Intracellular Cytokine Profiles of Cord and Adult Blood Lymphocytes

To the Editor:

In a recent issue of Blood, Chalmers et al1communicated their findings of intracellular cytokine profiles of cord and adult blood. Analyzing cytokine synthesis at a single-cell level, they showed that cord blood (CB) lymphocytes produced less interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) than adult peripheral blood (AB) lymphocytes. Further subset analysis indicated that in CB lymphocytes, the majority of cytokine-producing cells were CD4⁺ CD45RA⁺in contrast to AB lymphocyte, which were both CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺. Thus, they concluded that the reduced incidence of graft-versus-host disease (GVHD) in CB stem cell transplantation (CBSCT) could be partly due to the observed altered cytokine profile in CB lymphocytes.

Interestingly, we also recently conducted a similar study assessing intracellular cytokine profiles of purified CB CD4⁺ and CD8⁺ T-cell subsets in comparison with those of healthy older children and adults. We have found and reported IFN-γ production capability to be altered and significantly lower (<5% of total T cells) in CB compared with that of older children and adults.2 However, in contrast, our findings revealed normal and comparably high IL-2 production capability, at a single-cell level, relative to older children and adults with even significantly higher mean cell percentage values of the cytokine-producing cell populations in CB. In a concurrent assessment of CB mononuclear cell population able to produce IFN-γ, we found that a large proportion of natural killer (NK) cells had the ability to produce IFN-γ with virtually no CD4⁺ cells (<1%) being able to do so (Fig1). Thus, the few IFN-γ–producing CB mononuclear cells were almost exclusively not T but largely NK cells. We observed that CB T lymphocytes, as indicated by their almost exclusive expression of CD45RA antigen and sole production of IL-2, were comprised almost exclusively of T helper (Thp) and T cytotoxic (Tcp) precursor cells, explaining in part the observed altered low IFN-γ production. However, when CB mononuclear cells were cultured with anti-CD3 monoclonal antibody and exogenous IL-2, the IFN-γ–producing cells markedly increased, reaching levels comparable to those of AB (Fig 2). This suggests that CB T cells may not, as such, be hyporesponsive in an in vivo alloantigen setting such as during CBSCT. Indeed, recent reports3,4 together with our in vitro studies (manuscript in preparation) suggest that the neonatal T cell, though naive, is immunologically and comparably competent. Thus, factors other than altered cytokine profiles, not yet sought and investigated, may fully explain the reported infrequent GVHD associated with CBSCT.

Finally and understandably, the variation in intracellular IL-2 and IFN-γ values between the report of Chalmers et al and our report, among other factors, could be due to intracellular staining protocol modifications. Indeed, Jason and Larned5 have recently illustrated how slight protocol modification in intracellular cytokine flow cytometry can lead to diverse variations of fluorescence stain results. Chalmers et al used 5 ng/mL phorbol 12-myristate 13-acetate (PMA), whereas we used 50 nmol/L (30.8 ng/mL) to induce cytokine production. It is documented that a high concentration of PMA may be necessary to induce a more profound T-cell response, such as the expression of IL-2 receptor.6 However, despite the use of a high concentration of PMA in our study, CB IFN-γ–producing cells were consistently low in both CD4⁺ and CD8⁺T-cell subsets.