A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities

Abstract

Human cytomegalovirus (HCMV) can cause life-threatening disease in infected hosts. Immunization with human leukocyte antigen (HLA)-restricted immunodominant synthetic peptides and adoptive transfer of epitope-specific T cells have been envisaged to generate or boost HCMV-specific cellular immunity, thereby preventing HCMV infection or reactivation. However, induction or expansion of T cells effective against HCMV are limited by the need of utilizing peptides with defined HLA restrictions. We took advantage of a combination of seven predictive algorithms to identify immunogenic peptides of potential use in the prevention or treatment of HCMV infection or reactivation. Here we describe a pp65-derived peptide (pp65(340-355), RQYDPVAALFFFDIDL: RQY16-mer), characterized by peculiar features. First, RQY-16mer is able to stimulate HCMV pp65 specific responses in both CD4(+) and CD8(+) T cells, restricted by a wide range of HLA class I and II determinants. Second, RQY-16mer is able to induce an unusually wide range of effector functions in CD4(+) T cells, including proliferation, killing of autologous HCMV-infected target cells and cytokine production. Third, and most importantly, the RQY-16mer is able to stimulate CD4(+) and CD8(+) T-cell responses in pharmacologically immunosuppressed patients. These data suggest that a single reagent might qualify as synthetic immunogen for potentially large populations exposed to HCMV infection or reactivation.

Figure 1

Responsiveness of CD4⁺ and CD8⁺ T cells to immunostimulation by RQY‐16mer peptide irrespective of HLA typing. (A) CD4⁺ and CD8⁺ T cells from 14 seropositive healthy donors were cultured for 2 weeks in the presence of RQY‐16mer peptide, as detailed in the ‘Materials and methods’ section. Peptide‐induced expression of the indicated cytokine genes was then assessed following a 3‐hr incubation in the presence or absence of RQY‐16mer. Data are reported as cytokine gene relative quantification (2^−ΔΔCt) in RQY‐16mer triggered cells, as compared to cells incubated in the absence of peptide. Interquartile ± S.D. are reported for each single cytokine gene expressed by either CD4⁺ or CD8⁺ T cells upon RQY‐16mer induction. Negative cut‐off (dotted line) was set at 1.9‐fold increase based on average variation of cytokine gene expression in T cells cultured in the absence of antigenic peptide for 2 weeks and then challenged for 3 hrs in the presence or absence of RQY‐16mer peptide. RQY‐16mer stimulation of T cells from seronegative donors (n= 2) failed to induce increases in cytokine gene expression exceeding cut‐off values (see panels B–E, empty circles). (B–E) Specific RQY‐16mer T‐cell reactivation as an hallmark of HCMV latency. Numbers of IFN‐γ and TNF‐α gene transcript copies induced by RQY‐16mer stimulation in either CD4⁺ or CD8⁺ cultured T cells were correlated to those of pp65 DNA copies detectable in autologous fresh CD14⁺ cells for each of the 14 HCMV‐seropositive (full circles) and two HCMV‐seronegative (empty circles) donors under investigation. Categorical markers were analysed by Pearson's chi‐square test. A two‐tailed paired t‐test was used to define P‐values (B) CD4⁺/IFN‐γ: P= 0.0004; (C) CD8⁺/IFN‐γ: P= 0.002; (D) CD4⁺/TNF‐α: P= 0.003; (E) CD8⁺/TNF‐α: P= 0.004). (F) Antigen‐specific responsiveness to RQY‐16mer is not related to unspecific stimulatory effects. IFN‐γ gene expression was studied in cells from five cord blood specimens upon ex vivo RQY‐16mer peptide stimulation (median 1.28 folds, range 0.33–1.85) or, in HLA‐A*0201 cord blood specimens (n= 3) following stimulation with different HLA‐A*0201 restricted virus derived antigens (HCMV pp65_495−503, BKV large T antigen _579−587, EBV LMP‐2_426−434, influenza matrix₅₈₋₆₆; median 1.01, range 0.32–1.57) as compared to mitogenic phytoemagglutinin stimulation (median 14.52 folds, range 9.85–15.14; P= 0.0001). Data are reported as IFN‐γ gene expression relative quantification (2^−ΔΔCt) as compared to cells from the same donors pre‐incubated in the absence of stimuli.

