Administration of interleukin-7 increases CD4 T cells in idiopathic CD4 lymphocytopenia

Abstract

Idiopathic CD4 lymphopenia (ICL) is a rare syndrome defined by low CD4 T-cell counts (<300/µL) without evidence of HIV infection or other known cause of immunodeficiency. ICL confers an increased risk of opportunistic infections and has no established treatment. Interleukin-7 (IL-7) is fundamental for thymopoiesis, T-cell homeostasis, and survival of mature T cells, which provides a rationale for its potential use as an immunotherapeutic agent for ICL. We performed an open-label phase 1/2A dose-escalation trial of 3 subcutaneous doses of recombinant human IL-7 (rhIL-7) per week in patients with ICL who were at risk of disease progression. The primary objectives of the study were to assess safety and the immunomodulatory effects of rhIL-7 in ICL patients. Injection site reactions were the most frequently reported adverse events. One patient experienced a hypersensitivity reaction and developed non-neutralizing anti-IL-7 antibodies. Patients with autoimmune diseases that required systemic therapy at screening were excluded from the study; however, 1 participant developed systemic lupus erythematosus while on study and was excluded from further rhIL-7 dosing. Quantitatively, rhIL-7 led to an increase in the number of circulating CD4 and CD8 T cells and tissue-resident CD3 T cells in the gut mucosa and bone marrow. Functionally, these T cells were capable of producing cytokines after mitogenic stimulation. rhIL-7 was well tolerated at biologically active doses and may represent a promising therapeutic intervention in ICL. This trial was registered at www.clinicaltrials.gov as #NCT00839436.

Figure 1

Graphic overview of study schedule. (*) Six patients were excluded, 5 withdrew, 1 was not given rhIL-7 as a result of a study hold.

Figure 2

Changes in circulating T cells after rhIL-7 treatment. Fold change in (A) CD4 T cells and (C) CD8 T cells in the weeks after rhIL-7 administration. Each patient’s results are depicted by a color according to dose (orange, 3 µg/kg; green, 10 µg/kg; blue, 20 µg/kg) and by a unique symbol. The bold black line represents the median value for all patients. Absolute numbers of (B) CD4 T cells and (D) CD8 T cells in each patient over the first 24 weeks of the study using the same color code and symptoms as for (A). (E) Increase in CD4 T cells at week 5 (peak CD4 T-cell count) on the x-axis compared with week 1 (baseline) CD4 T-cell count on the y-axis. Each symbol represents an individual patient using the same color scheme and symbols as in (A). *P < .05; **P < .01.

Figure 3

Longitudinal CD4 T-cell counts after rhIL-7 treatment. PBMCs were assayed by flow cytometry. The blue lines in panels A-E represent CD4 T cells in the weeks after rhIL-7 initiation. rhIL-7 was administered as a series of injections once per week for 3 weeks. Each dotted vertical orange line represents a dose of rhIL-7. (A) Patient who experienced a hypersensitivity reaction after the sixth rhIL-7 dose at week 26, including the titer of non-neutralizing anti-IL-7 antibodies (Ab). (B-E) Patients received repeated rhIL-7 dosing after week 48 for CD4 T cells that remained at <350/μL.

Figure 4

Mechanism of CD4 T-cell pool expansion. PBMCs were assayed by flow cytometry. (A) Proportions of CD4 and (B) CD8 T cells expressing the proliferation marker Ki-67. The solid blue line represents median values and the dotted blue lines represent the upper and lower IQRs. Data from each of the 9 patients are included at each time point. rhIL-7 was administered as a series of injections once per week for 3 weeks. Each dotted vertical orange line represents a dose of rhIL-7. (C) Fold change in T-cell receptor excision circle (TREC) content. (D) Fold change in CD31 expression in PBMCs in the weeks after initiation of rhIL-7 therapy. Each patient’s results are depicted by color and symbol as in Figure 2.

Figure 5

Phenotypic T-cell changes after rhIL-7 treatment. Proportions of (A) CD4 and (B) CD8 T cells expressing CD127 (IL-7Rα). Mean fluorescence intensity (MFI) for CD127 in (C) CD4 and (D) CD8 T cells. (E) Proportion of Tregs (CD25⁺FoxP3⁺) CD4 T cells. In all five panels, the solid blue line represents median values and the dotted blue lines represent the upper and lower IQRs. Data from each of the 9 patients are included at each time point except for MFI data for CD8 T cells at week 1 for patient 10-002. rhIL-7 was administered as a series of injections once per week for 3 weeks. Each dotted vertical orange line represents a dose of rhIL-7.

Figure 6

Polyfunctionality of CD4 and CD8 T cells after rhIL-7 treatment. CD4 and CD8 T cells from HCs (n = 6) and study patients (n = 5) at weeks 0, 12, and 48 were stimulated with phorbol myristate acetate (PMA) and ionomycin. Stimulated cells were then analyzed by flow cytometry for the production of intracellular cytokines IL-2, tumor necrosis factor α (TNF-α), IL-17α, and interferon gamma (IFN-γ). Pie charts depict median values for HCs or individual values for each study patient at each time point, demonstrating increasing polyfunctionality (ie, increasing numbers of cytokines. The bar graphs adjacent to each pie chart demonstrate the proportion of CD4 T cells producing IL-2, TNF-α, IL-17α, and IFN-γ at the corresponding time point.

Figure 7

Tissue distribution of CD3⁺ T cells after rhIL-7 treatment. Eight study patients and 17 HCs underwent flexible sigmoidoscopy with rectosigmoid biopsy for immunohistochemical analysis. (A) Representative image of CD3⁺ staining of the lamina propria in a study patient before and after rIL-7 treatment. The black arrows indicate areas of positive CD3 staining. (B) Percentage of lamina propria surface area staining positive for CD3 in each of the 8 patients with rectosigmoid biopsies before (week 0) and after (week 24) 3 doses of rhIL-7 treatment as well as the values for the HCs. Each patient’s results are depicted by color and symbol, as in Figure 1. HCs are depicted as black diamonds, and the straight horizontal line shows the median value for the HCs. (C) Seven study patients and 10 HCs underwent bone marrow biopsy for immunohistochemical staining for CD3. (C) Representative image of CD3⁺staining in the bone marrow before and after rIL-7 in a study patient. The black arrows indicate areas of positive CD3 staining. (D) Mean number of CD3⁺ cells per high power field for each of the 7 patients with bone marrow biopsies before and after rhIL-7 treatment. The color scheme and symbols are the same as those used in (B).