IL-2 Complex Research Articles

Revisiting the role of IL‐2 in autoimmunity

IL-2 was discovered as a T-cell growth factor that promoted T-cell-dependent immune responses; however, more recent studies suggest that the essential role of IL-2 is to maintain functional Treg and thus control immune responses. These results are leading to new ideas about the potential of IL-2 as a therapeutic strategy in autoimmune diseases. In this issue of the European Journal of Immunology, a study further examines the role of IL-2 in immune regulation and shows for the first time that IL-2 complexes can ameliorate autoantibody-mediated autoimmunity. This commentary examines the current findings in relation to what we already know about IL-2 complexes.

Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC Binding Epitope

The cytokine interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Ralpha) and the common gamma-chain (gammaC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Ralpha, whereas the interaction between IL-21 and gammaC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling gammaC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues, which compose the gammaC binding epitope using homology modeling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants, which have significantly impaired gammaC affinity with undiminished IL-21Ralpha affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists.

IL-2/ IL-2 mAb treatment generates prolonged expansion and increased cytokine production by NKT cells (50.42)

Abstract Regulatory T cells can be dramatically boosted in vivo by stimulation with a complex of IL-2 and an IL-2 mAb (JES6-1). The swift 15-fold expansion creates a tolerogenic environment that significantly perturbs autoimmunity and allograft rejection. We recently observed that IL-2/mAb treatment also expands Natural Killer T (NKT) cells, a glycolipid-specific, CD1d-restricted T cell lineage implicated in both regulatory and immunogenic responses. Interestingly, the phenotype of the IL-2/mAb-expanded NKT cells differed from those expanded with the prototypic NKT ligand, alpha-galactosylceramide (α−GalCer). The IL-2/mAb-expanded NKT cells were relatively stable and contracted slowly. They were hyper-functional, with the capacity to rapidly produce cytokines for several days after stimulation. In comparison, α−GalCer stimulated NKT cells characteristically produce a quick burst of Th1 and Th2 cytokines, an ability that then dramatically declines along with their number in the following days. Interestingly, the range of cytokines produced by IL-2/mAb-NKT cells was broad and included not only IFNγ and IL-4, but also significantly high amounts of IL-10 and IL-13. IL-17, however, was notably reduced. These variations in cytokine production appeared to alter the phenotype of dendritic cells in their vicinity. These data suggest an alternate approach to the expansion of NKT cells, specifically within the context of a regulatory milieu that may be appropriate for tolerance induction.

Facilitating Engraftment After MHC-Matched, Allogeneic BMT by IL-2 / Anti IL-2 Complex Treatment Requires Targeting CD25 On, and Activation in Situ of, Residual CD4 Tregs.

Abstract 66We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. <1%). These findings demonstrate that IAC effects require host CD25+ Treg cells and we propose that facilitation of engraftment by this strategy was a direct result of Treg cell activation and expansion following engagement of IAC with CD25. To assess Treg activation, we examined these cells in our standard BALB.B à B6 HSCT model. Tregs isolated 7 days post-HCT from IAC-treated but not untreated recipients expressed readily demonstrable levels of pStat-5a expression (∼2X increase in IAC-treated vs. PBS controls). Moreover, culture of the former Tregs in the presence of rmIL-2 illustrated their heightened sensitivity to activation by this cytokine as virtually all Tregs from IAC-treated animals exhibited high levels of pStat-5a expression (3.2 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.6) compared to control mice (0.4 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.5). Following this activation, increased numbers of host CD4+ FoxP3+ Tregs were readily identified in the PB and spleen of IAC vs. PBS-treated recipients. We next directly examined functional capacity of residual host Tregs exposed to IAC post-5.5Gy TBI and-transplant. Eight days post-HSCT, host Tregs (0 – 25 × 103/well) were isolated and highly enriched populations assessed for suppression of TCONV in anti-CD3 activation assays. Tregs from IAC-treated recipients efficiently mediated suppression at least equivalent to that by normal, untreated Tregs in these assays. Additionally, these IAC treated residual B6 Tregs also effectively inhibited allogeneic MLR responses by B6 CD4+ CD25− responder cells. Therefore, the capacity of host Tregs to respond to IAC activation signals and suppress T cell activation remained intact following conditioning and HSCT. We conclude that host Treg cell activation/expansion is central to the suppression of host effector cell responses to donor hematopoietic antigens leading to the inhibition of HVG following IAC administration resulting in enhanced engraftment. Disclosures:No relevant conflicts of interest to declare.