Figure 2

Peptide‐specific immune stimulation induced by 9mer‐peptides nested within RQY‐16mer in CD8⁺ T cells following in vitro expansion driven by RQY‐16mer. (A) CD8⁺ T cells from representative HCMV seropositive healthy donors (D9‐D11, see Table 1) expressing the indicated HLA class I specificities were cultured in the presence of RQY‐16mer, as detailed in the ‘Materials and methods’ section. Cells were then stimulated for 3 hrs in the presence of 9mer‐peptides encompassed within RQY‐16mer sequence, and cytokine gene expression was assessed by qrt‐PCR. Data are reported as percentage of IFN‐γ gene expression, as compared to positive control phytoemagglutinin (PHA). Black bars identify stimulatory 9mer‐peptides inducing cytokine gene expression >2% of PHA triggered levels. The HLA‐A*0201 restricted pp65_495−503 (NLV) peptide was used as control. (B) Cytotoxic activity of RQY‐16mer stimulated CD8⁺ T cells against nested 9mer‐peptides. CD8⁺ T cells from the same experiments depicted in A were tested as effector cells in ⁵¹Cr release assays by using, as targets, autologous EBV‐transformed B cells upon pulsing with the indicated peptides. Data are reported as mean of delta specific lysis (ΔSL) ± S.D. at 10:1 E/T ratio. (C–H) Frequencies of HCMV epitope specific cells ex vivo or following peptide‐driven in vitro (i.v.) T‐cell expansion. Total T cells (C–E) or purified CD8⁺ T cells (F–H) from two HCMV‐seropositive donors (D10 and D9, respectively) expressing appropriate HLA specificities were stained ex vivo with RQY‐9mer/HLA‐A*0201‐ (C) and QYD‐9mer/HLA‐A*2402‐pentamers (F). Cells were considered positive if their mean fluorescence intensity (MFI) exceeded by at least 10‐fold that of multimer negative CD8⁺ cells. The same cell preparations were then stimulated for 2 weeks in the presence of RQY‐9mer (D), QYD‐9mer (G), or RQY‐16mer (E, H) and staining with RQY‐9mer/HLA‐A*0201‐ and QYD‐9mer/HLA‐A*2402‐pentamers was repeated on cultured cells. Cells expanded in the presence of specific 9mer‐peptides or RQY‐16mer were also used as effector cells in cytotoxicity assays utilizing, as targets autologous EBV‐transformed B cells upon pulsing with specific (squares) or a control (gp100_280−288, HLA‐A*0201 restricted melanoma associated epitope, diamonds) peptide. Data are reported as percentage‐specific lysis at the indicated E/T ratios. Standard deviations, never exceeding 10% of the reported values, were omitted.

Figure 3

Glutaraldehyde (Gla)‐fixation of dendritic cells jeopardizes the induction of RQY‐16mer peptide specific immune responses. (A) IFN‐γ gene expression by cultured CD4⁺ and CD8⁺ T cells is significantly (P < 0.05) impaired if RQY‐16mer peptide is presented by autologous Gla‐fixed (white bars) iDCs, as compared to RQY‐16mer treated live iDCs (black bars). Data are reported as IFN‐γ gene expression as related to the response in the absence of antigenic peptide. A two‐tailed paired t‐test was used to define P‐values (P= 0.05; CI 95%). (B) Gla‐fixation of iDCs (white squares) does not impair responsiveness of CD8⁺ T cells to HLA class I restricted 9mer‐peptides, as compared to reactivity upon peptide presentation by live iDCs (black squares). Instead, a significant increment of IFN‐γ gene expression is observed in CD8⁺ T cells co‐cultured with live iDCs in the presence of RQY‐16mer peptide, as compared to Gla‐fixed cells (514% for the first donor and 549% for the second donor).

