Research ArticleImmunotherapy

CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo

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Science Translational Medicine  29 Apr 2015:
Vol. 7, Issue 285, pp. 285ra63
DOI: 10.1126/scitranslmed.aaa2546
  • Fig. 1. CMV-specific T cells expanded from CMVneg donors.

    (A) Specificity of EBV-, CMV-, and adenovirus-specific T cells from CMVneg donors over 16 to 23 days as shown by an IFN-γ ELISPOT assay. SFC, spot-forming cells. (B) CMV specificity of T cells expanded by enriching for naïve T cells and using overlapping peptides of CMVpp65. Lines were considered positive if they were more than five spots above the negative control with confirmatory individual peptide pools when possible. (C) Phenotype of cells shown in (B). (D) Polyfunctionality of five of the responding T cell lines as determined by intracellular or surface staining. Positive cells were counted as >2% above background staining. The markers tested were IFN-γ, TNF-α, GM-CSF, CD40L, and IL-2. (E) Derivation of CMVpp65-specific T cells from the naïve population. Before stimulation, CD3+ T cells were sorted for CD45RA+/CCR7+ cells (naïve) and CD45RA/CCR7 (“non-naïve”) cells and were then stimulated as indicated and tested for specificity to CMVpp65 and EBV-LCLs. T cells derived from the naïve fraction are shown in black and the non-naïve fraction in red. Error bars represent the SD from the mean. *P < 0.005 versus non-naïve by two-tailed t test. In (A) to (C), each symbol represents a T cell line taken from 10 CMVneg donors, and the black bars indicate the mean.

  • Fig. 2. Comparison of recognition of typical and atypical epitopes of CMVpp65 by CMVpos versus seronegative donors.

    (A) Epitope specificity for the three CMVpos HLA-A2+ donors based on overlapping peptide pools of CMVpp65. The pp65 protein was divided into 20 amino acids overlapping by 11 amino acids. The peptides were then distributed into a total of 22 pools, with each peptide present in 2 pools. Pools 2 and 21 contain the 20-mer peptide 98, harboring the typical HLA-A2–restricted epitope NLV. The percentages of cells recognizing each pool from each of the three donors are shown. This was calculated by totaling the total number of spots across all pools and dividing by spots per pool. (B) Peptide pool recognition by four CMVneg HLA-A2+ donors.

  • Fig. 3. Recognition of typical and atypical epitopes by CMVpos donors.

    (A and B) Virus-specific T cells from five CMVpos donors were expanded against the entire CMVpp65 antigen. After three stimulations, they were tested for recognition of atypical (LQT and MLN) and typical (NLV) epitopes as determined by the IFN-γ ELISPOT assay (A) and pentamer analysis (B). (C and D) T cells from CMVpos donors were stimulated three times with DCs pulsed with the indicated peptide. The number of cells that secrete IFN-γ in response to stimulation with LQT (C) or MLN (D) peptide is shown on about day 21 of culture. Gray bars indicate mean n values. (E) Failure to generate NLV-specific T cells from CMVneg donors (gray diamonds, n = 3) or CB (black diamonds, n = 5). T cells were stimulated with DCs pulsed with the peptide NLV in the absence of other peptides. After three stimulations, the resulting cells were tested for their ability to recognize NLV. Gray bars indicate mean values. (F) T cells from CMVpos donors were stimulated as above with the peptide NLV or MLN and tested by limiting dilution for the mean one-half effective concentration (EC50). Error bars indicate the SD from the mean of triplicate wells; a representative result is shown (see Table 1 for the avidity of other lines). Values shown in (E) and (F) are the numbers of spots above background.

  • Fig. 4. Epitope recognition by T cells stimulated with live CMV.

