Soluble IL-7 Ralpha ( sCD 127 ) and Measurement of IL-7 in the Plasma of HIV Patients

To the Editor: Interleukin-7 (IL-7) is an essential cytokine that is in constant production by nonhematopoietic cells. One of its roles is to control thymopoiesis and the homeostasis of peripheral T lymphocytes. It also stimulates T-cell immune functions. It acts through a receptor that is composed of 2 chains: IL-7Ralpha or CD127 and gamma c or CD132. IL-7 plasma levels are increased in HIVinfected patients and this would seem to be related to the CD4 lymphopenia that gradually appears during the chronic phase of the disease. IL-7 levels are also elevated in other lymphopenic conditions, for example, chemotherapyinduced lymphopenia, acute lymphocytic leukemia, idiopathic lymphopenia, etc. The precise mechanism leading to this increase in IL-7 is unknown. Recently, sCD127 was detected by Western blotting in human plasma. We have confirmed and extended this finding by means of an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. Plasma samples are incubated in plates coated with goat anti-CD127 antibodies, bound sCD127 is detected by anti-CD127 monoclonal antibody (mAb) 40 131, biotinylated anti-mouse immunoglobulin, and streptavidin horse radish peroxidase (HRP). Recombinant human CD127-Fc chimera protein is used as a standard. All the reagents used in this study were obtained from R & D Systems, Minneapolis, MN. This ELISA technique was used to show that plasma sCD127 is far more abundant (23.6 G 3.5 ng/mL, n = 20) than IL-7, which is found to be in the picogram range. A full characterization of the sCD127 molecules will be reported later. We tested sCD127 interference on IL-7 and here discuss the putative consequences of our findings with regard to the mechanism that increases IL-7 in the plasma of HIV-positive patients and the function of the IL-7/IL-7 receptor system. We first determined the proportion of IL-7 bound to sCD127 in plasma samples taken from healthy individuals. Plasma (n = 6) were adsorbed onto rabbit anti-CD127 polyclonal antibodies coated on agarose beads. Rabbit polyclonal immunoglobulin-coated beads were used as a control. As seen in Figure 1A, most of the sCD127 molecules were specifically removed from the plasma by the antiCD127 beads. An assay was then conducted on the same untreated or adsorbed plasma samples to determine their IL-7 content. A significant fraction of the IL-7 was seen to be bound to sCD127, thus indicating that IL-7 has affinity for sCD127. In view of this result, we tested the putative interference caused by sCD127containing plasma on the assay of IL-7 (Quantikine HS, Human IL-7 & Immunoassay). Before the assay, the IL-7 was diluted either in the control solution provided by the supplier or in this same solution containing 0.5% plasma. As can be seen in Figure 1B, the amount of IL-7 detected was unaffected by the presence of sCD127 derived from the plasma of healthy individuals, even at very low IL-7 concentrations. This suggests that the affinity of the anti–IL-7 antibodies used in the assay is far higher than that of IL-7 for sCD127. More significantly, the results indicate that changes in the quantity of sCD127 in the plasma do not affect the assay of IL-7. We then compared IL-7 affinity for membrane-expressed CD127 with its affinity for plasma sCD127. CD127 is constitutively and highly expressed at the surface of resting CD4 T lymphocytes, whereas gamma c expression is low or undetectable. Most CD127 molecules are therefore free. The study was performed with allophycocyanin-labeled anti-CD4 mAb used to stain CD4 T lymphocytes from peripheral blood mononuclear cell. Two CD127-specific, phycoerythrinlabeled mAb were employed: mAb R 34 34 (Beckman Coulter, Marseille, France), whose binding is inhibited by IL-7, and mAb C 40 131 (see above), which recognizes an epitope independent of the IL-7 binding site. These 2 mAb were assayed at concentrations giving 50% maximal binding on CD4 T lymphocytes in the absence or presence of IL-7. As expected, IL-7 inhibited the binding of mAb R 34 34 measured by the mean fluorescence intensity (MFI) of CD127. Half maximal inhibition was obtained with about IL-7 10 ng/mL (Fig. 1C). By contrast, the binding of mAb C 40 131 was unaffected under the same experimental conditions (Fig. 1C). A similar experiment was then performed using our ELISA. The sCD127 was revealed either by mAb C 40 131, as described above, or mAb R 34 34 (Fig. 1D). Both were used at a concentration that gave 50% maximal response in the ELISA. IL-7 did not inhibit the binding of mAb R 34 34 in these tests, even at doses as high as 1 mg/mL (Fig. 1D). Therefore, under these experimental conditions, IL-7 did not show any detectable affinity for plasma sCD127. It is noteworthy here that the inhibition assays used are suitable for comparing the affinity of membrane and sCD127. Finally, we assayed sCD127 in plasma samples taken from 7 HIVinfected patients (CD4 count: 34 G 8 cells/mm, viral load: 303,061G 42,813 RNA copies/mL). Activation of the immune system in HIV-infected patients leads to decreased expression of membrane CD127. However, we did not find any significant change in the amount of sCD127 in the plasma of HIV patients (21.6 G 4.3 ng/mL). Our results therefore differ from those reported by Vranjkovic et al who found that sCD127 increased in 6 HIV patients. The accuracy of their measurements based on a densitometric analysis of Western blots is debatable. The molecular weight of the molecule detected does not correspond to a soluble form of CD127. Our ELISA, which was specifically designed to quantify sCD127, is based on the detection of a 55 kDa molecule (data not shown). Two main points warrant discussion. (1) The increased levels of plasma IL-7 in HIV-infected patients cannot result from changes in plasma sCD127 because this molecule does not interfere with IL-7 detection by the conventional immunoassays used in many laboratories and do not decrease in the plasma of HIV patients. Our results also indicate that the reduced expression of CD127 seen at the surface of CD8 T lymphocytes in