Structural reorganization of the interleukin-7 signaling complex

We report here an unliganded receptor structure in the common gamma-chain (γ_c) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an “X” geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γ_c/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4⁺ T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα–γ_c heterodimer to the active IL-7–IL-7Rα–γ_c ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γ_c from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7– and γ_c-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γ_c receptors that form inactive homodimers and heterodimers independent of their cytokines.     

HIV-infected long-term nonprogressors display a unique correlative pattern between the interleukin-7/interleukin-7 receptor circuit and T-cell homeostasis

Background

We hypothesized that there may be a correlation between the interleukin‐7 (IL‐7)/IL‐7 receptor (IL‐7R) regulatory system and parameters of T‐cell homeostasis in HIV‐infected long‐term nonprogressors (LTNPs) as compared with patients with disease progression.

Methods

The possibility of a correlation between T‐cell homeostatic parameters and IL‐7/IL‐7R was investigated in 22 LTNPs (CD4 count ≥500 cells/μL for >10 years) vs. HIV‐positive patients at different disease stages [12 early: CD4 count ≥400 cells/μL; 15 late (AIDS‐presenters): CD4 count ≤150 cells/μL].

Results

Compared with early‐stage HIV‐positive patients, LTNPs displayed a higher circulating IL‐7 concentration (P=0.05), which was positively associated with higher IL‐7Rα expression and a higher T‐cell receptor excision circle (TREC) content specifically within CD4 cells (P<0.05). Compared with late‐stage disease patients, early‐stage disease patients displayed a lower IL‐7 concentration (P<0.01) and higher percentages of IL‐7Rα⁺ CD4 and CD8 cells (P=0.05). IL‐7 was positively correlated with the percentage of TREC⁺ CD4 cells (P<0.01), which translated into a higher percentage of naïve CD4 cells in early‐stage disease patients than in late‐stage disease patients; however, the CD4 cells in early‐stage disease patients were less enriched in recent thymic emigrants (RTEs) compared with LTNPs (P<0.05). In late‐stage AIDS‐developing patients, substantially increased IL‐7 was correlated with a decreased percentage of IL‐7Rα⁺ CD4 cells (P=0.01), which resulted in these patients having a significantly lower percentage of naïve T cells (P<0.01) and a significantly lower content of TREC (P<0.01) than the other patients.

Conclusions

The maintenance of high CD4 cell counts in LTNPs was associated with a specific IL‐7/IL‐7R pattern characterized by increased IL‐7 and highest IL‐7Rα‐expressing CD4 cells relative to other patients. Compared with patients with late‐stage disease, LTNPs displayed a phenotypically naïve, less activated CD4 cell pool highly enriched in RTEs, suggesting the existence of a compensatory IL‐7‐mediated pathway specifically sustaining peripheral CD4 counts.

     

Abnormal interleukin-7 function in common variable immunodeficiency

Common variable immunodeficiency (CVID) is characterized by low levels of circulating immunoglobulins, leading to frequent infections, particularly of the respiratory tract. Frequently, T-cell abnormalities are observed. Interleukin-7 (IL-7) is involved in the homeostasis of lymphocytes, and may be elevated in lymphopenia. Mutations of genes related to IL-7 may lead to severe immunodeficiency disorders. We report elevated plasma levels of circulating IL-7 in a subgroup of CVID. These patients have increased numbers of circulating CD8+ T cells with decreased apoptosis and a predominance of CC chemokine receptor 7- (CCR7-) effector-memory T cells. Moreover, in some of these patients there is impaired response to IL-7 as assessed by in vitro proliferation and secretion of interferon gamma and transforming growth factor beta. These findings suggest novel pathogenic mechanisms and specific targets for further research in CVID.     

Effects of exogenous interleukin-7 on human thymus function

Immune reconstitution is a critical component of recovery after treatment of human immunodeficiency virus (HIV) infection, cancer chemotherapy, and hematopoietic stem cell transplantation. The ability to enhance T-cell production would benefit such treatment. We examined the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain reaction demonstrated that the highest level of TRECs (14 692 copies/10 000 cells) was present in the CD1a(+)CD3(-)CD4(+)CD8(+) stage in native thymus, suggesting that TREC generation occurred following the cellular division in this subpopulation. In a thymic organ culture system, exogenous IL-7 increased the TREC frequency in fetal as well as infant thymus, indicating increased T-cell receptor (TCR) rearrangement. Although this increase could be due to the effect of IL-7 to increase thymocyte proliferation and decrease apoptosis of immature CD3(-) cells, the in vivo experiments using NOD/LtSz-scid mice given transplants of human fetal thymus and liver suggested that IL-7 can also directly enhance TREC generation. Our results provide compelling evidence that IL-7 has a direct effect on increasing TCR-alphabeta rearrangement and indicate the potential use of IL-7 for enhancing de novo na?ve T-cell generation in immunocompromised patients.     