    (A) Comparison of response to CMVpp65 by T cells from HLA-A2+ CMVneg donors, CMVpos donors, and CB. The T cells were stimulated with CMV-infected HLA-A2+ fibroblasts that were cocultured with DCs and then tested with an IFN-γ ELISPOT assay. Mean ± SD values from triplicate wells are shown. (B) Typical epitope (HLA-A2–restricted NLV, B7-restricted TPR and RPH, or A24-restricted QYD) recognition of cells from a CMVpos donor line stimulated with CMV-infected fibroblasts or an adenoviral vector expressing CMVpp65 as described. (C) Typical epitope recognition by CB T cells stimulated with CMV-infected fibroblasts or the adenoviral pp65 vector. (D) Typical epitope recognition by CMVneg T cells stimulated with CMV-infected fibroblasts or the adenoviral pp65 vector. (E) Atypical epitope recognition by CB-derived T cells generated with CMV-infected fibroblasts. Pool 13 contained the atypical epitope LQT.

  • Fig. 5. CB-derived T cells for clinical use.

    (A) Phenotype of three CB-derived T cell lines manufactured from the 20% fraction of a clinical CB unit. (B) Reactivity of the CB line infused into patient P3275 to viral antigens and epitopes of pp65 including the typical epitope NLV and the atypical epitope LQT. (C) The specificity of the pp65 response of the CB line infused into patient P3275 was evaluated using overlapping pp65 peptide pools. (D) CMV load of P2891 (left) in relation to the number of reactive cells from the peripheral blood (right). The timing of T cell infusions is indicated by arrows.

  • Table 1. Avidity of CMVpos, CMVneg, and CB T cells recognizing typical and atypical epitopes.
    T cell sourceNLV
    [mean EC50 (ng/ml)]
    MLN
    [mean EC50 (ng/ml)]
    CMVpos A0.43.0
    CMVpos E0.08Not determined
    CMVpos B0.030.4
    CMVpos C0.411
    Mean0.23*4.8*
    CMVneg H0.3
    CMVneg C0.6
    Mean0.45
    CB line 10.7
    CB line 20.2
    CB line 30.8
    CB line 40.5
    CB line 50.4
    Mean0.52

    *P = 0.019679 of paired t test of log-transformed values from CMVpos A, CMVpos B, and CMVpos C.

    • Table 2. Identification of TCR sequences specific for typical and atypical epitopes in CB transplant recipients.

      CMVneg, CMV-seronegative recipient; CMVpos, CMV-seropositive recipient; CDR3, complementarity-determining region 3. # and * represent the time point at which the samples were taken.

      CB transplant recipients (% TCRs)
      CMVnegCMVpos
      121234567
      CMV reactivation?NoYesNoNoNoNoYesNoYes
      PeptideCDR3
      NLVCASSLDRVTGELFF0.00578
      CASSPGTGREQFF0.00536*
      CASSLTNEQFF0.004860.01544
      CASSLTSEQFF0.04603*0.00448
      CASSLAERSYEQYF
      CASSSVNEQFF0.33467#
      CASSLAPGATNEKLFF0.00026
      MLNCASSFGVNTEAFF0.00530.00614
      CASSLGLNYEQYF0.00027
      CSARDRDRGYEQYF0.00055
      CASRAVSTDTQYF
      CATSPTANTEAFF
      CASSPSGYNEQFF0.00921#0.00382
      CASSLDLGASTDTQYF0.00263
      CASSFRGDTEAFF0.0015
      LQTCASSPPGGSGNTIYF0.001270.00216
      Time points
      observed
      12 mo12 mo3mo*
      12 mo#
      12 mo3 + 12 mo*
      3 + 6 + 12 mo#
      6 mo12 mo12 mo12 mo
      Time points
      not observed
      3 moOnly time pt6 mo3 or 6 mo3 mo3 mo3 mo3 or 6 mo6 mo
    • Table 3. Characteristics of CB transplant recipients.

      CSA, cyclosporin A; MMF, mycophenolate mofetil; Pred, prednisone; Bu, busulfan; Cy, Cytoxan; Flu, fludarabine; TBI, total body irradiation.

