In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Although cytomegalovirus (CMV) infection in immunocompetent individuals is usually minimally symptomatic, CMV may cause fatal disease in hematopoietic cell transplant (HCT) recipients. Adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs), expanded from donor memory cells, can prevent CMV disease in HLA-identical HCT recipients.¹  Monocyte-derived dendritic cells (Mo/DCs) and other antigen-presenting cells (APCs) have been used to elicit CMV-specific CTLs^2-5  (for a review, see Bollard et al⁶ ). Although CMV-specific CTL generation is feasible in seropositive donors, it rarely succeeds in CMV-seronegative individuals.⁷ 

Antiviral CTLs have been unavailable for umbilical cord blood transplant (UCBT) recipients because cord blood (CB), like seronegative donors, contains antigen-inexperienced T cells. Additionally, compared to CD45RA⁺/CD28⁺ `naive' adult T cells, CB T cells are defective in the inducible expression of Th1/Tc1 cytokines, primarily IFN-γ, as a result of Th2/Tc2 skewing imprinted by placental factors (for a review, see Marchant and Goldman⁸ ). Independently, diminished IL-12 expression by neonatal DCs impairs Th1 immunity.^9,10  We now report that CMV-specific CTLs can be generated from CB along the Th1/Tc1 pathway.

Fresh mononuclear cells (MNCs) from discarded CB units obtained with Institutional Review Board approval were cultured in RPMI 1640 plus 10% pooled human serum (PHS; NABI, Miami, FL). CD34⁺ progenitors were selected from nonadherent cells¹¹  using CD34-PE (Becton Dickinson, San Jose, CA) and anti-PE microbeads (Miltenyi Biotech, Auburn, CA) over a MiniMACS column per the manufacturer's instruction. CD34^- cells were frozen. Immature Mo/DCs developed from adherent monocytes in RPMI 1640 plus 1% PHS, GM-CSF, and IL-4 over 5 to 6 days.¹¹  Unused MNCs were frozen and subsequently thawed weekly until exhausted to generate immature Mo/DCs as described except for the adherence media, LGM-3 plus 10% PHS (Clonetics, San Diego, CA). On “day –2” DCs were pulsed at 37°C with inactivated AD169 CMV lysate (5 μg/mL, Biodesign, Carmel, NY). One hour later 1 mg/mL CMV hyperimmune globulin (Medimmune, Gaithersburg, MD) was added for 6 to 8 hours, followed by maturation in TNF-α plus PGE₂^11,12  for 24 to 36 hours. Once Mo/DCs were exhausted CD34⁺-derived DCs were generated in AIM V (Gibco, Grand Island, NY) plus 3% PHS, 20 ng/mL SCF, 100 ng/mL Flt-3L (both from Amgen, Thousand Oaks, CA), GM-CSF, and IL-4. After 12 to 14 days, adherent cells were subcultured in GM-CSF plus IL-4 for 4 to 5 days. Nonadherent cells remained in the original cytokines until subcultured.

CTL cultures were started on “day 0” when CMV-immune complex (IC)-loaded mature Mo/DCs and unpulsed/control Mo/DCs were mixed with autologous lymphocytes/CTL responders. Responders were generated from CD34^- cells thawed on “day –3,” depleted of human IgG adherent cells,¹¹  and cytokine-primed in AIM V plus 3% PHS, 1 ng/mL IL-7, and 20 U/mL IL-12 (both from R&D Systems, Minneapolis, MN). CTL cultures were started at 1 × 10⁶ cells/mL at 10:1 responder/DC ratio in AIM V plus 3% PHS plus IL-1β plus IL-2 plus IL-4¹³  and 1 ng/mL IL-7 and 20 U/mL IL-12 with or without 1 μg/mL CMV lysate. Media/cytokines, except for IL-1 and IL-4, were renewed on days 3 and 5. After day 7, 10 IU/mL IL-2 (R&D Systems) was supplemented besides IL-7 and IL-12, with or without CMV lysate. Media/cytokines were renewed at 2 to 3 per week and freshly prepared DCs with or without CMV lysate were added weekly.

Cells (10⁵ cells/100 μL) from weekly restimulated CTL cultures was pulsed 3 days later with ³H-thymidine.¹¹  Stimulation index (SI) calculates the ratio of mean CMV-stimulated/unstimulated counts.

Immunophenotyping was performed weekly as described.¹⁴  For cytokine secretion DC plus responder cocultures were incubated with anti-CD28 and anti-CD49d antibodies (1 μg/mL) with or without 5 μg/mL CMV lysate (Advanced Biotechnologies, Columbia, MD) for 10 to 12 hours. Brefeldin A was present the last 8 to 10 hours.¹⁴  Reagents and antibodies were purchased from Becton Dickinson. CTL activity was quantitated by a 5-hour LDH-release assay per the manufacturer's instructions (Cytotox96; Promega, Southampton, United Kingdom) against autologous immature CD34⁺-derived DC targets with or without CMV lysate.

