Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV’s immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT.

Introduction Human Cytomegalovirus (CMV), a β-herpesvirus, causes lifelong latent infections in humans, reaching a seroprevalence of 50-90% [1,2]. CMV-infection of immunocompetent individuals takes usually a subclinical course, but reactivation or primary infection in immunocompromised patients after solid organ transplantation (SOT) or hematopoietic stem cell transplantation (HSCT) can lead to severe morbidity and mortality [3,4]. The introduction of potent antiviral agents reduced the incidence of CMV manifestations, but these drugs are associated with limiting side effects like interstitial transplant fibrosis in kidney transplant recipients [5] and bone marrow suppression after HSCT [6]. As CMV-specific cytotoxic T cells play an essential role in viral control [7,8], alternative strategies such as adoptive transfer of CMV-specific T cells have been intensively investigated during the last years [9][10][11][12][13]. Interestingly, selection of early-differentiated memory T cells could be advantageous for sustained reconstitution in HSCT patients, in particular if applied prophylactically [14,15]. Additionally, it was shown that the use of reversible MHC Streptamers enables clinical purification of minimally manipulated CMV-specific T cells to high purity, avoiding complex regulatory requirements for advanced therapy medicinal products (ATMPs) [16][17][18][19]. Finally, T cell responses mediated by HLA-I molecules not belonging to HLA-A and-B alleles could play an important role in viral control. One promising CMV epitope is the immediate early-1 (IE-1) peptide 309-317 , which is restricted to HLA-C Ã 07:02 [20]. HLA-C Ã 07:02 is an inhibitory ligand for Killer-cell immunoglobulin-like receptor (KIR) 2DL2/3, which inhibits Natural Killer (NK) cell-mediated killing [20][21][22]. Interestingly, HLA-C Ã 07:02 is much less susceptible to viral immune evasive strategies than CMV epitope-presenting HLA-A or-B molecules [20], presumably to avoid NK cell cytotoxicity. Furthermore, a possible evolutionarily beneficial inheritance from the Neandertalian genome [23] and its high allelic frequency of approximately 15% within the Caucasian population [24] hint both to a putative selection advantage of HLA-C Ã 07:02 [25]. This makes the HLA-C Ã 07:02-presented CMV epitope IE-1 309−317 also an interesting new target for vaccination, adoptive T cell therapy and immune monitoring. In order to learn more about its role within CMV-specific immunity, we developed suitable MHC multimer staining protocols, intracellular cytokine staining (ICS) and magnetic serial enrichment with recently generated reversible MHC multimers [19,20]. Our results demonstrate that HLA-C Ã 07:02-restricted IE-1 309−317 CD8 + T cell responses are detectable with high frequencies in CMV-seropositive donors and have some characteristic differentiation patterns. Depletion of T cell subsets mediating epitope-independent KIR-binding allowed to develop a novel enrichment protocol for robust and highly efficient enrichment of HLA-C Ã 07:02-restricted CMV IE-1-specific T cell populations.
consent approving the presented work reported in this manuscript. Here, one exemplary HLA-typed patient was used for characterization of HLA-C Ã 07:02/IE-1-restricted CMV-specific CD8 + T cells.

Quantification of absolute cell counts and statistical analysis
For the calculation of absolute CMV-specific T cells, the BD TM Trucount kit (BD Biosciences) was used. Statistical analysis were performed with the Mann-Whitney U test and calculated by GraphPad Prism 5 (GraphPad Software, La Jolla, USA) for Windows.

