Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb-Mar;39(2):71-80.
doi: 10.1097/CJI.0000000000000107.

Generating Peripheral Blood Derived Lymphocytes Reacting Against Autologous Primary AML Blasts

Rohtesh S Mehta  1 , Xiaohua Chen, Jeyaraj Antony, Michael Boyiadzis, Paul Szabolcs
Affiliations

Generating Peripheral Blood Derived Lymphocytes Reacting Against Autologous Primary AML Blasts

Rohtesh S Mehta et al. J Immunother. 2016 Feb-Mar.

Abstract

Expanding on our prior studies with cord blood T cells, we hypothesized that primary acute myeloid leukemia (AML)-reactive autologous T cells could be generated ex vivo under immunomodulatory conditions. We purified AML and T cells from 8 newly diagnosed high-risk patients. After 2 weeks expansion, T cells were stimulated with interferon-γ-treated autologous AML weekly × 3, interleukin-15, and agonistic anti-CD28 antibody. Cytotoxic T cells and ELISpot assays tested functionality; reverse transcriptase quantitative polymerase chain reaction tested AML and T-cell gene expression profiles. On the basis of combined positive ELIspot and cytotoxic T cells assays, T cells reactive against AML were generated in 5 of 8 patients. Treg proportion declined after cocultures in reactive T-cell samples. AML-reactive T cells displayed an activated gene expression profile. "Resistant" AML blasts displayed genes associated with immunosuppressive myeloid-derived suppressor cells. We discuss our approach to creating primary AML-reactive autologous T cell and limitations that require further work. Our study provides a platform for future research targeting on generating autologous leukemia-reactive T cells.

PubMed Disclaimer

Conflict of interest statement

Disclosures: The authors have no financial disclosures or conflicts of interest to declare.

Figures

Figure 1
Figure 1. Study schema demonstrating the process of reactive T cell generation
PBMCs were obtained from patients with AML presenting with hyperleukocytosis by leukapheresis. T cells were expanded from thawed samples (n=8) with anti-CD3/CD28 beads in culture media as we previously reported . The “negative fraction” obtained after T cell extraction contained primarily AML blasts, was cultured on a layer of bone marrow derived MSCs in serum-free media and cytokine cocktail. After 2 weeks of T cell expansion, autologous AML blasts were used for co-cultures of expanded T cells for 3 weeks. At the end of 3 weeks, cytotoxicity was assessed using EuTDA assay and IFN-γ ELISpot assay. T cell activation and inhibitory molecules were analyzed before and after co-cultures using multi-color flow cytometry and q-PCR gene expression. Tregs were analyzed using flow cytometry. q-PCR gene expression studies were used to assess MDSC-like phenotype in AML blasts.
Figure 2
Figure 2. Mean (SEM) fold expansion of autologous T cells
Figure depicts mean (SEM) fold expansion at the end of 14-day expansion period and with each subsequent week of co-cultures.
Figure 3
Figure 3. IFN-γ ELISpot assay performed at the end of third week of co-cultures
Figure shows the number of IFN-γ spot forming cells/50,000 responders (>95% CD3+ T cells) against autologous AML blasts, irrelevant target (U937), negative control (media alone) and positive control (anti-CD3/CD28 beads). Figure 3A depicts the 4 AML-reactive samples and figure 3B presents the cohort of 4 AML non-reactive samples.
Figure 3
Figure 3. IFN-γ ELISpot assay performed at the end of third week of co-cultures
Figure shows the number of IFN-γ spot forming cells/50,000 responders (>95% CD3+ T cells) against autologous AML blasts, irrelevant target (U937), negative control (media alone) and positive control (anti-CD3/CD28 beads). Figure 3A depicts the 4 AML-reactive samples and figure 3B presents the cohort of 4 AML non-reactive samples.
Figure 4
Figure 4. The EuTDA cytotoxicity assay performed at the end of third week of co-cultures
Ex vivo expanded T cells were re-stimulated with autologous irradiated AML blasts weekly X 3. At the end of week 3, cytotoxicity was assessed using EuTDA assay using labeled autologous AML blasts and irrelevant target (U937 cell line), as described. The X-axis demonstrates mean (SEM) percentage specific release against autologous AML blasts (solid line) and non-specific targets – U937 (grey line) at effector: target ratios of 40:1, 20:1, 10:1 and 5:1 (Y-axis). P-values compare specific release against AML vs U-937.
Figure 5
Figure 5
Percentage of CD4+CD25brightFoxP3+Tregs (% of total CD4 population) before and after co-cultures in AML-reactive cultures (n=4).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Teague RM, Kline J. Immune evasion in acute myeloid leukemia: current concepts and future directions. J Immunother Cancer. 2013;1(13) - PMC - PubMed
    1. Weber G, Gerdemann U, Caruana I, et al. Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant. Leukemia. 2013;27(7):1538–1547. - PMC - PubMed
    1. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509–1518. - PMC - PubMed
    1. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371(16):1507–1517. - PMC - PubMed
    1. Amrolia PJ, Reid SD, Gao L, et al. Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity. Blood. 2003;101(3):1007–1014. - PubMed

Publication types