The paired 2-tailed Student t test analyzed parallel culture conditions.

DCs are uniquely capable to endocytose and process soluble antigens and present major histocompatibility class I-restricted peptides to CD8⁺ T cells in a process coined “cross-presentation.”¹⁵  Antigens opsonized with specific antibodies form ICs that can gain entry into DCs via Fcγ receptors¹⁶  resulting in superior antigen presentation.¹⁵  Immature Mo/DCs were generated from CB and pulsed with CMV lysate and CMV-ICs. Although CMV-IC generation was not verified experimentally, high concentrations of CMV antigens and CMV-hyperimmune globulin should inevitably lead to IC formation.¹⁷  Wild-type CMV infection may impair DC phenotype/function¹⁸ ; however, CMV-IC-loaded Mo/DCs expressed potent T cell-stimulatory phenotype (Figure 1).

To overcome the type 2 bias of neonatal T cells imparted by reduced tryptophan levels and high progesterone, IL-10, and IL-4 at the maternal-fetal interface,^8,19  CB T cells were cytokine-primed before encountering DCs. After about 5 weeks in CTL culture there was greater expansion (2.5 ×; range, 1.7-4.5 ×) in the CMV-pulsed cultures (P = .04, n = 5) than control (0.5 ×; range, 0.2-1.7 ×) despite identical cytokine milieu. CMV-pulsed T-cell proliferation exceeded controls (mean SI, 3.0 ± 3.7, P = .003, 24 time points/5 experiments). SI increased further within each experiment as cell death exceeded proliferation in controls while CMV-loaded cultures kept expanding (not shown).

Beyond 4 weeks CD45RO⁺/RA^-/“memory” T cells dominated in CMV-pulsed cultures (Table 1). By week 6, CD45RO replaced CD45RA among about 90% of T cells, depleting CD45RA⁺/CD62L⁺/“recent thymic emigrants” (< 10%, P = .003, n = 10 between weeks 4 and 6; not shown). “Memory CD8⁺” cells/(CD45RA^-/CD27⁺/CD8⁺) expanded significantly in CMV-IC-pulsed cultures compared to control (P = .03, n = 9 time points; Table 1). CD57 and CD28 expression remained unchanged, as all T cells regardless of culture conditions retained the CD28⁺/CD57^- phenotype. CD27 expression was retained in more than 95% of T cells (not shown).

CMV-specific, IFN-γ-producing cells appeared by the fourth week of stimulation and by week 5 they exceeded more than 10% of the total T-cell pool in 5 of 5 consecutive experiments (median, 26%). Similarly, TNF-α and IL-2 secretion was detected in response to CMV (Table 1; Figure 2A). Importantly, IFN-γ and TNF-α, which are critical for effective antiviral immunity, were produced by CD8⁺ and CD4⁺ cells. Nevertheless, under these conditions CD4⁺ responses dominated (TNF-α, P = .001; IFN-γ, P = .065, n = 7) corroborating other reports on viral lysate stimulation.²⁰  CD4⁺ T cells may contribute to viral control independently.^21,22  After at least 4 weeks of culture CMV-specific lysis could be detected (Figure 2B) provided CMV-specific IFN-γ- and TNF-α-secreting T cells were enriched to 10% or more. T cells from the CMV-IC-pulsed cultures killed CMV-pulsed autologous DCs significantly better than unmanipulated DCs (P = .03, n = 4).

Cytokine-priming and continued exposure of CB T cells and DCs to Th1/Tc1-promoting cytokines is probably essential for CTL generation from CB. IL-12 may be the most critical because it can induce 4-1BB expression on naive CB T cells to receive CD28-independent costimulation, vital for acquisition of CTL function²³  besides correcting neonatal DC impairment. The overall phenotype in our CMV-IC-loaded cultures was similar to the IFN-γ-, TNF-α-secreting CD45RA^-/CD28⁺/CD27⁺ cells described in healthy and HIV⁺ individuals during the acute phase of protective immunity to CMV. ²⁴  Starting with about 1 million T cells present in 2 to 3 mLCB,¹⁴  at most 100 to 1000 T cells may possess CMV reactivity.²⁵  Following about 4 weeks in the described CTL culture if CMV-reactive T cells comprise about 25% of 2.5 × 10⁶ cells, then about 600 000 CMV-specific lymphocytes could be generated. Clinical responses were reported after infusing 350 to 400 CMV-specific T cells/kg expanded from seropositive donors.² 

In conclusion, this report documents the in vitro generation of virus-specific CTLs from naive human CB lymphocytes. Although currently time and labor intensive, our approach may lead to effective clinical strategies to prevent or treat opportunistic viral infections in the CB transplantation setting.

The authors thank Eli Gilboa for his critical review of the manuscript and Young-June Kim for helpful discussions.