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
. 2013 May 30;8(5):e65212.
doi: 10.1371/journal.pone.0065212. Print 2013.

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells

Marleen M van Loenen  1 , Renate S Hagedoorn, Renate de Boer, J H Frederik Falkenburg, Mirjam H M Heemskerk
Affiliations

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells

Marleen M van Loenen et al. PLoS One. .

Abstract

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HLA-I-TCR td CD4+ T-cells co-transferred with wtCD8αß or intracellularly modified CD8αß demonstrate equal effector functions.
To study the minimal part of CD8 needed for optimal co-receptor function in HLA-I-TCR td CD4+ T-cells, HA-2-TCR td CMV-specific CD4+ T-cells (A) co-transferred with wtCD8αα or wtCD8αß co-receptor, or (B) co-transferred with either wtCD8α,ΔCD8α or CD8α Lck in combination with either wtCD8ß or ΔCD8ß were purified and used in a stimulation assay. Td T-cell populations were tested against HLA-DR1+ LCL-CBH either unpulsed (grey striped bars) or pulsed with pp65 peptide (grey bars), or against HLA-A2+ HA-2− LCL-IZA either unpulsed (white bars) or pulsed with HA-2 peptide (black bars), or against HLA-A2+ HA-2+ LCL-JYW (black striped bars). IFN-γ production was measured after 18 h of stimulation in duplicate, and a representative experiment out of 3 is depicted. The IFN-γ production of the different CD8αß expressing TCR td T-cells was compared to the IFN-γ production of CD8αα expressing TCR td T-cells within their group using students' t-test. P-values <0.05 are indicated with an asterisk. (C) To study whether co-transfer of CD8 would also result in polyfunctional helper functions of TCR td CMV-specific CD4+ T-cells, both mock and HA-2-TCR td CMV-specific CD4+ T-cells with or without co-transfer of different CD8 subunits as indicated in the figure were stimulated with HLA-DR1+ LCL-CBH pulsed with pp65 peptide (grey bars; pp65 pep), unpulsed HLA-A2+ HA-2− LCL-IZA (white bars; control), HA-2 peptide pulsed HLA-A2+ HA-2− LCL-IZA (black bars; HA-2 pep) or HLA-A2+ HA-2+ LCL-JYW (striped bars; HA-2 endogenous). After 5 h of stimulation, T-cells were stained with anti-IFN-γ, anti-TNF-α, anti-CD40L and anti-IL-2 mAbs and were analyzed using flow cytometry. The percentage of IFN-γ, TNF-α and IL-2 producing or CD40L expressing T-cells after stimulation is depicted. The percentages of cytokine producing and CD40L upregulating CD8αß expressing TCR td T-cells that were significantly higher than CD8 negative and CD8αα expressing TCR td T-cells (p-values <0.05) are indicated with an asterisk. (D/E) To study differences in avidity between HLA-I-TCR td CD4+ T-cells co-transferred with the different CD8α and CD8ß constructs, HA-2 tetramer staining was analyzed. (D) Mock or (E) HA-2-TCR td CD4+ T-cells co-transferred with either wtCD8α-T2A-wtCD8ß (wtCD8 T2A; left dot plots) or ΔCD8α-T2A-ΔCD8ß (ΔCD8 T2A, right dot plots) were stained with anti-CD8α and ß mAbs and HA-2-tetramers and analyzed using flow cytometry. Populations were gated on CD8αß positive expression and HA-2 tetramer staining is depicted for the gated populations. Percentages of HA-2-tetramer positive T-cells are indicated in the upper right and MFI of the HA-2-tetramer staining in the upper left of the dot plots. Data shown are representative for 2 independent experiments.
Figure 2
Figure 2. Improved HLA-class I restricted avidity of CD8αß expressing HA-2-TCR td CD4+ T-cells results in improved proliferation.
(A) To study whether co-transfer of CD8 would also improve the peptide sensitivity of CD4+ T-cells transduced with a next generation HA-2-TCR, both mock and HA-2-TCR td CMV-specific CD4+ T-cells with or without co-transfer of different CD8 subunits as indicated in the figure were purified using flow cytometry based cell sorting and stimulated with unpulsed HLA-A2+ HA-2− LCL IZA (white bars; LCL IZA), HLA-A2+ HA-2− LCL-IZA pulsed with decreasing concentrations of HA-2 peptide (range 1 µM-10 pM) or HLA-A2+ HA-2+ LCL JYW (striped bars; LCL JYW). IFN-γ production was measured after 18 h of stimulation in duplicate, and a representative experiment out of 2 is depicted. The IFN-γ production of ΔCD8αß and wtCD8αß expressing HA-2-TCRCC td CD4+ T-cells significantly higher (p-values <0.05) than CD8 negative or CD8αα expressing HA-2-TCRCC td CD4+ T-cells is indicated with an asterisk. (B) To investigate their proliferative capacity, both mock and HA-2-TCR td CD4+ T-cells without CD8 or co-transferred with wtCD8α, wtCD8αß, or ΔCD8αß were purified based on markergene expression and CD8 cell surface expression and were either not stimulated (filled histograms) or stimulated with HLA-A2+ HA-2+ LCL-JYW (thick black line). Histograms depict PKH dilution measured 5 days after stimulation, and a representative example of 2 independent experiments is depicted.
Figure 3
Figure 3. In general, co-transfer of the extracellular domains of CD8α and ß is required and sufficient.
To confirm the generality of the previous data, total CD4+ T-cells were transduced with codon optimized and cysteine modified HA-1-, HA-2- or PRAME-TCR (transduction efficiency 48%, 48% and 22%, respectively) either with or without co-transfer of different CD8 molecules, as indicated in the figure. One week after transduction, non-purified TCR td CD4+ T-cells were stimulated and tested for cytokine production using flow cytometry. HA-1- or HA-2-TCR td CD4+ T-cells were stimulated either with HA-1 or HA-2 peptide pulsed or unpulsed HLA-A2+ HA-1- HA-2− LCL-IZA, or HLA-A2+ HA-1+ HA-2+ LCL-MRJ, and PRAME-TCR td CD4+ T-cells were stimulated either with PRAME peptide pulsed or unpulsed HLA-A2+ PRAME− melanoma cells, or HLA-A2+ PRAME+ melanoma cells. 5 h After stimulation, T-cells were permeabilized and stained with anti-NGF-R in combination with either anti-IFN-γ (upper panel), anti-IL-2 (middle panel) or anti-TNF-α (lower panel), and analyzed using flow cytometry. The percentage of markergene positive and CD8 positive T-cells producing cytokines after stimulation with antigen-negative cells (white bars; control), peptide pulsed cells (grey bars; pulsed peptide) or antigen-positive cells (black bars; endogenous peptide) is indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, et al. (2008) Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol 26: 5233–5239. - PMC - PubMed
    1. Collins RH Jr, Shpilberg O, Drobyski WR, Porter DL, Giralt S, et al. (1997) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15: 433–444. - PubMed
    1. Kolb HJ, Mittermuller J, Clemm C, Holler E, Ledderose G, et al. (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76: 2462–2465. - PubMed
    1. Porter DL, Collins RH Jr, Hardy C, Kernan NA, Drobyski WR, et al. (2000) Treatment of relapsed leukemia after unrelated donor marrow transplantation with unrelated donor leukocyte infusions. Blood 95: 1214–1221. - PubMed
    1. Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, et al. (1999) Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol 163: 507–513. - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by the Dutch Cancer Society (NKB 2007-3927) and ZonMw (grant ZonMw 433.00.001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.