Strong and functional expansion of HLA-C Ã 07:02/IE-1-restricted T cells may contribute to virus-control in a CMV-reactivating kidney transplant recipient
In order to analyze the expansion capacity and phenotype of HLA-C Ã 07:02/IE-1-restricted T cells in a clinically relevant setting, we monitored a CMV-seropositive kidney transplant recipient with confirmed CMV-reactivation in the absence of antiviral prophylaxis. Both HLA-C Ã 07:02/IE-1 MAGEand HLA-B Ã 07:02/pp65-specific T cells were in the beginning of CMV reactivation hardly detectable, which correlated with low absolute CD3 + T cell levels. Proliferation of CMV-specific CTLs for both epitopes was associated with decreased viral load, suggesting the establishment of protective immunity (Fig 5A). Interestingly, HLA-C Ã 07:02/IE-1 MAGE--specific T cells showed an even more intense proliferation starting with 0.278% on day 36 and peak CTL levels of 9.82% on day 56, in parallel to viral control (Fig 5B). HLA-B Ã 07:02/pp65-specific CD8 + T cells expanded in similar kinetics, but less intensively (Fig 5B). In a second step, we analyzed the phenotype of both specificities and were able to detect CMV-specific CD8 + central memory T cells (CCR7 + / CD45RO + ) for both specificities. After viral clearance, high numbers of virus-specific central memory T cells remained detectable, indicating the generation of a long lasting protective T cell memory (Fig 5C). In concordance with the MHC multimer results, we detected expanding levels of IFNγ-producing CD8 + T cells for both specificities (Fig 5D and 5E). This clinical example suggests a contribution of HLA-C Ã 07:02/IE-1-specific T cells to the control of CMV reactivation and underlines the potential value of this population as a constituent of predictive diagnostic panels as well as a target for adoptive T cell therapy.

Magnetic purification of HLA-C Ã 07:02/IE-1-specific T cells
TCR-independent, KIR2DL2/3-mediated enrichment during magnetic-bead-coupled HLA-C Ã 07:02/IE-1 Streptamer purification could lead to a substantial contamination with potentially alloreactive T cells (Fig 6A and 6B). This would limit significantly the clinical use of this otherwise very interesting CMV-specific T cell product. Therefore, we conceived a serial magnetic enrichment protocol to isolate KIR2DL2/3-depleted, HLA-C Ã 07:02/IE-1 Streptamer-purified T cells. In a proof-of-concept experiment we used magnetic-bead coupled, KIR2DL2/3-specific antibodies to deplete first KIR2DL2/3-expressing T cells. Prior to depletion, the donor had a frequency of 6.9% KIR2DL2/3 + cells and 3.13% of KIR-adjusted HLA-C Ã 07:02/IE-1-restricted T cells (Fig 6C). The depletion of KIR-expressing cells was highly efficient with a reduction of 93%, which left only a negligible fraction of 0.512% KIR2DL2/3 + cells in the intermediate cell product (Fig 6D). Subsequently, we conducted a second, conventional positive enrichment step with magnetic bead-coupled HLA-C Ã 07:02/IE-1 Streptamers. This led to a purity of 88.3% for HLA-C Ã 07:02/IE-1-restricted T cells in the final T cell product (Fig 6E). This promising approach can be further developed to a GMP compatible process and should preserve, with the verified reversibility of HLA-C Ã 07:02/IE-1 Streptamers (S4 Fig), the functional and regulatory advantages of minimally manipulated MHC Streptamer-purified T cell products.

