific T cells per se in case of viral control. But at least this enables already the co-transfer of these cells in a recently started clinical trial, called

”Prophylactic Central Memory Transfer” (PACT; EudraCT-No. 2015-001522-41). For the first time, in this clinical trial the HSCT recipient receives 30 days after primary stem cell transplantation a prophylactic application of low-dose, but purely enriched central memory T cells in order to prevent or attenuate potential pathogen-induced malignancies. The transferred product comprises the complete CD4⁺ and CD8⁺ central memory T cell compartment including all different pathogen specificities. Therefore, due to the high central memory proportion of HLA-C*0702/IE-1-specific T cells with the transferred TCM

compartment, this study will allow a detailed characterization of HLA-C*0702/IE-1-specific T cells in vivo and an analysis of their protective capacities in these highly vulnerable HCST recipients.

Even though the phenotypical characteristics of the HLA-C*0702/IE-1-specific T cell are promising, the protective capacities of CMV-restricted IE-1-specific CD8⁺ T cells are still unclear. Immunmonitoring analysis of a kidney transplanted patient with CMV-reactivation indicated that both specificities (HLA-C*0702/IE-1 and HLA-B*0702/pp65) expanded strongly and were associated with further protection. As both specificities reached similar sizes and functional profiles, a prediction of the protective capacities of HLA-C*0702/IE-1-specific T cells was not possible. So far mainly pp65-specific T cells were used to treat severe CMV-replications in HSCT recipients or a combination of pp65- and IE-1-specific T cells was used (Cobbold et al., 2005; Einsele et al., 2002; Leen et al., 2006; Micklethwaite et al., 2007; Peggs

& Mackinnon, 2004; Riddell et al., 1992; Schmitt et al., 2011; Uhlin et al., 2012; Walter et al., 1995). Even though the transfer of TCR-selected IE-1-specific T cells in mice was associated with a highly functional and long-lasting CMV-specific immunity (Pahl-Seibert et al., 2005), the transfer of IE-1-specific T cells might be more potent in a prophylactic setting. As IE-1 is one of the first proteins expressed by CMV (Crough & Khanna, 2009; Moscarski et al., 2013), transferred IE-1-specific T cells might be able to inhibit lytic reactivations within the tissue in the first place, thereby preventing serious uncontrolled CMV-reactivation, whereas

pp65-specific T cells might be more helpful in case of an acute viral replication when the tegument protein pp65 is already widely expressed. Therefore, the generated HLA-C*0702/IE-1 StrepTamers might be useful for the isolation of IE-1-specific T cells and the prophylactic administration of these CMV-specific T cells, similar, to the different virus- and tumor-specificities used in the prophylactic administration in the ongoing clinical trial “T-control”. So far, the epitopes used for this trial are mainly focused on HLA-A*0201, but in a potential extension of this trial, HLA-C*0702/IE-1-specific T cells might be an interesting target.

In addition to the transfer of the IE-1-specific T cells, further characterization of this T cell population might already give new insights into their potential protective capacities. One highly interesting new method is the “flow cytometry-based TCR-ligand Koff-rate assay”

(Nauerth et al., 2016). This method would enable the measurement of the dissociation time of HLA-C*0702/IE-1-specific T cells, which is linked to the avidity of the TCR-ligand interaction.

In previous studies, a high avidity was associated with increased pathogen or tumor protection (Derby et al., 2001; Nauerth et al., 2013; Zeh et al., 1999). Therefore, the avidity of HLA-C*0702/IE-1-specific T cells might correlate with viral control. In order to analyze the Koff-rate, only a minimal manipulation of the vector sequence is necessary, enabling the fusion of the Alexa488 maleimide fluorophore to the MHC-StrepTamer and is therefore currently under investigation.

Taken together, HLA-C*0702/IE-1-specific T cells are a large and polyfunctional CMV-specific T cell population. The high proportion among TCM cells in combination with the strong proliferation capacities upon CMV-reactivation identifies HLA-C*0702/IE-1-specific T cells as an interesting target for ACT. Furthermore, the high allelic frequency in the Caucasian population (30%) and the immuno dominance of the IE-1309-317 epitope facilitates the usage in ACT (Ameres et al., 2013; Schmidt et al., 2009). In addition, the reversibility in combination with MHC- and Fab-StrepTamers enables potentially the clinical grade isolation of these cells in order to transfer minimally manipulated CMV-specific T cells, most likely in a prophylactic setting.

4.2 Characterization of CMV-specific T cell responses after kidney transplantation

In addition to the generation of a reversible HLA-C*0702/IE-1-specific StrepTamer, a non-interventional clinical trial encompassing patients after kidney transplantation was

conducted. The aim of this study was the characterization of CMV-specific immune responses in order to establish a risk-stratification for the preemptive antiviral treatment of these patients. So far, only the serostatus (CMV IgG) of the patients was used in order to determine future clinical interventions (KIDGO, 2009; Kotton, 2010). Based on this CMV-specific serostatus, three different risk groups were defined: low risk patients (D-/R-), standard risk patients (D+/R+; D-/R+), and high risk patients (D+/R-) (Kotton, 2013; Kotton et al., 2013).