A non-human primate model for analysis of safety, persistence, and function of adoptively transferred T cells

Abstract

Background: Adoptive immunotherapy with antigen-specific effector T-cell (T(E) ) clones is often limited by poor survival of the transferred cells. We describe here a Macaca nemestrina model for studying transfer of T-cell immunity.

Methods: We derived, expanded, and genetically marked CMV-specific CD8(+) T(E) clones with surface markers expressed on B cells. T(E) cells were adoptively transferred, and toxicity, persistence, retention of introduced cell-surface markers, and phenotype of the persisting T cells were evaluated.

Results: CD8(+) T(E) clones were efficiently isolated from distinct memory precursors and gene-marking with CD19 or CD20 permitted in vivo tracking by quantitative PCR. CD19 was a more stable surface marker for tracking cells in vivo and was used to re-isolate cells for functional analysis. Clonally derived CD8(+) T(E) cells differentiated in vivo to phenotypically and functionally heterogeneous memory T-cell subsets.

Conclusions: These studies demonstrate the utility of Macaca nemestrina for establishing principles for T-cell therapeutics applicable to humans.

Fig. 1. Identification of CMV-reactive CD8⁺ T cells in peripheral blood lymphocytes from M. nemestrina

(A) Arrangement of 137 15-mer peptides spanning the sequence of the rhCMV IE-1 protein. The shaded areas correspond to the peptides present in the two positive pools (8; 18) in a representative epitope mapping experiment. PBMC obtained from a 23 macaques were examined for the presence of CMV-specific T-cell responses by CFC. (B) CFC detects IFN-γ production by CMV-specific CD8⁺ T cells after stimulation of PBMC with CMV IE-1 peptides in pool 8 (upper right panel) and 18 (lower left panel). Stimulation of PBMC with the corresponding peptide 68 (lower right panel) confirmed that sequences within the single peptide shared by both pools 8 and 18 stimulated IFN-γ-production by CD8⁺ T cells. PBMC stimulated with medium alone (upper left panel) served as a negative control. Data are gated on CD3⁺CD8⁺ cells.

Fig. 2. Derivation of macaque CMV-specific CD8⁺ T cells from T_EM or T_CM subsets

(A) Flow cytometry showing CD8⁺ T-cell subsets in macaque PBMC including T_CM (CCR7⁺CD95⁺), T_EM (CCR7⁻CD95⁺), and T_N (CCR7⁺CD95⁻). (B) Frequency of CD8⁺ T_CM, CD8⁺ T_EM, and CD8⁺ T_N (%) in peripheral blood CD8⁺ lymphocytes. Aliquots of PBMC obtained from 18 healthy macaques were stained with mAbs binding to CD8, CD3, CD95, and CCR7 and examined by flow cytometry after gating on CD3⁺CD8⁺ T cells. Mean and SD is shown. (C) CMV IE-specific T cells are present in distinct CD8⁺ T_M subsets. CFC assay for IFN-γ⁺CD8⁺ T cells specific for CMV in sort-purified CD8⁺CD62L⁺ and CD8⁺CD62L⁻ T-cell subsets obtained from 4 macaques. The absolute number of CMV-specific CD8⁺ T_CM or T_EM/μL peripheral blood was determined by calculating the absolute number of CD3⁺CD8⁺ T cells/μL of blood (% of CD8⁺ T cells/lymphocyte subset × lymphocyte count/μL blood/100). Subsequently, the absolute number of the T_EM, T_CM, and T_N subset was derived (% subset × number of CD3⁺CD8⁺/μL blood/100). Finally, we calculated the absolute number of CMV-specific CD8⁺ T cells/μL in each subset (% IFN-γ⁺ cells × absolute number of CD3⁺CD8⁺ T_CM+T_N/μL or CD3⁺CD8⁺ T_EM/μL blood/100). (D, E) CFC assay of macaque CMV-specific T-cell lines detects IFN-γ⁺CD8⁺ CMV-specific T cells in sort-purified CD8⁺CD62L⁺ cells containing T_CM (upper panels) and CD8⁺CD62L⁻ T_EM subsets (lower panels). Sort-purified subsets from 2 representative macaques were stimulated with autologous CMV peptide-pulsed antigen-presenting cells and assayed by CFC for production of IFN-γ after stimulation with medium alone (left panels), or CMV peptide antigen (right panels). Data are gated on CD3⁺CD8⁺ cells.

