Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients

Abstract

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (T_N) vs. effector (T_E) T cells, T_N cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the T_N/T_E ratio of CART cells.

Materials and methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays.

Results: IL-7/IL-15 preferentially induced differentiation into T_N, stem cell memory (T_SCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (T_EM), CD56+ and CD4+ T regulatory (T_Reg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CART_N cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CART_N cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CART_N cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CART_N cells in untreated CLL patients. Final T_N/T_E ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CART_N expansion.

Conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of T_N among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the T_N/T_E ratio within the CART product.

Keywords: CD19; T cell expansion; T cell subpopulations; chimeric antigen receptor; chronic lymphocytic leukemia; cytokines; immunotherapy; naive T cells.

Figure 1

Net amplification of all chimeric antigen receptor T (CART) cells during culture. CART cells were generated from cryopreserved peripheral blood mononuclear cells of three healthy donors (n = 3, orange lines) and three untreated chronic lymphocytic leukemia patients (n = 3, gray lines) after activation of T cells with anti-CD3/anti-CD28 antibodies (days 0–3), transduction with a CD19.CAR-CD28-CD137zeta retroviral vector (day 3), and culture with either interleukin (IL)-7/IL-15 (n = 3, solid lines) or IL-2 (n = 3, hatched lines) (days 2–20). The net amplification of all CD3+ CART cells were determined by flow cytometry for each treatment condition and time point as indicated. Mean values of all twelve cell samples ± SDs are plotted.

Figure 2

Net amplification of chimeric antigen receptor T (CART) subpopulations during culture. The net amplification of T_N (CD45RA+ CCR7+), T_CM (CD45RA − CCR7+), T_EM (CD45RA − CCR7−), and T_E (CD45RA+ CCR7−) CART cells from healthy donors (n = 3, colored lines) and untreated chronic lymphocytic leukemia patients (n = 3, gray lines) was determined. The cellular subsets were also compared according to the mode of stimulation, either with interleukin (IL)-7/IL-15 (n = 3, solid lines) or with IL-2 (n = 3, hashed lines). Mean values of the net amplifications of the various cellular subsets were calculated for each group and are represented with SD. Significance is represented as * for p < 0.05 and ** for p < 0.01. Exact data are specified in the text.

Figure 3

Net amplification of CD4+ and CD8+ naive chimeric antigen receptor T (CART) cells during culture. The net amplification of the CD4+ and CD8+ naive CART subtypes from healthy donors (n = 3, blue lines) and untreated chronic lymphocytic leukemia patients (n = 3, gray lines) was determined. The cellular subsets were also compared according to the mode of stimulation, either with interleukin (IL)-7/IL-15 (n = 3, solid lines) or with IL-2 (n = 3, hashed lines). Mean values of the net amplifications of the various cellular subsets were calculated for each group and are represented with SD. Significance is represented as ** for p < 0.01 and *** for p < 0.001. Exact data are specified in the text.

Figure 4

Analysis for different subpopulations of chimeric antigen receptor T (CART) cells from healthy donors (HDs) vs. patients on day 17 of culture with interleukin (IL)-7/IL-15 vs. IL-2. (A) The absolute cell number of specific cell subpopulations after stimulation with IL-7/IL-15 vs. IL-2 was compared for each donor independently (n = 6). Each unit on the x-axis represents one donor. Fields are highlighted with blue or red color according to the highest cell number of CART cells for each subtype, i.e., blue when proliferation resulted in higher absolute cell numbers under culture conditions with IL-7/IL-15 and red when proliferation resulted in higher absolute cell numbers under culture conditions with IL-2. Light colors were used for HDs, dark colors for patients. (B) The mean values of the percentage of CART cells with a specific phenotype on day 17 are represented and grouped into four categories: blue for stimulation with IL-7/IL-15, red for stimulation with IL-2 (HDs: light blue/red; patients: dark blue/red).

Figure 5

Functional characterization of chimeric antigen receptor T (CART) cells: cytotoxicity and cytokine expression. (A) The cytotoxic efficacy of CART cells against CD19+ Daudi cells (fresh n = 3, freshly thawed n = 3, non-transduced n = 3) was assessed after stimulation with interleukin (IL)-7/IL-15 vs. IL-2. The average lysis of CD19+ Daudi cells for different effector:target ratios (10:1, 5:1, 1:1) was determined by release of radioactive chromium-51 from labeled target cells. (B) Differences in the absolute cell number of CART cells expressing TNF-α, IFN-γ, and IL-2 after stimulation with IL-7/IL-15 vs. IL-2 are represented for each donor (n = 3). Fields are highlighted with blue or red color according to the highest percentage of CART cells for each subtype, i.e., blue for IL-7/IL-15 and red for IL-2. (C) The mean values of the percentage of CART cells with a specific phenotype on day 15 are represented (blue for stimulation with IL-7/IL-15, n = 3; red for stimulation with IL-2, n = 3). Significance is represented as * for p < 0.05 and ** for p < 0.01.

Figure 6

Exhaustion of naive T cell subpopulation for untreated chronic lymphocytic leukemia (CLL) patients in comparison to healthy donors (HDs). PD-1 expression on the surface of all chimeric antigen receptor T cells and the subpopulations T_N, T_CM, T_EM, and T_E on day 10, for HDs (n = 3, light colors) and untreated CLL patients (n = 3, dark colors). The exhaustion marker expression was also compared according to the mode of stimulation, either with interleukin (IL)-7/IL-15 (blue) or with IL-2 (red). Significance is represented as * for p < 0.05 and ** for p < 0.01.

Figure 7

Naive to effector chimeric antigen receptor T (CART) ratio (T_N/T_E) during culture. Representation of the T_N/T_E ratio for CART cells from healthy donors (n = 3, light colors) vs. untreated chronic lymphocytic leukemia patients (n = 3, dark colors) during culture. Stimulation with interleukin (IL)-7/IL-15 is marked in blue, stimulation with IL-2 in red. Significance is represented as ** for p < 0.01 and *** for p < 0.001.

Figure 8

Illustration of the differentiation and killing process of chimeric antigen receptor T (CART) subpopulations. After administration of CART cells into the patient (A), the more differentiated T_EM and T_E CART cells are responsible for the first killing of CD19+ cells (B). As they become anergic, the less-differentiated T_N and T_CM subsets build up a second line of effector CART cells (C). They differentiate into T_EM and T_E and are able to kill remaining malignant cells, which evaded the immune defense beforehand. Ideally, these cells eventually mediate a complete response within the patient.