Selective Targeting of IL-15Rα Is Sufficient to Reduce Inflammation.

Cytokines are crucial molecules for maintaining the proper functioning of the immune system. Nevertheless, a dysregulation of cytokine expression could be involved in the pathogenesis of autoimmune diseases. Interleukin (IL)-15 is a key factor for natural killer cells (NK) and CD8 T cells homeostasis, necessary to fight cancer and infections but could also be considered as a pro-inflammatory cytokine involved in autoimmune inflammatory disease, including rheumatoid arthritis, psoriasis, along with tumor necrosis factor alpha (TNF-α), IL-6, and IL-1ß. The molecular mechanisms by which IL-15 exerts its inflammatory function in these diseases are still unclear. In this study, we generated an IL-15-derived molecule called NANTIL-15 (New ANTagonist of IL-15), designed to selectively inhibit the action of IL-15 through the high-affinity trimeric IL-15Rα/IL-2Rß/γc receptor while leaving IL-15 signaling through the dimeric IL-2Rß/γc receptor unaffected. Administrating of NANTIL-15 in healthy mice did not affect the IL-15-dependent cell populations such as NK and CD8 T cells. In contrast, we found that NANTIL-15 efficiently reduced signs of inflammation in a collagen-induced arthritis model. These observations demonstrate that the inflammatory properties of IL-15 are linked to its action through the trimeric IL-15Rα/IL-2Rß/γc receptor, highlighting the interest of selectively targeting this receptor.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

IL-15 superagonist RLI has potent immunostimulatory properties on NK cells: implications for antimetastatic treatment.

BACKGROUND: As the immune system is compromised in patients with cancer, therapeutic strategies to stimulate immunity appear promising, to avoid relapse and increase long-term overall survival. Interleukin-15 (IL-15) has similar properties to IL-2, but does not cause activation-induced cell death nor activation and proliferation of regulatory T cells (Treg), which makes it a serious candidate for anticancer immunotherapy. However, IL-15 has a short half-life and high doses are needed to achieve responses. Designed to enhance its activity, receptor-linker-IL-15 (RLI) (SO-C101) is a fusion molecule of human IL-15 covalently linked to the human IL-15Rα sushi+ domain currently assessed in a phase I/Ib clinical trial on patients with advanced/metastatic solid cancer. METHODS: We investigated the antimetastatic activity of RLI in a 4T1 mouse mammary carcinoma that spontaneously metastasizes and evaluated its immunomodulatory role in the metastatic lung microenvironment. We further characterized the proliferation, maturation and cytotoxic functions of natural killer (NK) cells in tumor-free mice treated with RLI. Finally, we explored the effect of RLI on human NK cells from healthy donors and patients with non-small cell lung cancer (NSCLC). RESULTS: RLI treatment displayed antimetastatic properties in the 4T1 mouse model. By characterizing the lung microenvironment, we observed that RLI restored the balance between NK cells and neutrophils (CD11b+ Ly6Ghigh Ly6Clow) that massively infiltrate lungs of 4T1-tumor bearing mice. In addition, the ratio between NK cells and Treg was strongly increased by RLI treatment. Further pharmacodynamic studies in tumor-free mice revealed superior proliferative and cytotoxic functions on NK cells after RLI treatment compared with IL-15 alone. Characterization of the maturation stage of NK cells demonstrated that RLI favored accumulation of CD11b+ CD27high KLRG1+ mature NK cells. Finally, RLI demonstrated potent immunostimulatory properties on human NK cells by inducing proliferation and activation of NK cells from healthy donors and enhancing cytotoxic responses to NKp30 crosslinking in NK cells from patients with NSCLC. CONCLUSIONS: Collectively, our work demonstrates superior activity of RLI compared with rhIL-15 in modulating and activating NK cells and provides additional evidences for a therapeutic strategy using RLI as antimetastatic molecule.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1µg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

IL-15Rα membrane anchorage in either cis or trans is required for stabilization of IL-15 and optimal signaling.

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.

Emergence of NK Cell Hyporesponsiveness after Two IL-15 Stimulation Cycles.

IL-15 is a cytokine playing a crucial role in the function of immune cells, including NK and CD8 T cells. In this study, we demonstrated that in vivo, in mice, IL-15-prestimulated NK cells were no longer able to respond to a second cycle of IL-15 stimulation. This was illustrated by defects in cell maturation, proliferation, and activation, seemingly linked to the environment surrounding NK cells but not related to the presence of CD4 regulatory T cells, TGF-ß, or IL-10. Moreover, NK cells from immunodeficient mice could respond to two cycles of IL-15 stimulation, whereas an adoptive transfer of CD44+CD8+ cells impaired their responsiveness to the second cycle. Conversely, in immunocompetent mice, NK cell responsiveness to a second IL-15 stimulation was restored by the depletion of CD8+ cells. These biological findings refine our understanding of the complex mode of action of NK cells in vivo, and they should be taken into consideration for IL-15-based therapy.