Direct interaction between Kit and the interleukin-7 receptor

In vivo analyses of thymopoiesis in mice defective in signaling through Kit and c or Kit and IL-7R demonstrate synergy and partial complementation of c or IL-7–mediated signaling by the Kit signaling pathway. Our molecular analysis in T-lymphoid cells as well as in nonhematopoietic cells shows that Kit and IL-7R signaling pathways directly interact. KL-mediated activation of Kit induced strong tyrosine phosphorylation of c and IL-7R in the absence of IL-7. Activated Kit formed a complex with either IL-7R or c, and tyrosine phosphorylation of both subunits occurred independently of Jak3, suggesting that c and IL-7R are each direct substrates of Kit. Kit activated Jak3 in an IL-7R–dependent manner. Moreover, deficient Stat5 activation of the Kit mutant YY567/569FF lacking intrinsic Src activation capacity was partially reconstituted in the presence of IL-7R and Jak3. Based on the molecular data, we propose a model of Kit-mediated functional activation of c-containing receptors such as IL-7R, similar to the interaction between Kit and Epo-R. Such indirect activation of the Jak-Stat pathway induced by the interaction between an RTK and type I cytokine receptor could be the underlying mechanism for a context-specific signaling repertoire of a pleiotropic RTK-like Kit.     

Murine thymocytes proliferate in direct response to interleukin-7

The ability of interleukin-7 (IL-7) to stimulate murine thymocyte proliferation was investigated. IL-7, either alone or in concert with lectin, induced proliferation of adult thymocytes as well as day 13 fetal and adult CD4-/CD8-thymocytes. The IL-7-induced proliferative response of unfractionated thymocytes could not be inhibited by antibodies to IL-2, or IL-4, IL-6, or the IL-2 receptor. In addition, IL-2, IL-4, and IL-6 were not produced by thymocytes activated with IL- 7, as judged by the absence of biologically active cytokine in IL-7- stimulated culture supernatants. IL-7 could act in concert with IL-2 and IL-4 or with IL-4 to enhance the proliferative response of thymocyte cultures. Thus, IL-7 may cause proliferation of thymocytes directly, not indirectly, through production of IL-2, IL-4, or IL-6. IL- 7 may then play a significant role in differentiation of T lymphocytes.     

A potential role for interleukin-7 in T-cell homeostasis

Interleukin (IL)-7 is known to up-regulate thymopoietic pathways of T-cell regeneration. Recent work also has shown it to potently enhance thymic-independent peripheral expansion and to restore immunocompetence in athymic T-cell-depleted hosts. We hypothesized that endogenous IL-7 could contribute to the restoration of T-cell homeostasis following T-cell depletion. To analyze this, we evaluated circulating IL-7 levels and lymphocyte subsets in multiple clinical cohorts with T-cell depletion of varying etiologies. In pediatric (n = 41) and adult (n = 51) human immunodeficiency virus-infected CD4-depleted patients, there were strong inverse correlations between IL-7 levels and CD4 counts (r = -0.77, P <.0001, and r = -0.68, P <.0001). Declines in IL-7 were temporally correlated with recovery of CD4 counts. Similar patterns were observed in CD4-depleted patients receiving cancer chemotherapy (r = -0.65, P =.009). Therefore, in 2 disparate clinical scenarios involving CD4 depletion, IL-7 levels dynamically respond to changes in CD4 T-cell number, making this cytokine uniquely suited as a candidate regulator of T-cell homeostasis. Furthermore, in patients with idiopathic CD4 lymphopenia, a much weaker relationship between IL-7 levels and peripheral blood CD4 counts was observed, suggesting that an impaired IL-7 response to CD4 depletion may contribute to the impaired lymphocyte homeostasis observed in this population. In light of the known effects of IL-7 on T-cell regeneration, we postulate that increased availability of IL-7 could play a critical role in restoring T-cell homeostasis following T-cell depletion.     

Priming of T cells to Fas-mediated proliferative signals by interleukin-7

T-cell depletion associated with HIV infection or cytoreductive therapies triggers potential T-cell regenerative mechanisms such as peripheral T-lymphocyte expansion to weak antigenic stimuli and the increased availability of interleukin-7 (IL-7), a cytokine with potent antiapoptotic and proliferative activities. Deleterious mechanisms also associated with lymphopenia, such as increased Fas expression and apoptosis of T cell, however, may result in opposing effects. In this study, we show that Fas molecules, primarily associated with T-cell depletion in lymphopenic settings, may also contribute to compensatory T-cell expansion through transmitting costimulatory signals to suboptimally activated T cells. Proliferation of T lymphocytes in response to concomitant Fas and T-cell receptor (TCR) triggering was shown to be increased in HIV-infected individuals compared with noninfected controls. As IL-7 levels are often elevated in lymphopenic individuals in association with increased Fas expression, we analyzed whether IL-7 would influence Fas-mediated proliferative signals in T cells. We show that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. Thus, high IL-7 levels associated with lymphopenic conditions may simultaneously induce sensitivity to Fas-mediated apoptosis in nonactivated T cells and increase Fas-induced costimulatory signals in T cells recognizing low-affinity antigens.     

Identification of a novel low-affinity receptor for human interleukin-7.