IL-2 + Anti-IL2 Complex in Situ Stimulation of Host Tregs Combined with Absence of Donor B7.1 / B7.2: A Novel Approach to Facilitate Chimerism in RIC MHC-Matched Miha-Mismatched BMT Recipients.

Abstract 2441Poster Board II-418Reducing and ultimately eliminating conditioning while enabling donor cell engraftment remains an elusive goal of allogeneic hematopoietic stem cell transplantation. The control of host resistance (HVG) to donor allogeneic transplantation antigens is pivotal for engraftment and the successful induction and maintenance of immune tolerance. Using a well characterized minor histocompatibility antigen (MiHA) matched allogeneic (HSCT) model, we have demonstrated a requirement for CD80/CD86 expression on donor APC in order to elicit resistance under reduced intensity conditioning (RIC) of 5.5Gy TBI. Based on this observation, we hypothesize that expansion of host Treg cells which may engage donor APC – the site of host anti-donor Tconv activation - is an advantageous strategy to effectively inhibit HVG responses. To address this hypothesis, we reduced TBI almost 30% to 4.0Gy, and found that even recipients transplanted with 4×106 CD80-/-CD86-/- T cell depleted donor BMC failed to engraft. Experiments were therefore initiated to target and rapidly expand host Tregs by administering IL-2 + anti-IL2 (IAC) complex to further strengthen suppression of HVG. C3H.SW (H2b) recipient mice were conditioned with 4.0Gy TBI (D-1) and B6-CD80-/-CD86-/- TCD-BM (H2b, 7×106) infused on the following day (D0). IAC was introduced on the day of conditioning (D-1) and then daily on days D 0, 1 and 2. We reported that anti-IL-2 + IL-2 complex can expand residual host Treg cells in RIC recipients (Blood, 113:733-743, 2009; Biol. Blood Marrow Transplantation. 15: 785-794, 2009). One month later, 1/3 of mice receiving complex – but not untreated controls demonstrated significant levels (15% B220+) of hematopoietic chimerism. These results, while encouraging, indicated that stimulation of the endogenous Treg compartment alone in 4.0 Gy recipients was not sufficient to regulate the HVG response. We then modified our approach by infusing 1×106 recipient Tregs obtained from normal C3H.SW mice one day following 4.0Gy RIC, i.e. at D0 and began expanding these cells with IAC injected early on D0 and again on D1 prior to the B6-CD80-/-CD86-/- TCD-BM graft – delayed one day and administered on D1. 100% of these recipients (n=3/gr) exhibited high donor chimerism (43.7+/-10.8 SD) 6 weeks post-HSCT, whereas all non-Treg infused control recipients rejected these marrow allografts. Results of 4 independent transplant experiments demonstrated 10/12 recipients contained similar levels of B220+ chimerism (mean donor: 53.9%) and significant T cell chimerism (mean donor: CD4, 19.8%; CD8, 35%). When both host Tregs and IAC were employed, strong chimerism was observed using 4 or 7×106 CD80-/-86-/- TCD-BM grafts. Notably, addition of 1×106 donor (i.e. B6-wt) Tregs and IAC treatment did not augment chimerism vs. IAC only in 4.0Gy conditioned C3H.SW recipients. These results suggest that the introduction of host Tregs 2 days prior to the BM graft allowed adequate time for IAC to mediate Treg expansion in the lymphopenic environment to generate sufficient Treg levels and together with the absence of donor APC B7 signaling to host Tconv cells overcome, i.e. suppressed HVG responses. Transplants were then performed using the identical Treg and IAC protocol with B6-wt TCD-BM which is readily rejected in 4.0 Gy TBI conditioned recipients. Despite the presence of B71/2 on B6-wt donor APC, one-third of the recipients receiving an infusion of host Tregs and IAC treatment exhibited chimerism, although lower (6.3%) than observed with B7.1/2 deficient donors. Transplants of wild type TCD-BM into MiHA mismatched recipients will determine how anti-B7.1/2 mab blockade, together with rapid in situ expansion of infused host Tregs post-TBI and pre-HSCT can be utilized to further diminish immunosuppressive TBI conditioning (<4.0 Gy) regimens. Disclosures:No relevant conflicts of interest to declare.