Discovery of a Small-Molecule Inhibitor of Interleukin 15: Pharmacophore-Based Virtual Screening and Hit Optimization.

Interleukin (IL)-15 is a pleiotropic cytokine, which is structurally close to IL-2 and shares with it the IL-2 ß and γ receptor (R) subunits. By promoting the activation and proliferation of NK, NK-T, and CD8+ T cells, IL-15 plays important roles in innate and adaptative immunity. Moreover, the association of high levels of IL-15 expression with inflammatory and autoimmune diseases has led to the development of various antagonistic approaches targeting IL-15. This study is an original approach aimed at discovering small-molecule inhibitors impeding IL-15/IL-15R interaction. A pharmacophore and docking-based virtual screening of compound libraries led to the selection of 240 high-scoring compounds, 36 of which were found to bind IL-15, to inhibit the binding of IL-15 to the IL-2Rß chain or the proliferation of IL-15-dependent cells or both. One of them was selected as a hit and optimized by a structure-activity relationship approach, leading to the first small-molecule IL-15 inhibitor with sub-micromolar activity.

Cutting Edge: Differential Fine-Tuning of IL-2- and IL-15-Dependent Functions by Targeting Their Common IL-2/15Rß/γc Receptor.

Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rß/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15- and IL-2-dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2-dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.

[Interleukin 2 revival: a revisited model and new therapeutic applications]. / Le renouveau de l'interleukine 2 - Modèle revisité et nouvelles applications thérapeutiques.

Interleukin-2, a cytokine identified as T-cell growth factor, has long been regarded as central to the development and effector activities of immune responses. Several gene knockout mouse studies and observations in humans, however, have undermined that vision, and the discovery of regulatory T cells showed that IL-2, in contrast to the accepted dogma, has the essential function of promoting (1) homeostasis and (2) the function of these T regulator cells the which, limit the action of the effector cells, in particular to prevent the autoimmune reaction drifts. This new paradigm has major implications on the use of IL-2 in therapy, and creates new strategies to manipulate the Teffectors/Tregulators balance.

IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.

Syndecan-1 regulates the biological activities of interleukin-34.

IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPß/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPß/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse.

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.

IL-15.IL-15Rα complex shedding following trans-presentation is essential for the survival of IL-15 responding NK and T cells.

Interleukin (IL)-15 and its specific receptor chain, IL-15Rα, support the development of various effector cells, including NK and CD8 T cells via a mechanism called trans-presentation. Whereas the dynamic of trans-presentation has been shown to involve the recycling of IL-15Rα by presenting cells, the way responding cells integrate, or take advantage of this process has not been evaluated yet. To address this question, we set up a trans-presentation model using a membrane-bound IL-15.IL-15Rα fusion protein, and found that IL-15 is detectable within responding cells following IL-15 trans-presentation. The role of the proteolytic cleavage of IL-15Rα in this process was investigated by generating an uncleavable form of IL-15Rα. We showed that IL-15 entry into responding cells necessitates the cleavage of IL-15.IL-15Rα complex from the surface of IL-15 presenting cells, and observed that IL-15Rα cleavage is associated with a decrease of the duration of Stat5 signaling. Once separated from presenting cells, responding cells are able to recycle IL-15.IL-15Rα complexes via intracellular compartments, for residual proliferation in a time-limited manner. These studies define an unprecedented cytokine pathway in which the IL-15.IL-15Rα complex cleaved from presenting cells allows responding cells to internalize, store and use IL-15.IL-15Rα complex for their own proliferation and survival.

Regulation of CD4(+)NKG2D(+) Th1 cells in patients with metastatic melanoma treated with sorafenib: role of IL-15Rα and NKG2D triggering.

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4(+)NKG2D(+) T cells. Hence, the increase of blood CD4(+)NKG2D(+) T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4(+)NKG2D(+) subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHC class I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D(+) Th1 cells.

Antitumor activity of an immunocytokine composed of an anti-GD2 antibody and the IL-15 superagonist RLI.

The use of interleukin (IL)-15 or the IL-15 superagonist RLI as immunological adjuvants presents many advantages over that of IL-2, including a reduced toxicity and an improved efficacy. We have generated an immunocytokine that specifically targets RLI to a tumor-associated antigen, namely, disialoganglioside GD2. This agent displayed robust antitumor activity in 2 syngeneic murine models of malignant disease.

Tumor targeting of the IL-15 superagonist RLI by an anti-GD2 antibody strongly enhances its antitumor potency.

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Multiplexed infectious protein microarray immunoassay suitable for the study of the specificity of monoclonal immunoglobulins.