Human recombinant interleukin-7 (IL-7) was labeled with biotin and used to examine IL-7 receptor (IL-7R) expression and regulation on human primary hematopoietic cells, the monocytoid line THP1, and a range of B- and pre-B-celi lines by flow cytometry. A strong intensity of staining was observed using relatively high (greater than 1 x 10(-7) mol/L) concentrations of biotinylated IL-7 on the majority of cell types examined. This reactivity, which could be effectively competed with excess unlabeled IL-7, did not correlate with either mRNA levels for the cloned receptor or with estimates of IL-7R expression determined by [125I]IL-7 binding. Staining of cells with a titration of biotinylated IL-7 showed, at concentrations greater than 1 x 10(-7) mol/L binding with a Ka in the range of 1 x 10(6) mol/L-1, to 1 x 10(7) mol/L-1, an affinity 100 to 1,000 times lower than that reported for the cloned IL-7 receptor. Further data suggesting the existence of a distinct low- affinity IL-7R were provided by two antibodies specific for the cloned IL-7R. Staining with these monoclonal antibodies (MoAbs) correlated with both IL-7R mRNA levels and receptor expression determined by [125I]IL-7 binding, but was not compatible with the distribution of reactivity seen with biotinylated IL-7. Using tritiated biotin to label IL-7, it was estimated that the total number of IL-7 binding sites on the cell lines examined ranged from 1 x 10(4) to at least 5 x 10(5)/cell. Cross-linking studies showed that [125I]IL-7 associated with two major proteins of approximately 62 Kd and 70 Kd on the surface of RPMI 1788 and THP1 cells, in contrast to the 75- to 80 Kd molecule characteristic of the previously cloned receptor, expressed on the surface of Daudi cells. Proliferation of THP1 cells, expressing only the low-affinity form of IL-7R and lacking detectable IL-7R mRNA, could be inhibited by the addition of IL-7 in a concentration-dependent fashion, indicating that, at least on this cell line, binding of IL-7 with a Ka of 1 x 10(6) mol/L-1 to 1 x 10(7) mol/L-1 can transduce a biological signal. Taken together, the data contained in this report demonstrate the existence of a low-affinity IL-7R, expressed in high numbers on hematopoietic cells of different lineages, which is the product of a gene distinct from that encoding the cloned IL-7R.     

Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7

Bone marrow transplantation (BMT) is followed by a period of profound immune deficiency, during which new T lymphocytes are generated from either stem cells or immature thymic progenitors. Interleukin-7 (IL-7) induces proliferation and differentiation of immature thymocytes. We examined whether the in vivo administration of IL-7 to mice receiving BMT would alter thymic reconstitution. Lethally irradiated C57BL/6 mice received syngeneic BMT, followed by either IL-7 or placebo from days 5 to 18 post-BMT. At day 28, BMT recipients that had not received IL-7 had profound thymic hypoplasia (< 5% of normal), with relative increases in the numbers of immature thymocytes, decreased numbers of mature peripheral (splenic) T lymphocytes, and severely impaired T- and B-cell function. In contrast, transplanted mice treated with IL-7 had normalization of thymic cellularity, with normal proportions of thymic subsets and T-cell receptor beta variable gene (TCRV beta) usage, normal numbers of peripheral CD4+ T lymphocytes, and improved antigen- specific T- and B-cell function. In the BMT-IL-7 mice, there was an eightfold increase in the number of immature CD3-CD4-CD8- thymocytes in G2-M of the cell cycle, indicating that restoration of thymic cellularity was due to enhanced proliferation of immature thymic progenitors. Similar effects following IL-7 administration were also observed when donor bone marrow was depleted of mature T lymphocytes, indicating that IL-7 administration affected immature hematopoietic progenitors. IL-7 promotes thymic reconstitution after BMT, and may be useful in preventing post-BMT immune deficiency.     

Growth inhibitory and agonistic signals of interleukin-7 (IL-7) can be mediated through the CDw127 IL-7 receptor

The present study was aimed at identifying surface-membrane molecules involved in the regulation of human B-cell ontogeny. For this purpose, murine monoclonal antibodies (MoAbs) were generated against Pre-Alp, a pre-B acute lymphoblastic leukemia (ALL) cell line, and MoAb R34.34 was selected for further characterization. R34.34 recognized a molecule expressed on normal B-cell precursors (BCP) but not on mature B cells. The antibody also reacted with T lymphocytes, a subpopulation of monocytes from peripheral blood, and a subset of CD34+ cells. Immunoprecipitation analysis indicated that R34.34 recognizes an 80-kD molecular weight antigen. Antibody R34.34 was further found to be directed against an epitope interfering with binding of interleukin-7 (IL-7) to Pre-Alp cells. Expression cloning from a Pre-Alp cDNA library showed that R34.34 antigen is CDw127, the 75- to 80-kD IL-7 receptor. Proliferation of the B-lineage ALL cell lines Reh and Mieliki was inhibited by IL-7, and this effect was specifically reverted by MoAb R34.34. In addition, antibody R34.34 specifically inhibited IL-7- dependent proliferation of normal BCP, Pre-Alp cells, and peripheral T cells. These results imply that both inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. R34.34 antibody should be important for the analysis of signal transduction through CDw127.