Fig. 3. Gene-marking of macaque CD8⁺ T_E with non-immunogenic B-cell lineage marker

(A) Efficient transduction and selection of macaque CD8⁺ T_E with the ΔCD19 or CD20 marker. Macaque CD8⁺ T_E were stimulated with anti-CD3/CD28 mAbs, transduced with ΔCD19 or CD20, and enriched by immunomagnetic selection. Aliquots of the unselected (□) or selected () T cells were examined by flow cytometry after staining with mAbs against CD3, CD8, and CD19 or CD20 mAbs. Shown are mean and range of the results with the ΔCD19 (n=7) or CD20 (n=3) marker. (B) In vitro growth of gene-modified T_E clones. Left panel: Representative T_E clone either unmodified (◇) or ΔCD19⁺ (◆). Right panel: Representative T_E clone either unmodified (◇) or CD20⁺ (●). Aliquots of T cells unmodified or transduced with ΔCD19⁺ (left panel) or CD20⁺ (right panel) were restimulated with anti-CD3/CD28 mAbs, irradiated feeder cells and IL-2, and numeric expansion was measured by counting viable cells on the indicated days. Data are representative of results with ΔCD19⁺ or CD20⁺ T cells obtained from each three macaques. (C) Gene-marked CMV-specific CD8⁺ T_E clones retain CMV-specific reactivity. Aliquots of CMV-specific CD8⁺ T_E either unmodified or either ΔCD19⁺ (left panel) and CD20⁺ (right panel) were restimulated in vitro and examined in a chromium release assay for recognition of autologous target cells, either unpulsed (□) or pulsed with the CMV cognate peptide at an effector-to-target (E/T) ratio of 10:1 (■), 5:1(), 2.5:1 (), or 1.25:1 (). Data are representative of results with ΔCD19⁺ or CD20⁺ T cells from each three macaques. (D, E) Stability of the marker-gene expression in macaque CD8⁺ T cells. The ΔCD19⁺ (D) or CD20⁺ (E) T cells were stimulated with anti-CD3/CD28 mAbs and examined by flow cytometry on day 6 (ii) and 14 (iii) of the stimulation cycle for ΔCD19 or CD20 expression. Unmodified T cells served as negative control (i). Aliquots of T cells were also rested and the ΔCD19 or CD20 expression was assessed by flow cytometry after 4 weeks of rest (iv) or 6 days after restimulation with anti-CD3/CD28 mAbs (v). Inset values show the % of CD3⁺CD8⁺ T cells positive for ΔCD19 or CD20 and the MFI is indicated for each time point. Data showing the difference in the stability of expression of ΔCD19 and CD20 at the cell surface is representative for experiments with ΔCD19 and CD20-modified T cells from 3 animals, and was observed in eight ΔCD19⁺ and CD20⁺ T_E clones.

Fig. 4. Tracking of ΔCD19⁺ or CD20⁺ CMV-specific CD8⁺ T_E clones following adoptive transfer by qPCR

(A) Schematic design of retroviral vector constructs encoding for macaque B-cell lineage marker genes and location of primer and fluorescent probe (red bar) used for the qPCR assay. Abbreviations: MPSV-LTR, myeloproliferative sarcoma virus retroviral long terminal repeat; PRE, woodchuck hepatitis virus post-transcriptional regulatory element. (B) Detection of ΔCD19 or CD20-marked T cells within PBMC by qPCR. Samples of titrated numbers of ΔCD19⁺ () or CD20⁺ T cells (■) were spiked into aliquots of pre-infusion PBMC (each 10⁵ PBMC/reaction) and examined by real-time qPCR for detection of marker-positive T cells. Data are representative of each 3 assays with ΔCD19⁺ or CD20⁺ T cells. (C) Enumeration of transferred ΔCD19⁺ or CD20⁺ T cells determined by real-time qPCR for vector sequences. Autologous ΔCD19⁺ () or CD20⁺ (■) T_CM-derived T_E clones were expanded in vitro and transferred to each one of the macaques at a dose of 5×10⁸/kg. DNA was isolated from samples of PBMC obtained before and at indicated time-points after the T-cell infusion and examined by real-time qPCR for detection of marker-positive T cells.