In Vivo Expansion of Activated Naive CD8+ T Cells and NK Cells Driven by Complexes of IL-2 and Anti-IL-2 Monoclonal Antibody As Novel Approach of Cancer Immunotherapy

IL-2 is potent imunostimulatory molecule that plays a key role in T and NK cell activation and expansion. IL-2 is approved by the FDA to treat metastatic renal cancer and melanoma, but its extremely short half-life and serious toxicities are significant limitations of its use. It was reported that in vivo biological activity of IL-2 can be increased by association of IL-2 with anti-IL-2 mAb (S4B6). IL-2/S4B6 mAb immunocomplexes were described to be highly stimulatory for NK and memory CD8(+) T cells and intermediately also for regulatory T cells. IL-2/JES6-1 mAb immunocomplexes are stimulatory solely for regulatory T cells. In this study we show that although both mentioned IL-2 immunocomplexes are less potent than free IL-2 in vitro, they possess extremely high stimulatory activity to expand activated naive CD8(+) T cells in vivo. IL-2 immunocomplexes expand activated naive CD8(+) T cells several hundred-fold times after four doses and more than 1000-fold times after six doses (1.5 microg/dose of IL-2), whereas free IL-2 given at the same dosage shows negligible activity. IL-2/S4B6 mAb immunocomplexes also induce massive expansion of NK cells (40% of DX5(+)NK1.1(+) cells in spleen). Importantly, activated naive CD8(+) T cells expanded by IL-2 immunocomplexes form robust population of functional memory cells. We also demonstrate in two distinct tumor models that IL-2/S4B6 mAb immunocomplexes possess considerable antitumor activity. Finally, by using radioactively labeled IL-2, we provide for first time direct evidence that IL-2 immunocomplexes have much longer half-life in circulation than free IL-2, being approximately 3 h vs <15 min, respectively.

In Situ Activation and Expansion of Host Tregs: A New Approach to Enhance Donor Chimerism and Stable Engraftment in Major Histocompatibility Complex-Matched Allogeneic Hematopoietic Cell Transplantation

Host antidonor effector T cells represent a major barrier to the successful engraftment of allogeneic donor hematopoietic progenitor and stem cells. Here, administration of a complex of IL-2 and anti-IL 2 antibodies (IAC) significantly enhanced donor chimerism early as well as long-term engraftment following reduced-intensity conditioning (RIC) and allogeneic major histocompatibility complex (MHC)-matched hematopoietic cell transplantion (HCT). Timing of administration of this complex was crucial: administration of IAC post-HCT more efficiently facilitated marrow engraftment than pre-HCT treatment. Donor chimerism persisted to >6 months post-HCT. Importantly, this approach clearly suppressed the emergence of host antidonor CD8 T cells 2 to 3 weeks post-HCT as assessed by tetramer staining. Following in vivo reactivation of IAC-treated and control recipients at >5 months post-HCT with donor antigen, only PBS-treated control marrow allograft recipients responded with tetramer-binding CD8 cells. In total, the present findings support the notion that the transient activation and expansion of host Tregs in situ post-HCT can be explored as a new approach to regulate host alloreactivity posttransplant. Interestingly, direct stimulation of recipient Treg cells in RIC recipients obviated a requirement for exogenous Treg cell transfusion in this model and may represent a viable alternative to, and/or complement the adaptive transfer of Treg populations in clinical HCT.