Discussion
CMV is still the most common viral infection after solid organ or stem cell transplantation [3,4] and can be treated by adoptive transfer of CMV-specific T cells in the setting of HSCT [7][8][9][10][11][12]. Here we show that HLA-C Ã 07:02/IE-1-specific T cells represent a large, functional and T CM -rich T cell population, which could play a beneficial role for optimized immune monitoring and adoptive T cell therapy.
Our analysis of donors with the HLA allele C Ã 07:02, which is very common in Caucasians [24], indicated a broad availability of HLA-C Ã 07:02/IE-1-specific T cells. After exclusion of epitope-independent binding via MHC multimer double staining they were found in high frequencies among CD8 + T cells containing stable fractions of early differentiation phenotypes. This observation underlines previous findings supporting an immunodominance of this epitope [20]. As there is a possible link between population size and enrichment efficiency in apheresis products from healthy donors [16], the prominence of HLA-C Ã 07:02/IE-1-specific T cell populations could have a beneficial role for the processing of these cells with regard to purity and availability of sufficient CMV-specific T cells for adoptive T cell therapy.
The interaction of HLA-C Ã 07:02 and KIR2DL2/3 prohibiting NK cell mediated killing [20][21][22] is potentially responsible for reduced immune evasion of HLA-C Ã 07:02-expressing CMVinfected cells [20]. Interestingly, this well-known interaction of HLA-C1 receptors with KIR2DL2/3 [22,28,30] was obviously strong enough to allow epitope-independent staining with HLA-C Ã 07:02 Streptamers. In previous analyses (data not shown) using MHC multimers containing HLA-A24, a member of the Bw4 group [28], we could not detect KIR (KIR3DL1)associated binding. This indicates that specifically HLA-C Ã 07:02-and potentially also other HLA-C multimers have a sufficiently strong binding capacity for KIR molecules expressed on T cells, which requires the adaptation of the well-established single-step magnetic MHCenrichment protocols for the generation of primary antigen-specific CD8 + T cells [12,16]. In the clinical setting of adoptive T cell transfer after allo-HSCT, depletion of KIR-expressing epitope-unspecific T cells is necessary to prevent enrichment of potentially alloreactive T cells, which could mediate graft-versus-host disease. As a depletion step with HLA-C Ã 07:02/ MAGE Streptamers was found to be technically challenging (data not shown), we used instead a KIR2DL3-specific antibody for depletion before the HLA-C Ã 07:02/IE-1 Streptamer purification. This procedure worked sufficiently well, but for clinical applications a fully reversible Fab Streptamer for KIR2DL2/3 [31] would be desirable. This could prevent regulatory issues that would be raised with the transfer of not completely depleted KIR2DL3 antibody-coated cells. Fortunately, full reversibility of HLA-C Ã 07:02/IE-1 Streptamers also needed in this context could be demonstrated by addition of D-biotin, as it has been shown earlier for HLA-A and HLA-B Streptamers (Knabel et al. 2002).
In addition, we were able to test the newly generated HLA-C Streptamer in a clinical monitoring setting. In several clinical trials the adaptive immunity in context of CMV has been analyzed by MHC tetramer staining and cytokine secretion assays and there is consensus that CMV-specific T cells play a major role in viral control. Phenotype, proliferation, absolute numbers and cytokine profiles are crucial for protection, as well as prediction of CMV-associated complications [18,[32][33][34][35][36][37][38]. Our newly developed combinatorial HLA-C multimer staining approach enables now the characterization of HLA-C Ã 07:02/IE-1-specific T cells in a clinical setting. They showed strong proliferation and intense cytokine production after stimulation both being indicative for robust CMV immunity and qualifying them as an interesting target for the immune monitoring of transplanted patients. If, based on the stable expression of HLA-C7 on CMV-infected cells [20], HLA-C Ã 07:02/IE-1-specific T cells have advantages in viral control still needs to be clinically elucidated, e.g. by the use of this T cell population in adoptive transfer trials.
Furthermore, we and others have described the beneficial role of early differentiated T cells in adoptive T cell therapy due to protracted survival and increased proliferation capacities [11,14,39,40]. We could detect high numbers of central and effector memory T cells with HLA-C Ã 07:02/IE-1-specificity, underlining the possible potential of this population. We recently initiated an interventional clinical trial in patients after HSCT in which recipients receive low doses of Fab Streptamer-selected central memory T cells (T CM ) containing the complete CD4 + and CD8 + donor T cell repertoire (PACT, EudraCT-No. 2015-001522-41). A large majority of HLA-C Ã 07:02-positive donor-derived T CM products will be likely to comprise HLA-C Ã 07:02/IE-1-specific T cells allowing precise clinical characterization of this novel T cell population in the context of prophylactic adoptive T cell transfer.

S1 Fig. Gating strategy for CMV-specific T cell staining. (A)
Gating strategy for multimer staining. After selecting for living CD3 + CD8 + lymphocytes, multimer frequencies were assessed. If applicable, the differentiation phenotype of multimer + T cells was analyzed. (B) Gating strategy for ICS. After selecting for living CD3 + IFNγ + lymphocytes and CD8 + T cells, cytokine production was analyzed. 1x10 6 PBMCs/ staining were used for both flow cytometric analyses.