Fig. 5. Tracking of transferred ΔCD19⁺ or CD20⁺ CD8⁺ T_E clones by flow cytometry

(A) An autologous CMV-specific T_E clone modified to express ΔCD19 was expanded in vitro and transferred back to a macaque at a cell dose of 5×10⁸/kg. PBMC were collected before (pre) and on day 1 and 3, and at week 3 after infusion and examined by flow cytometry after staining with mAbs to CD3, CD8, and CD19. Data are gated on CD3⁺CD8⁺ T cells and are representative for results in three animals. Inset values show the frequency (%) of CD3⁺CD8⁺ T_E positive for ΔCD19. (B) An autologous CD20⁺ CMV-specific T_E clone was given to a macaque at a dose of 5×10⁸/kg. PBMC were collected at the indicated times and analyzed by flow cytometry for the frequency (%) of CD20⁺ T cells within the CD3⁺CD8⁺ T-cell subset. (C) Activation-dependence of the CD20-expression in persisting CD20⁺ T cells. Flow cytometry analysis of PBMC obtained before (left panel) and 6 days after the CD20⁺ T-cell infusion (second right panel) after staining with mAbs to CD3, CD8, and CD20. After 4 days of activation with anti-CD3/CD28 mAbs, aliquots of the activated pre-infusion PBMC (second left panel) and ‘Day 6’ PBMC (right panel) were examined by flow cytometry for cell-surface expression of CD20 after gating on CD3⁺CD8⁺ T cells. Inset values show the frequency (%) of CD3⁺CD8⁺ T_E positive for CD20. The MFI of the CD20⁺ T_E (R1) is shown.

Fig. 6. Analysis of phenotype and function of adoptively transferred ΔCD19⁺ T_CM-derived T_E clones

(A) Multiparameter flow cytometry of macaque PBMC obtained 4 weeks after a ΔCD19⁺ T_E infusion. Transferred ΔCD19⁺ T cells were identified in PBMC after staining with mAbs to CD3, CD8, and CD19. (B) The expression of T_M marker on CD3⁺CD8⁺ΔCD19⁺ T cells was determined by flow cytometry after co-staining with CD62L, CCR7, CD28, CD127 or CD95. (C) Aliquots of macaque PBMC were obtained 8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone. CCR7⁺CD95⁺ T_CM and CCR7⁻CD95⁺ T_EM subsets either in the CD3⁺CD8⁺ΔCD19⁻ or CD3⁺CD8⁺ΔCD19⁺ cells were identified as described in (A) and (B). Cells were then stained for intracellular expression of Ki-67. Inset values show the frequency (%) of Ki-67⁺ T cells. (D) Analysis of intracellular Ki-67 in PBMC obtained from 2 macaques 6-8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone, and in samples of BM and LN obtained from 3 macaques 2 weeks after the infusion. PBMC: mean; BM and LN: mean ± SD. (E) Function of re-isolated ΔCD19⁺ T_E obtained from T_CM or T_EM subsets. Aliquots of post-infusion PBMC were sort-purified in CD8⁺ΔCD19⁺CD62L⁺ and CD8⁺ΔCD19⁺CD62L⁻ subsets, restimulated in vitro using anti-CD3/CD28 mAbs, and examined in a chromium release assay for recognition of unpulsed (□) or peptide-pulsed target cells. E/T (■) 2.5:1, () 1.25:1. The transferred ΔCD19⁺ T_E clone served as control. (F) Aliquots of the infused ΔCD19⁺ T_E clone (■) and re-isolated ΔCD19⁺ T_E either obtained from T_CM () or T_EM (□) were stimulated with medium or CMV peptide-pulsed antigen-presenting cells, and examined by CFC for production of IFN-γ, TNF-α, and IL-2 after gating on CD3⁺CD8⁺ T cells.