Insight into Mechanism of IL-2-Induced Toxicity Provides Rationale for Improved Treatment Strategy using IL-2/mAb Complexes (38.8)

Abstract IL-2 immunotherapy is used against certain metastatic cancers and for patients infected with human immunodeficiency virus. However, administration of IL-2 can lead to serious side effects including pulmonary edema, the pathogenesis of which is ill-defined. Here, we provide data showing that IL-2 may bind directly to CD25 on lung endothelial cells and thus cause toxicity and pulmonary edema. Thus, lung endothelial cells express CD25 under steady-state conditions, and expression levels of CD25 on these cells increase in vivo upon injection of mice with IL-2. Activation of CD25+ lung endothelial cells and of IL-2 receptor-positive NK cells and T cells leads to pulmonary edema upon IL-2 treatment. Significantly, co-administration of anti-CD25 antibody along with IL-2 treatment is able to completely abrogate IL-2-induced pulmonary edema despite considerable proliferation of NK cells and T cells. Similarly, knocking out CD25 on non-immune cells or obscuring the CD25-binding epitope of IL-2 by the use of selective immune complexes of IL-2 plus anti-IL-2 antibody (IL-2/mAb) is able to prevent IL-2-induced pulmonary edema. Notably, IL-2-mediated toxic effects are also evident on a human lung cell line and can be abrogated using the anti-CD25 antibody daclizumab or IL-2/mAb complexes. Moreover, IL-2/mAb complexes are very efficient in generating anti-tumor responses in vivo against syngeneic local or metastatic tumor nodules. Thus, CD25+ endothelial cells play a crucial role in IL-2-mediated vascular leak syndrome and this can be circumvented by the use of IL-2/mAb complexes.

Suppression of Murine Allergic Airway Disease by IL-2:Anti-IL-2 Monoclonal Antibody-Induced Regulatory T Cells

Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10(+)CD4(+)CD25(+) and Foxp3(+)CD4(+)CD25(+)) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10(+/+), but not IL-10(-/-) Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.

IL-2/Anti-IL-2 Antibody Complex Enhances Vaccine-Mediated Antigen-Specific CD8+ T Cell Responses and Increases the Ratio of Effector/Memory CD8+ T Cells to Regulatory T Cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.

Rapid generation of a functional NK-cell compartment

AbstractBone marrow transplants are an important therapeutic tool for treating certain types of cancer as well as genetic diseases affecting the hematopoietic system. Until the transferred stem cells differentiate and reconstitute the immune system, recipients are at increased risk from opportunistic infections. We report the rapid generation of a functional natural killer (NK) compartment in lethally irradiated mice that received bone marrow cells from a syngeneic donor by treatment with IL-2/anti–IL-2 antibody complexes. We demonstrate that IL-2 complexes specifically expand the donor but not the host NK population and discuss the implications of this finding in the context of graft-versus-host disease and tumor relapse. Finally, we show that NK cells rapidly generated by IL-2 complexes kill MHC class I–deficient cells effectively in vivo. These data underline the unique therapeutic potential of IL-2 complexes.

The Existence of Multiple Conformers of Interleukin-21 Directs Engineering of a Superpotent Analogue

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, alpha-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

Naive CD8+ T cells differentiate into protective memory-like cells after IL-2–anti–IL-2 complex treatment in vivo

An optimal CD8+ T cell response requires signals from the T cell receptor (TCR), co-stimulatory molecules, and cytokines. In most cases, the relative contribution of these signals to CD8+ T cell proliferation, accumulation, effector function, and differentiation to memory is unknown. Recent work (Boyman, O., M. Kovar, M.P. Rubinstein, C.D. Surh, and J. Sprent. 2006. Science. 311:1924–1927; Kamimura, D., Y. Sawa, M. Sato, E. Agung, T. Hirano, and M. Murakami. 2006. J. Immunol. 177:306–314) has shown that anti–interleukin (IL) 2 monoclonal antibodies that are neutralizing in vitro enhance the potency of IL-2 in vivo. We investigated the role of IL-2 signals in driving CD8+ T cell proliferation in the absence of TCR stimulation by foreign antigen. IL-2 signals induced rapid activation of signal transducer and activator of transcription 5 in all CD8+ T cells, both naive and memory phenotype, and promoted the differentiation of naive CD8+ T cells into effector cells. IL-2–anti–IL-2 complexes induced proliferation of naive CD8+ T cells in an environment with limited access to self–major histocompatibility complex (MHC) and when competition for self-MHC ligands was severe. After transfer into wild-type animals, IL-2–activated CD8+ T cells attained and maintained a central memory phenotype and protected against lethal bacterial infection. IL-2–anti–IL-2 complex–driven memory-like CD8+ T cells had incomplete cellular fitness compared with antigen-driven memory cells regarding homeostatic turnover and cytokine production. These results suggest that intense IL-2 signals, with limited contribution from the TCR, program the differentiation of protective memory-like CD8+ cells but are insufficient to guarantee overall cellular fitness.

