The ability to obtain high transduction efficiencies in large batches of activated PBMCs for human cell therapy8
makes it possible to limit the ex vivo expansion period while achieving numbers of tumor antigen-specific lymphocytes that are in proportional keeping with the cell transfer load required to induce tumor responses in mice.17
The limitation of ex vivo manipulation is important since earlier lymphocytic populations with shorter ex vivo expansions are likely to optimize in vivo function. Hence, this work was aimed at defining key features of activated lymphocytes that may enable the identification of optimized conditions for the generation of clinical grade T cell therapies. Our major findings were that ex vivo activation of human PBMCs demonstrated defined and predictable phases under all tested protocols, and that lymphocytes early on in culture demonstrated larger sizes, greater metabolic activity, higher lentiviral transduction efficiency, and unique immunophenotypic signatures that may be more advantageous for recipients of ACT therapy. The generability of the findings from this study is limited by the fact that cells from only one healthy donor were used throughout this work. Even though all studies were internally controlled and included multiple replicates and overlapping readouts, it is possible that a certain activation protocol may be more effective in PBMC from some subjects and not others.
Lymphocyte size is an essential attribute that reflects cellular fitness and function.33, 34
Cell size, as demonstrated with larger cell diameter noted early on in the ex vivo activation process, and cell cycle, as shown with the rapid proliferation starting two days after the ex vivo activation, are coordinated but distinct processes.35
During the early activation phase, anti-CD2/3/28 activation beads generated cells that acquired a larger cell size phenotype and subsequently demonstrated enhanced proliferative rate. However, this did not translate into major differences in metabolic tracer uptake or in their phenotype. The observation of increased retention of [3
H]-3′FLT and [3
H]-FAC by lymphocytes activated with OKT3-containing regimens could be explained by a relative decrease in mass per given cell in the anti-CD2/CD3/CD28 bead group, and hence less retention of radioactive tracer since they experienced greater proliferative rate. However, the findings with early FLT uptake and decreased during exponential growth are unexpected. FLT uptake most accurately reflects human thymidine kinase-1 (hTK1) activity, an enzyme expressed during the DNA synthesis phase of the cell cycle. TK1 activity has been reported to be highest in proliferating cells and low in quiescent cells.36
However, our data suggest it may be highest in the initial culture period prior to and in preparation for (building up stores) lymphocyte proliferation. The same pattern was noted with FAC uptake, which reflects deoxycytidine kinase (DCK) activity, the enzyme responsible for the initial phosphorylation and intracellular trapping of this PET tracer.30
An additional explanation for the higher accumulation of FLT and FDG before exponential proliferation is that the surface expression of transporters for these nucleoside analogues may be higher during the initial culture period; this possibility was not explored in this work. It is of particular interest that peak lentiviral vector-mediated transduction efficiency was noted during the early stage of cell proliferation concordant with the higher metabolic tracer uptake.
CD4/CD8 skewing seen with varying activation protocols and on different days may result in lymphocytes with distinct phenotype and function for ACT. Preparations with a higher percentage of CD4+ T cells may be better equipped for supporting CD8+ T cells in vivo.37
In this regard, the anti-CD2/3/28 bead protocol without the addition of IL-15 generated higher absolute numbers and proportions of CD4+ T cells during the first two weeks of culture. Therefore, it would be interesting to test this activation protocol in ACT experiments. We expected anti-CD2/3/28 activation beads to costimulate as well as provide TCR signaling by non-specific CD3 engagement, resulting in markedly different lymphocyte phenotypes when compared to activation with OKT3 (lacking the CD28 costimulation). However, our findings suggested that the days spent in culture had a greater influence on immunophenotype than the specific T cell activation and expansion conditions. Of note, the high concentrations of IL-2 used in all the tested conditions may have an overriding effect to potential benefits that may be derived from adding IL-15. It is certainly possible that lower concentrations of IL-2 would have allowed noticing beneficial effects of adding IL-15, which was not evident in our studies.
The day 2 immunophenotype was markedly different compared to day 0, suggesting a rapid change in cell surface marker expression upon ex vivo expansion. It is interesting to note that PD-1, a marker associated with T cells that have progressed beyond their effector phenotype and display evidence of exhaustion38
did not consistently increase with the progression of the cultures until day 21. Both CD4+ and CD8+ T cells almost uniformly lacked the lymph node homing marker CCR7 by culture day 2, but this marker increased on the surface of the CD8 subset gradually and peaked at 3 weeks. Although there are several models to explain the progression between effector and memory cell phenotypes, 39
this observation with ex vivo activated and expanded cultures would suggest that peripherally-circulating TEFF cells can acquire a phenotype more consistent with centrally circulating lymphocytes with long term cultures.
There is limited published data to date on the phenotype of TCR transgenic lymphocytes administered to patients. The most complete is from Johnson et al,8
where cells used for ACT had low CD27, intermediate CD28, low CD45RA and high CD45RO. Our analysis did not readily detect a significant population of cells with this phenotype (see ). However, their approach included the addition of a rapid expansion protocol (REP) involving expanded cultures in IL-2 and allogeneic feeder cells after the initial culture in OKT3 and IL-2. The REP protocol38
results in further expansion of cells by one to two logs, which may generate functional phenotypes different from those described herein. Interestingly, in the Johnson et al.
work, the ratio of CD45 isoforms changed in TCR transgenic cells recovered from the peripheral blood of patients, with the majority reverting to a CD45RA+ phenotype. This suggests that the functional phenotype of cells prior to ACT changes with cell expansion in a lymphodepleted host, as previously described in experiences analyzing the process of homeostatic proliferation in murine models.39
Finally, the advent of multicolor flow cytometry poses challenges in conceptualizing and depicting the concomitant analysis of multiple markers simultaneously expressed by single cells analyzed by flow cytometry. If depicted and followed individually or in pairs, the added value of detecting multicolor staining in single cells is lost. With our data, we found that heat map representations of unsupervised hierarchical clustering revealed how cell surface phenotypes segregated based on culture conditions and time. Using this approach, we concluded that the time spent in culture has a greater impact on cell phenotype than the four activation and expansion protocols tested here. This surface phenotype data may aid in the future in interpreting the functional phenotype of adoptively transferred cells to patients.
This work was undertaken in preparation for a clinical program using TCR transgenic lymphocytes for ACT therapy in patients with melanoma (clinical trial registration number NCT00910650). Based on the data presented herein we made decisions for the ex vivo expansion and viral vector transduction of PBMC, with the caveat that data from a single healthy donor may not fully recapitulate what can be encountered when activating and gene modifying PBMC from a series of patients with advanced melanoma. Viral vector transduction to insert TCR genes into lymphocytes is performed on days 2 and 3 after initiating ex vivo PBMC expansion to optimize transduction efficiency. The observation that the time in culture was the major determinant of T cell functional phenotype, as opposed to a particular ex vivo expansion protocol, led us to decide to use OKT3 and IL-2 as the activation protocol given its prior successful use in clinical trials of TCR engineering ACT. Since the production of clinical-grade OKT3 has been recently discontinued, we are using OKT3 from a commercial vendor with additional lot release testing for purity and potency as defined in the investigator new drug (IND) application filed with the FDA. The marked changes with expanded culture in vitro and the emergence of cellular populations with non-physiological phenotypes led us to select a limited (7-day) expansion ex vivo with the hopes to optimize in vivo proliferation after ACT to lymphopenic hosts.