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. 2002 May;76(10):5071-81.
doi: 10.1128/jvi.76.10.5071-5081.2002.

Primary immune responses by cord blood CD4(+) T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro

A Douglas Wilson  1 , Andrew J Morgan
Affiliations

Primary immune responses by cord blood CD4(+) T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro

A Douglas Wilson et al. J Virol. 2002 May.

Erratum in

  • J Virol 2002 Aug;76(16):8504

Abstract

Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 10(5.9). We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4(+) T cells and CD16(+) CD56(+) NK cells. After virus challenge both CD4(+) T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16(+) CD69(+) cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4(+) T cells and CD16(+) NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4(+) T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4(+) T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4(+) T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.

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Figures

FIG. 1.
FIG. 1.
Photomicrograph of fetal cord blood 4 days after infection with EBV. Large clumps of cells have formed throughout the well, indicating that many cell types become activated and adhere to one another in association with EBV-infected B cells.
FIG. 2.
FIG. 2.
Bar graph showing the transformation titers of EBV on five separate samples of unfractionated cord blood. The titer of virus was lower when measured on purified B cells. The titer was restored by addition of monocytes (Mφ) to the purified B cells. In contrast, addition of T cells to purified B cells reduced the virus titer.
FIG. 3.
FIG. 3.
Flow cytometry plots of fetal cord blood stained with different combinations of fluorescent antibodies. (a) CD4-PE and CD8-FITC staining of fresh fetal cord blood, showing the typical pattern of CD4high T cells and CD4low monocytes on the y axis versus CD8high T cells and CD8low NK cells on the x axis. Third-color staining with CD3-PE-Cy5 confirmed the phenotype of the T cells (data not shown). (b) CD45 expression on freshly isolated cord blood. Many of the cells express the CD45RA isoform, which is typical of naive lymphocytes; only a few cells express the CD45RO isoform, present on memory cells. A considerable number of cells do not express either isoform of CD45. (c) There is a slight increase in the proportion of fetal cord blood cells expressing CD45RO after 7 days of culture, indicating that some cells have become activated and developed a memory phenotype. (d) There is a greater increase in the proportion of cells expressing CD45RO 7 days after EBV challenge of fetal cord blood, indicating that many cells have become activated and developed a memory phenotype.
FIG. 4.
FIG. 4.
(a through c) Expression of CD4 and CD69 (a), CD8 and CD69 (b), or CD16 and CD69 (c) on fetal cord blood cells after 7 days in culture with FCS alone. (d through f) Expression of CD4 and CD69 (d), CD8 and CD69 (e), or CD16 and CD69 (f) on fetal cord blood cells 7 days after challenge with EBV.
FIG. 5.
FIG. 5.
Significant negative correlation between the transformation titer of EBV obtained with each cord blood sample and the percentage of CD16+ cells present in cultures of the same cord blood sample 7 days after virus challenge.
FIG. 6.
FIG. 6.
Cytotoxic activities in 100-μl aliquots of cells taken from cord blood cultures 7 days post-virus challenge. The percentage of CD16+ NK cells in each culture is given. All cultures had some cytotoxicity for the NK K562 target cells. The cultures with high percentages of CD16+ cells had the highest NK activities; they were also able to kill the Burkitt's lymphoma cell line Daudi, which is a target for lymphokine-activated killer cells, as well as autologous LCL.
FIG. 7.
FIG. 7.
Comparison of cytotoxic activities in three samples of cord blood before virus challenge and on day 7 after virus challenge. Open rectangles, NK activity of fresh cord blood against the MHC class I-negative cell line 221; filled rectangles, NK activity of the cells against 221 target cells on day 7; open triangles, cytotoxic activity of fresh cord blood against autologous LCL; filled triangles, cytotoxic activity of the same sample against autologous LCL 7 days after virus challenge. (a) Cells from a cord blood sample with a low virus transformation titer of <101; (b) cells from a sample with an intermediate titer of 103.9; (c) cells from a cord blood sample with a high titer of 105.9. All of the resting samples had NK activity but showed no significant lysis of autologous LCL. After 7 days of culture, there was an increase in NK activity in all the samples; this was greatest for samples a and b, with a low or intermediate virus titer, respectively. This increase in NK activity was accompanied by the development of cytotoxicity toward autologous LCL, which was most marked in the samples with lower virus titers.
FIG. 8.
FIG. 8.
Phenotypes of NK cells in resting fetal cord blood and cultured fetal cord blood 7 days post-virus challenge. (a) Dual staining of fresh fetal cord blood showing two populations of CD16+ and CD56+ cells. The CD56+ CD16low population was shown to be large and more granular, consistent with their being NK cells. CD16 was also found on CD14+ monocytes (data not shown). (b) Seven days after virus challenge, there was a mixed population of CD16+ and CD56+ cells. CD14+ monocytes were absent from these cultures (data not shown). (c through e) The CD16+cells also expressed low levels of CD8 (c) but were CD3− (d) and CD4− (e). (f) The CD8low cells did not express CD3, but the CD8high cells were CD3+ T cells. The data indicate that the CD16+ cells present in day-7 cultures have an NK cell phenotype.
FIG. 9.
FIG. 9.
Cytotoxic activities of CD16+ and CD4+ populations purified by FACS. (a) Cytotoxic activity of CD16+ and CD4+ cell populations derived from cultures of fetal cord blood 7 days after virus challenge and FACS purification. The MHC class I-negative 221 target cells were killed most effectively by the CD16+ population. They were only slightly affected by purified CD4+ T cells. (b) In contrast, autologous LCL were killed both by CD16+ and by CD4+ populations of effector cells.
FIG. 10.
FIG. 10.
Flow cytometry of intracellular cytokines in T-cell blasts derived from fetal cord blood. Cells were stained with anti-CD4-FITC and anti-CD3-Cy5 plus control IgG-PE or anti-cytokine-PE. Plots show the gated CD4+ CD3+ population. (a) Cord blood cells were stimulated with PHA for 3 days and then washed and cultured in fresh medium with IL-2 until day 7, at which time they were restimulated with PMA and ionomycin for 6 h in the presence of monensin prior to intracellular staining. (b) Cord blood cells cultured for 7 days after infection with EBV. Prior to staining, cells were restimulated with PMA and ionomycin in the presence of monensin for 6 h. The results show that PHA blasts retained a naive phenotype and produced only IL-2 upon restimulation. In contrast, EBV-stimulated blasts produced both IL-2 and IFN-γ, but not IL-4.
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