Rapid Generation of a Functional NK Cell Compartment (95.12)

Abstract Anti-IL-2 antibodies have been used with the premise that they block IL-2 signaling in vitro and in vivo, but recent findings demonstrate that anti-IL-2 antibodies form a complex with IL-2 in vivo that actually enhances IL-2 activity. NK cells are one cell type affected by these IL-2 complexes. We asked at what stage IL-2 complexes act on NK cells and whether they could be utilized to generate functional NK cells with potential therapeutic value. We found that the administration of IL-2 complexes resulted in the rapid generation of NK cells in IL-15−/− animals. IL-15 is required for the development and survival of NK cells. Mature, transferred NK cells survived in IL-15−/− animals treated with IL-2 complexes, but proliferated only modestly. These data suggest that the IL-2 complex preferentially expands an immature subset of NK cells. To determine whether this expansion results in the generation of functional NK cells, we treated lethally irradiated, bone marrow reconstituted mice with IL-2 complexes and assessed the activity of NK cells by an in vivo killing assay. Bone marrow chimeras that were treated with IL-2 complexes displayed greatly enhanced NK lytic activity compared to control chimeras 4 days after initiation of treatment. We conclude that IL-2 complex has the potential to rapidly generate functional NK cells that may reduce the risk of viral infections and influence GVHD processes.

Potential use of IL-2/anti-IL-2 antibody immune complexes for the treatment of cancer and autoimmune disease

Initially discovered as a potent T cell proliferation factor, IL-2 was soon used for cancer immunotherapy, especially for metastatic melanoma and renal cell carcinoma; however, the severe side effects of IL-2 therapy, plus the negative role of IL-2 in maintaining of CD4+ CD25+ T regulatory cells (Tregs), has somewhat dampened enthusiasm for using IL-2 in immunotherapy. This opinion article discusses the possibility of combining IL-2 with certain anti-IL-2 antibodies for reducing the dose of IL-2 needed and preferentially stimulating effector T cells, but not Tregs, an approach that might provide an improved strategy for anticancer immunotherapy. Alternatively, complexes of IL-2 with other anti-IL-2 antibodies can selectively stimulate Tregs and could, therefore, be useful for treating autoimmune diseases.

Compensatory Energetic Mechanisms Mediating the Assembly of Signaling Complexes Between Interleukin-2 and its α, β, and γ c Receptors

Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the α, β and the “common γ” (γ c) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor–ligand, and receptor–receptor interactions that are necessary for formation of the high-affinity IL-2/αβγ c signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its α receptor, a moderately entropically favorable low affinity interaction between IL-2 and its β receptor, and no interaction between IL-2 and the shared receptor, γ c. Formation of the stable intermediate trimolecular complexes of IL-2 with α and β receptors, as well as IL-2 with β and γ c receptors proceeds through enthalpy–entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor α and β chains, suggesting that a preformed αβ complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Rαβγ c high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by γ c contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.

Compensatory Energetic Mechanisms Mediating the Assembly of Signaling Complexes Between Interleukin-2 and its $alpha;, $beta;, and $gamma;c Receptors

Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the α, β and the “common γ” (γc) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor–ligand, and receptor–receptor interactions that are necessary for formation of the high-affinity IL-2/αβγc signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its α receptor, a moderately entropically favorable low affinity interaction between IL-2 and its β receptor, and no interaction between IL-2 and the shared receptor, γc. Formation of the stable intermediate trimolecular complexes of IL-2 with α and β receptors, as well as IL-2 with β and γc receptors proceeds through enthalpy–entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor α and β chains, suggesting that a preformed αβ complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Rαβγc high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by γc contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.

