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CD19 chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated dramatic results for the treatment of B cell malignancies such as chronic lymphocytic leukemia (CLL). As T cell defects are common in patients with CLL, we compared the T cells from these patients with healthy donors (HDs), and subsequently the CD19 CAR T cells produced from patients and HDs. Despite initial differences when comparing the phenotype of circulating T cells in patients with CLL and HDs, the CD19 CAR T cells manufactured from patients' or HDs' cells showed a similar phenotype (effector memory or terminally differentiated), both were specifically activated by and killed CD19 target cells, and secreted cytokines (ie, IL-2, TNF, and IFN-γ). The frequency of CD19 CAR T cells producing IFN-γ was significantly higher in cells produced from patients as compared with those produced from HDs. Furthermore, our data showed that the polyfunctional profile of CD19 CAR T cells was differently modulated by CD19 K562 cells and autologous B cells. The increased IFN-γ production by CD19 CAR T cells produced from patients with CLL after in vitro stimulation, may if this is also the case in vivo, contribute to a higher risk of a cytokine release syndrome in patients. The different impact by CD19 target cells on the polyfunctional profile of CD19 CAR T cells in vitro underlines the importance of the choice of CD19 target cells when assessing CD19 CAR T cells functions.

We examined the immunologic effects of allogeneic hematopoietic stem cell transplantation (HSCT) in the treatment of pancreatic ductal adenocarcinoma, a deadly disease with a median survival of 24 months for resected tumors and a 5-year survival rate of 6%. After adjuvant chemotherapy, 2 patients with resected pancreatic ductal adenocarcinoma underwent HSCT with HLA-identical sibling donors. Comparable patients who underwent radical surgery, but did not have a donor, served as controls (n=6). Both patients developed humoral and cellular (ie, HLA-A*01:01-restricted) immune responses directed against 2 novel tumor-associated antigens (TAAs), INO80E and UCLH3 after HSCT. Both TAAs were highly expressed in the original tumor tissue suggesting that HSCT promoted a clinically relevant, long-lasting cellular immune response. In contrast to untreated controls, who succumbed to progressive disease, both patients are tumor-free 9 years after diagnosis. Radical surgery combined with HSCT may cure pancreatic adenocarcinoma and change the cellular immune repertoire capable of responding to clinically and biologically relevant TAAs.

Human TCRαβ(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vβ CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.

One disadvantage with umbilical cord blood transplantation is that donor lymphocyte infusion (DLI) for treatment of threatening rejection or relapse of underlying malignant disease is not available. We have previously expanded T cells from the cord blood graft in clinical setting using anti-CD3/CD28 paramagnetic beads and interleukin (IL)-2 for possible future DLI. Here we studied the effect of adding clinical-grade IL-7 to the expansion protocol. T cells were positively selected with anti-CD3/CD28 paramagnetic beads and cultured in increasing concentrations of IL-2 with and without IL-7 (20 ng/mL). After 7 days of expansion, the T cells were analyzed for proliferative capacity and investigated with flow cytometry and Luminex to determine phenotype, cytokine production, and responsiveness to mitogenic stimulus. Cultures with IL-7 had significantly greater proliferation rate, higher CD4/CD8 ratio, a lower percentage of central memory T cells (CD45ROCCR7), and a higher percentage of effector memory T cells (CD45ROCCR7). We assessed the production of IL-2, tumor necrosis factor-α, interferon-γ, and CD107a and found a higher percentage of polyfunctional T cells (positive for 3 to 4 factors) in cells cultured with IL-7. The addition of IL-7 gives a proliferative advantage, also in cultures with a lower dose of IL-2. This could prove advantageous in T-cell culture for adoptive transfer to decrease the risk of apoptosis and other negative effects of cytokine deprivation in vivo. Addition of IL-7 also had an effect on the differentiation status of the cord blood-derived T cells. T cells cultured in IL-7 had more polyfunctional traits, possibly increasing the activity of a putative future umbilical cord blood DLI.

CD8+ T-cell memory formation has recently been demonstrated to be associated with CD8alphaalpha homodimer expression by T-cells in mice. Up to now, the knowledge about the clinical significance of CD8alphaalpha+ T-cells in humans is limited. We assessed in longitudinally collected blood samples from patients with melanoma, who underwent a peptide-based vaccination, the role of CD8alphaalpha+ T-cells in tumor-specific cellular immune responses. Phenotypic analysis showed that the expression of CD8alphaalpha+ by T-cells was stable over time and associated with a CD45RA+/-CCR7- effector-memory profile. Melan-A/MART-1-specific T-cells were identified in the CD8alphaalpha+ T-cell compartment by tetramer technology. Detection of intracellular cytokine production (interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) upon phorbol 12-myristate 13-acetate-ionomycin stimulation in CD8alphaalpha+ and CD8alphabeta+ T-cells revealed that CD8alphaalpha+ T-cells show a unique cytokine production pattern (tumor necrosis factor-alpha and interferon-gamma production) as compared with CD8alphabeta+ T-cells. T-cell receptor-CDR3 length analysis revealed that Melan-A/MART-1-specific CD8alphaalpha+ T-cells showed a similar T-cell receptor-repertoire as compared with Melan-A/MART-1-specific CD8alphabeta+ T-cells. Our results show that CD8alphaalpha+ T-cells represent a compartment of CD45RA+/- effector-memory cells in the peripheral circulation of patients with melanoma and suggest that CD8alphaalpha T-cells may originate from CD8+ T-cells that have down-regulated the expression of the CD8beta chain. CD8alphaalpha+ and tetramer-specific T-cells may represent a valuable marker to gauge long-term antigen-specific T-cell memory.