      P2891P3010P3275
      SexMMM
      Age (years)521
      DiseaseFanconi anemiaALLSCID
      Donor HLAA*23:01,30:01;
      B*58:01,57:01;
      C*07:18,07:01;
      DRB1*15:03,15:02;
      DQB1*08:02,06:01
      A*02:AGA,33:01;
      B*07:ANVB,14:02;
      C*05:01,08:02;
      DRB1*03:01,15:01;
      DQB1*02:01,06:02
      A*01:BMMP,02:01;
      B*08:01:01,39:05;
      C*07:WTR,07:WCP;
      DRB1:
      03:01,04:07:01;
      DQB1:
      02:01:01,03:02:01
      HLA match5 of 65 of 66 of 6
      Conditioning
      regimen
      Flu/Cy/TBIFlu/Cy/TBIBu/Cy/Flu
      GVHD
      prophylaxis
      CSA and MMFCSAPred and MMF
      Immune
      suppression
      at time of
      T cell infusion
      CSACSAPred and MMF
      Recipient
      CMV
      serostatus
      PositivePositiveNot determined but
      PCR-negative
      Viral
      infections
      CMV, adenovirusNoneNone
      Cell dose
      (cells/m2)
      5 × 1065 × 1061 × 107
      Concurrent
      antiviral
      drugs
      Foscarnet →
      ganciclovir
      NoneNone
      Viral outcomeCMV reactivation
      and adenovirus
      infection resolved
      No viral
      reactivations
      No viral
      reactivations
      Cell dose
      (cells/m2)
      5 × 1065 × 1061 × 107
      Duration of
      cell culture
      (days)
      575055

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/285/285ra63/DC1

      Fig. S1. Identifying atypical epitopes from CMVpp65.

      Fig. S2. Polyfunctionality of pp65-specific T cells derived from naïve T cells.

      Fig. S3. Atypical epitope recognition in patient receiving CB-derived virus-specific T cells.

      Fig. S4. Shared TCRs in the CB-derived virus-specific T cells and patient P2891 6 months after T cell infusion.

      Table S1. Epitope recognition by CMVneg donors.

      Table S2. Polyclonality of T cells from CMVneg and CMVpos donors that recognize typical and atypical epitopes.

      Table S3. Precursor frequencies of TCRs recognizing NLV and MLN from CB.

      Table S4. Inhibition of CMV dissemination by T cells recognizing typical and atypical epitopes.

      Source data

    • Supplementary Material for:

      CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo

      Patrick J. Hanley, Jan J. Melenhorst, Sarah Nikiforow, Phillip Scheinberg, James W. Blaney, Gail Demmler-Harrison, C. Russell Cruz, Sharon Lam, Robert A. Krance, Kathryn S. Leung, Caridad A. Martinez, Hao Liu, Daniel C. Douek, Helen E. Heslop, Cliona M. Rooney, Elizabeth J. Shpall, A. John Barrett, John R. Rodgers, Catherine M. Bollard*

      *Corresponding author. E-mail: cbollard{at}cnmc.org

      Published 29 April 2015, Sci. Transl. Med. 7, 285ra63 (2015)
      DOI: 10.1126/scitranslmed.aaa2546

      This PDF file includes:

      • Fig. S1. Identifying atypical epitopes from CMVpp65.
      • Fig. S2. Polyfunctionality of pp65-specific T cells derived from naïve T cells.
      • Fig. S3. Atypical epitope recognition in patient receiving CB-derived virus-specific T cells.
      • Fig. S4. Shared TCRs in the CB-derived virus-specific T cells and patient P2891 6 months after T cell infusion.
      • Table S1. Epitope recognition by CMVneg donors.
      • Table S2. Polyclonality of T cells from CMVneg and CMVpos donors that recognize typical and atypical epitopes.
      • Table S3. Precursor frequencies of TCRs recognizing NLV and MLN from CB.
      • Table S4. Inhibition of CMV dissemination by T cells recognizing typical and atypical epitopes.
      • Source data

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