IL-2 gene knockout affects T lymphocyte trafficking and the microglial response to regenerating facial motor neurons

Following facial nerve axotomy in mice, T cells cross the intact blood–brain barrier (BBB), home to nerve cell bodies in the facial motor nucleus (FMN), and augment neuroregenerative processes. The pivotal T cell immunoregulatory cytokine, IL-2, appears to have bidirectional effects on neuronal and microglial cell function, suggesting rival hypotheses that IL-2 could either enhance or disrupt processes associated with regeneration of axotomized facial motor neurons. We tested these competing hypotheses by comparing the effect of facial nerve axotomy on C57BL/6-IL-2 −/− knockout and C57BL/6-IL-2 +/+ wild-type littermates. Since IL-2 may also be produced endogenously in the brain, we also sought to determine whether differences between the knockout and wild-type mice were attributable to loss of IL-2 gene expression in the CNS, loss of peripheral sources of IL-2 and the associated effects on T cell function, or a combination of these factors. To address this question, we bred novel congenic mice with the SCID mutation (mice lacking T cell derived IL-2) that were homozygous for either the IL-2 knockout or wild-type gene alleles (C57BL/6 scid-IL-2 −/− and C57BL/6 scid-IL-2 +/+ littermates, respectively). Groups were assessed for differences in (1) T lymphocytes entering the axotomized FMN; (2) perineuronal CD11b + microglial phagocytic clusters, a measure of motor neuron death; and (3) activated microglial cells as measured by MHC-II positivity. C57BL/6-IL-2 −/− knockout mice had significantly higher numbers of T cells and lower numbers of activated MHC-II-positive microglial cells in the regenerating FMN than wild-type littermates, although the number of CD11b + phagocytic microglia clusters did not differ. Thus, despite the significant impairment of T cell function known to be associated with loss of peripheral IL-2, the increased number of T cells entering the axotomized FMN appears to have sufficient activity to support neuroregenerative processes. Congenic C57BL/6 scid-IL-2 −/− knockout mice had lower numbers of CD11b + microglial phagocytic clusters than congenic C57BL/6 scid-IL-2 +/+ wild-type littermates, suggesting that loss of the IL-2 gene in the CNS (and possibly the loss of other unknown sources of the gene) enhanced neuronal regeneration. Further study of IL-2's complex actions in neuronal injury may provide greater understanding of key variables that determine whether or not immunological processes in the brain are proregenerative.

Changes in Immunologic Cell Surface Markers during Cocaine Withdrawal in Pregnant Women

To evaluate the effects of acute cocaine withdrawal on the immune system of pregnant women, we analyzed changes in a panel of cell surface markers and plasma proteins that have immunological importance. The cell surface markers included complement receptors [CR1 (CD35), CR2 (CD21), CR3 (CD11b, CD18)], immunoglobulin Fc receptors [FcγRII (CD32), FcγRIII (CD16)], proteins important for lymphocyte function [CD3, CD4, CD8, CD19, CD25, CD45RA], and the framework antigen HLA-ABC. We also measured levels of the plasma proteins C3, C4, IgG, IgM, and IgA, along with the cytokine interleukin-2, soluble lymphocyte markers sCD25, sCD4, sCD8, IL-2, and soluble immune complexes. While no significant changes were seen in the levels of plasma proteins, changes paralleling the course of acute withdrawal were seen in complement receptors and immunoglobulin Fc receptors on leukocyte subpopulations. By contrast, proteins important for lymphocyte function were relatively unperturbed. There was an increase in receptor expression at the onset of withdrawal that peaked 3–5 days after last cocaine use, followed by a decrease in expression to initial (pre-withdrawal) levels. These changes in cell surface receptors may reflect altered immune function in the women who were withdrawing from cocaine.

Role of α chain - IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor

Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta chains in the dynamics of the formation of high-affinity IL-2 binding in T cells.