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Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens - PubMed

  • ️Fri Jan 01 2016

Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens

Andreas Kirschner et al. Oncotarget. 2016.

Abstract

Aim: Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells.

Methods: We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells.

Results: Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319.

Conclusion: Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells.

Keywords: CD107a; TCR transgenic T cells; amino-acid exchange scan; cross reactivity; fratricide.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. ADRB3 is an ES associated target antigen

A. ADRB3 is over-expressed in Ewing Sarcoma compared to a Normal Body Atlas and B. compared to other tumor entities. C. Doxycyclin-induced knockdown of EWSR1-FLI1 in shows an EWSR1-FLI1 dependent expression of ADRB3 in A673/TR/shEF1 cells [37, 52].

Figure 2
Figure 2. Wild type T cell clone ADRB3-1F4 specifically recognizes and kills HLA-A*02:01/ADRB3295 presenting ES cell lines

A. Flow-cytometry confirms HLA-A*02:01/ADRB3295 binding on T2 cells. ADRB3-1F4 shows peptide specificity and HLA-A*02:01 restriction against B. peptide loaded T2 cells and against C. HLA-A*02:01/ADRB3+ ES cell lines compared to controls in IFNγ ELISpot assays. D. Dose-dependent killing of HLA-A*02:01/ADRB3+ ES cell lines compared to controls determined by ELISpot granzyme B release. E. T cell clone ADRB3-1F4 recognizes ADRB3295 peptide pulsed on T2 cells in an IFNγ ELISpot assay in a dose dependent manner. IFNγ release diminishes at a threshold of <0.01 μM. Data are presented as mean and SEM. A673, EW7 and TC-71; HLA-A*02:01+ ES, SK-N-MC: HLA-A*02:01− ES, K562: MHC− NK cell control. Error bars represent standard deviation of triplicate experiments. Asterisks indicate significance levels. p values < 0.05 were considered statistically significant (*p < 0.05; **p < 0.005; ***p < 0.0005).

Figure 3
Figure 3. Functional evaluation of ADRB3295-TCR transgenic T cells

A. ADRB3295-TCR transgenic T cells specifically recognize ADRB3295 pulsed T2 cells and B. HLA-A*02:01/ADRB3+ ES cell lines in contrast to controls. C. ADRB3295-TCR transgenic T cells specifically kill HLA-A*02:01/ADRB3+ ES cell line A673 in an xCELLigence assay compared to controls. D. ADRB3295-TCR transgenic T cells recognize ADRB3295 peptide pulsed on T2 cells in an IFNγ ELISpot assay in a dose dependent manner. IFNγ release diminishes at a threshold of <0.01 μM. Data are presented as mean and SD. A673, EW7 and TC-71; HLA-A*02:01+ ES, SK-N-MC: HLA-A*02:01− ES, K562: MHC− NK cell control. E/T ratio for ELISpot assay: (A and D) 1:4, (B) 1:2. Error bars represent standard deviation of triplicate experiments. Asterisks indicate significance levels. *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 4
Figure 4. CD107a/annexin positivity is a sensitive early marker of ADRB3295-TCR transgenic T cell committed fratricide

A. In PBMC donors #1 and #2 ADRB3295-TCR transgenic T cell expansion is hampered compared to CHM1319-TCR transgenic T cells as measured by total cell counts at expansion day 3 and day 7, respectively; B. Flow cytometry reveals correlation of CD107a/annexin expression with expansion impairment of ADRB3295-TCR transgenic T cells in contrast to CHM1319-TCR transgenic T cells. Asterisks indicate significance levels. *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 5
Figure 5. Amino-acid exchange scans yield only limited power to predict cross-reactivity of CD8+ TCR transgenic T cells in contrast to in silico HLA-A*02:01 binding prediction

A + B. ADRB3295-TCR transgenic T cells as well as CHM1319-TCR transgenic T cells reveal unspecific recognition upon selective amino-acid exchange of respective target antigens to (A) alanine/serine and (B) alanine/threonine in IFNγ ELISpot assays. C. The number of peptides carrying the ADRB3 specific core motive is much higher in comparison to peptides carrying the CHM1 specific core motive. D. Peptides with similar ADRB3295 motive patterns (n=780) show higher binding scores for HLA-A*02:01 in comparison to the peptides with similar CHM1316 motive (n=33). *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 6
Figure 6. Various LCL cell lines and HLA-A*02 blocking of ES target cell lines to distinguish T cell cross-reactivity

A. Recognition of LCLs was limited to two lines expressing the HLA-A*02 superfamily. Further pulsing the cells with specific ADRB3295 peptide also gave no further hints for cross-reactivity. ES cell line A673 served as a positive control. B. Blocking of the HLA-A*02 molecules on ES cell lines shows reduced IFNγ release in ELISpot assays. E/T ratio for ELISpot assay: 1:2. Error bars represent standard deviation of triplicate experiments.

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References

    1. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med. 1988;319:1676–1680. - PubMed
    1. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, Citrin DE, Restifo NP, Robbins PF, Wunderlich JR, Morton KE, Laurencot CM, Steinberg SM, White DE, Dudley ME. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011;17:4550–4557. - PMC - PubMed
    1. Yannelli JR, Hyatt C, McConnell S, Hines K, Jacknin L, Parker L, Sanders M, Rosenberg SA. Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience. Int J Cancer. 1996;65:413–421. - PubMed
    1. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371:1507–1517. - PMC - PubMed
    1. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Levy CL, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011;29:917–924. - PMC - PubMed

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