Depletion of In-Vivo Activated Regulatory T Cells Followed by Synchronized Specific In-Vitro Activation Allows the Rapid Isolation of Leukemia-Reactive T Cells for Immunotherapy.
Authors: Jedema I. , Steeneveld E. , Hoogendoorn M. , Willemze R. , Falkenburg J.H.F. 
Published: April 2020

Abstract
By CD40 crosslinking in the presence of cytokines leukemic cells can be modified into good antigen presenting cells (APC) expressing costimulatory molecules CD40, CD80, CD86, and CD83. Previously, primary alloreactive T cell responses from HLA-matched donors have been generated using these leukemic-APC as stimulator cells against acute and chronic myeloid leukemia (AML&CML), and acute and chronic lymphocytic leukemias (ALL&CLL). However, the likelihood of generating a good immune response is highly unpredictable and long-term culture in the presence of high dose IL-2 is needed to enrich for leukemia-reactive T cells. Since the length of the in-vitro culture period has been shown to be inversely correlated with the potential of cells to survive and expand in-vivo, we developed a method facilitating early activation, detection and rapid isolation of leukemia-reactive T cells based on their interferon-gamma (IFNg) secretion using the cytokine capture assay (Miltenyi). In order to enrich for leukemia-reactive T cells and to synchronize the production of IFNg, T cells were first stimulated with the leukemic APC with addition of low dose IL-2 (10 IU/mL) at day 7, resulting in re-entry of the majority of the T cells into a quiescent state after 14 days of culture. Then, the cells were specifically restimulated resulting in synchronized production of IFNg and allowing efficient isolation. Using this method we were able to isolate T cell populations containing a high frequency of leukemia-reactive T cells against CLL, ALL, AML, and CML in 11 donor/patient pairs. Using a CFSE-based cytotoxicity assay (Jedema, Blood2004; 103: 2677) as read-out we were able to demonstrate 20–80% lysis of the primary leukemic blasts by the IFNg+ T cells at very low E/T ratios (3/1-0.3/1) in the majority of the responses, whereas the IFNg- fractions induced only 5–30% lysis. Single cell sorting of the IFNg producing T cells revealed that 15–30% of the T cell clones was capable of exerting minor antigen specific cytotoxic activity against the patient cells in an HLA-restricted fashion. However, in individual cases despite minor antigen disparities between donor and patient no specific anti-leukemia immune response could be detected. Prior to exposure to the leukemic-APC in-vivo activated T cells were observed in the responder T cell population of these donors that contained a high frequency of regulatory T cells defined as CD4+/CD25+, CD4+/CD152+, and CD8+/CD28−. We hypothesized that these regulatory T cells might actively inhibit the induction of an anti-leukemic T cell response. Therefore, in a donor/CLL patient pair, in which we were not able to induce a cytotoxic immune response against the CLL-APC we removed the in-vivo activated T cells from the responder material prior to the initial activation with the CLL-APC. Whereas no cytotoxic activity could be isolated from unmodified responder material (only 1/288 clones was cytotoxic), the IFNg+ T cells isolated from the response induced after depletion of the in-vivo activated T cells was capable of exerting massive cytotoxicity against both the primary CLL (55%) and the CLL-APC (70%). Single cell cloning of this response revealed that 35/129 T cell clones (&gt;25%, 26 CD8+, 9 CD4+) exerted HLA-restricted CLL-specific cytotoxicity. From these results we conclude that the likelihood of generating a primary anti-leukemic immune response is not only determined by the frequency of precursor CTLs, but also by the frequency of inhibitory regulatory T cells at the onset of the immune response.