Figure 4

RQY‐16mer is a promiscuous epitope stimulating cytokine production by CD4⁺ T cells across a wide range of HLA class II restrictions. (A) Percentages of CD4⁺ T cells producing pro‐inflammatory cytokines (IFN‐γ: circles; TNF‐α: triangles; IL‐2: diamonds) upon RQY‐16mer induction were assessed by ICS in cells from seven donors expressing 90% of the HLA‐DRB1* alleles represented in our group. Previously cultured for 2 weeks in the presence of the peptide, as detailed in the ‘Materials and methods’ section. Each symbol represents the average of two independent experiments performed by using cells from the same donor. Cut‐off limit was set at 0.18% (dotted line) based on RQY‐16mer peptide‐induced cytokine production by CD4⁺ T cells expanded for 2 weeks in the presence of mDC and IL‐2, but in the absence of peptide. Quadrants were set based on an IgG₁ isotype control. Median percentages of responding cells are indicated by bars. (B) Representative ICS of RQY‐16mer stimulated cytokine production from HCMV‐seropositive donors. Data reported in the quadrants refer to percentages of CD4⁺ T cells showing evidence of the production of the indicated cytokines induced by RQY‐16mer in CD4⁺ T cells from different donors expanded for 2 weeks in the presence of the peptide. For each donor, HLA‐DRB1* tissue typing data and predicted netMHCIIpan algorithm associations of core sequences embedded within RQY‐16mer with defined allelic products are also reported (characters in bold, right column).

Figure 5

Functional features of RQY‐16mer induced CD4⁺ T cells. (A) Proliferation of CD4⁺ T cells upon RQY‐16mer stimulation. Freshly isolated PBMCs from four HCMV seropositive donors and one seronegative donor were cultured for 1 week in the presence (black bars) or absence (white bars) of RQY‐16mer, as detailed in material and methods. Proliferation was measured by ³H‐Thymidine incorporation. Results are reported as mean of cpm ± S.D. of triplicate wells. Two‐tailed paired t‐test (stimulation versus non‐stimulation) was used to define P‐values (P= 0.05; CI 95%). Significant increases in RQY‐16mer induced proliferation were observed in seropositive donors (DRB1*04,14 P= 0.04; DRB1*01,13 P= 0.05; DRB1*04,12 P= 0.03; DRB1*03,15 P= 0.004), but not in the seronegative donor (DRB1*07,14, P= 0.4). (B) Caspase‐3 production by HCMV‐infected iDCs co‐cultured with RQY‐16mer expanded CD4⁺ T cells. Following RQY‐16mer driven expansion (see ‘Materials and methods’ section), CD4⁺ T cells were co‐cultured at 10:1 E/T ratio with autologous iDCs infected at a MOI of 10 with HCMV VR1814 or mock infected. Caspase‐3 production, as detected in CD1a+ DCs was used as read‐out. The histogram shows a significant increase of caspase‐3 production in either mock‐infected (middle red peak) or HCMV‐infected iDCs (right peak), upon co‐culture with RQY‐16mer expanded CD4⁺ T cells, as compared to that detectable in negative control iDCs in the absence of effectors (left peak). A 60% increase of caspase‐3 production, as detectable by specific mean fluorescence intensity (MFI), in HCMV‐infected iDCs as compared to mock‐infected counterparts was observed in the presence of RQY‐16mer expanded CD4⁺ T cells. (C) Caspase‐3 production by HCMV infected versus mock‐infected iDCs following co‐culture with CD4⁺ T cells expanded in the presence of RQY‐16mer, as observed in three independent experiments by using cells from different HCMV seropositive donors. The fluorescence index was calculated by the following formula: (MFI experimental sample – MFI background)/(MFI background)%. Statistical significance (P= 0.0073) was analysed by two‐tailed paired t‐test.