Enzyme-linked immunosorbent assays (ELISAs) used to detect antibodies specific for common infectious agents such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), Toxoplasma gondii (T. gondii), and hepatitis C virus (HCV) are time-consuming and require large volumes of samples, which restrict their use. We propose a new assay based on a multiplexed infectious protein (MIP) microarray combining different epitopes representative of the four germs. Antigens and lysates were printed on nitrocellulose slides to constitute the microarray. First, the microarray was incubated with human serum samples. Then, the suitability of the microarray for analysis of the specificity of purified monoclonal immunoglobulin (mc Ig) was assessed using serum and mc Ig of HCV-positive patients. Bound human immunoglobulin G (IgG) was detected using fluorescently labeled secondary antibodies, and the signals were quantified. Results obtained in serum samples with the new MIP microarray immunoassay were compared with ELISAs; we observed concordances of 95% for EBV, 93% for CMV, 91% for T. gondii, and 100% for HCV. Regarding purified mc Ig of HCV-positive patients, 3 of 3 recognized antigens printed on the microarray. Hence, the novel EBV/CMV/T. gondii/HCV MIP microarray allows simultaneous diagnosis of polyclonal and monoclonal immune response to infectious diseases using very small volume samples.

Autonomous and extrinsic regulation of thymopoiesis in human immune system (HIS) mice.

Human Immune System (HIS) mice represent a novel biotechnology platform to dissect human haematopoiesis and immune responses. However, the limited human T-cell development that is observed in HIS mice restricts its utility for these applications. Here, we address whether reduced thymopoiesis in HIS mice reflects an autonomous defect in T-cell precursors and/or a defect in the murine thymic niche. Human thymocyte precursors seed the mouse thymus and their reciprocal interactions with murine thymic epithelial cells (TECs) led to both T-cell and TEC maturation. The human thymocyte subsets observed in HIS mice demonstrated survival, proliferative and phenotypic characteristics of their normal human counterparts, suggesting that the intrinsic developmental program of human thymocytes unfolds normally in this xenograft setting. We observed that exogenous administration of human IL-15/IL-15Rα agonistic complexes induced the survival, proliferation and absolute numbers of immature human thymocyte subsets, without any obvious effect on cell-surface phenotype or TCR Vß usage amongst the newly selected mature single-positive (SP) thymocytes. Finally, when IL-15 was administered early after stem cell transplantation, we noted accelerated thymopoiesis resulting in the more rapid appearance of peripheral naïve T cells. Our results highlight the functional capacity of murine thymic stroma cells in promoting human thymopoiesis in HIS mice but suggest that the "cross-talk" between murine thymic stroma and human haematopoietic precursors may be suboptimal. As IL-15 immunotherapy promotes early thymopoiesis, this novel approach could be used to reduce the period of T-cell immunodeficiency in the post-transplant clinical setting.

Despite an impaired response to IL-7, CD4+EM T cells from HIV-positive patients proliferate normally in response to IL-15 and its superagonist, RLI.

OBJECTIVE: In phase I/II trials, IL-7 immunotherapy has been shown to expand CD4(+) T cells. However, expression of the IL-7 receptor α-chain, CD127, is reduced on CD4(+) T cells from HIV-positive patients, and defects in CD127 signaling have also been reported. To refine and improve cytokine immunotherapy, it is important to identify stimuli that can restore proliferation of CD4(+) cells with defective responses to IL-7. DESIGN: Observational study comparing viremic HIV-positive patients with HIV-negative controls. METHODS: Peripheral blood mononuclear cells were cultured in the presence of 1 nmol/l IL-2, IL-7, IL-15 or RLI (an IL-15Rα/IL-15 fusion protein). Proliferation of different T-cell subsets was assessed by carboxyfluorescein succinimidyl ester fluorescence. Expression of CD127 on CD4(+) T-cell subsets was also analyzed. RESULTS: In HIV-positive patients, CD127 expression was correlated with CD4(+) T-cell count in the CD4(+)(N) (R(2) = 0.36; P < 0.01) and CD4(+)(CM) (R(2) = 0.45; P < 0.001) populations, whereas CD127 expression on CD4(+)(EM) cells was significantly reduced in HIV-positive individuals compared with controls (P = 0.001) independently of CD4(+) T-cell count. In patients with high CD4(+) T-cell counts, proliferation in response to IL-7 was significantly reduced only in CD4(+)(EM) cells (P < 0.05). RLI, and to a lesser extent IL-15, induced strong proliferation of CD4(+)(EM) cells from both HIV-positive patients and controls. Neither agent stimulated proliferation of CD4(+)(N) or CD4(+)(CM) cells. CONCLUSION: In HIV-positive patients, CD4(+)(EM) cells are deficient in both CD127 expression and proliferation in response to IL-7. RLI and IL-15 specifically induced proliferation of CD4(+)(EM) cells, suggesting that they may have a unique potential to complement IL-7 immunotherapy.