Tissue-resident memory T cell maintenance during antigen persistence requires both cognate antigen and interleukin-15.

Our understanding of tissue-resident memory T (TRM) cell biology has been largely developed from acute infection models in which antigen is cleared and sterilizing immunity is achieved. Less is known about TRM cells in the context of chronic antigen persistence and inflammation. We investigated factors that underlie TRM maintenance in a kidney transplantation model in which TRM cells drive rejection. In contrast to acute infection, we found that TRM cells declined markedly in the absence of cognate antigen, antigen presentation, or antigen sensing by the T cells. Depletion of graft-infiltrating dendritic cells or interruption of antigen presentation after TRM cells were established was sufficient to disrupt TRM maintenance and reduce allograft pathology. Likewise, removal of IL-15 transpresentation or of the IL-15 receptor on T cells during TRM maintenance led to a decline in TRM cells, and IL-15 receptor blockade prevented chronic rejection. Therefore, antigen and IL-15 presented by dendritic cells play nonredundant key roles in CD8 TRM cell maintenance in settings of antigen persistence and inflammation. These findings provide insights that could lead to improved treatment of chronic transplant rejection and autoimmunity.

Blockade of CD122 on memory T cells in the skin suppresses sclerodermatous graft-versus-host disease.

BACKGROUND: Antigen-stimulated naïve T cells differentiate into effector and memory T cells, of which resident memory T (TRM) cells reside permanently in organ tissues. Involvement of TRM cells has been indicated in pathological conditions of various skin diseases. CD122, which is the ß chain subunit of interleukin (IL)- 2 and IL-15 receptors, is expressed on immune cells including TRM cells. OBJECTIVE: To investigate whether CD122 signaling in skin CD8+ TRM cells mediates the development of mucocutaneous graft-versus-host disease (GVHD). METHODS: We used a genetically modified mouse expressing a membrane-bound form of chicken ovalbumin (OVA) under the control of the keratin 14 promoter, which develops GVHD-like erosive mucocutaneous disease resulting in sclerodermatous disease after transfer of OVA-specific T cell-receptor-transgenic CD8+ OT-I cells. Mice with mucocutaneous GVHD were treated with an anti-CD122 blocking antibody. RESULTS: Administration of an anti-CD122 blocking antibody suppresses the development of acute/chronic GVHD-like mucocutaneous disease in our murine model via the reduction of CD122-expressing memory CD8+ T cells, including skin, memory autoaggressive CD8+ T cells. Moreover, blockade of CD122, even after the establishment of acute GVHD, inhibited the development of chronic GVHD-like sclerodermatous disease via the reduction of epidermal and dermal TRM autoaggressive CD8+ T cells. CONCLUSION: Skin memory CD8+ T cells in particular mediate the development of mucocutaneous GVHD, and blockade of CD122 may be an effective treatment strategy, especially for sclerodermatous GVHD.

CD122 signaling in CD8+ memory T cells drives costimulation-independent rejection.

Interrupting T cell costimulatory signals as a strategy to control undesired immune responses, such as occur in autoimmunity or transplantation, has the potential to alleviate many of the unwanted side effects associated with current immunosuppressive therapies. Belatacept, a high-affinity version of CTLA4-Ig that blocks ligand ligation to CD28, has been approved for use in kidney transplant recipients. Despite the long-term benefits associated with its use, such as improved renal function and lower cardiovascular risk, a subset of patients treated with belatacept experience elevated rates of acute T cell-mediated rejection, tempering enthusiasm for its use. Here we demonstrate that costimulation-independent T cell alloreactivity relies on signaling through CD122, the shared IL-2 and IL-15 receptor ß-chain. Combined costimulatory and CD122 blockade improved survival of transplanted tissue in mice and nonhuman primates by controlling proliferation and effector function of CD8+ T cells. The high-affinity IL-2 receptor was dispensable for memory CD8+ T cell responses, whereas signaling through CD122 as a component of the high-affinity IL-15 receptor was critical for costimulation-independent memory CD8+ T cell recall, distinguishing specific roles for IL-2 and IL-15 in T cell activation. These studies outline a novel approach for clinical optimization of costimulatory blockade strategies in transplantation by targeting CD122.

Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.

Vitiligo is an autoimmune disease of the skin mediated by CD8+ T cells that kill melanocytes and create white spots. Skin lesions in vitiligo frequently return after discontinuing conventional treatments, supporting the hypothesis that autoimmune memory is formed at these locations. We found that lesional T cells in mice and humans with vitiligo display a resident memory (TRM) phenotype, similar to those that provide rapid, localized protection against reinfection from skin and mucosal-tropic viruses. Interleukin-15 (IL-15)-deficient mice reportedly have impaired TRM formation, and IL-15 promotes TRM function ex vivo. We found that both human and mouse TRM express the CD122 subunit of the IL-15 receptor and that keratinocytes up-regulate CD215, the subunit required to display the cytokine on their surface to promote activation of T cells. Targeting IL-15 signaling with an anti-CD122 antibody reverses disease in mice with established vitiligo. Short-term treatment with anti-CD122 inhibits TRM production of interferon-γ (IFNγ), and long-term treatment depletes TRM from skin lesions. Short-term treatment with anti-CD122 can provide durable repigmentation when administered either systemically or locally in the skin. On the basis of these data, we propose that targeting CD122 may be a highly effective and even durable treatment strategy for vitiligo and other tissue-specific autoimmune diseases involving TRM.

CD122 blockade restores immunological tolerance in autoimmune type 1 diabetes via multiple mechanisms.

Signaling through IL-2/IL-15Rß (CD122) is essential for the differentiation and function of T cells and NK cells. A mAb against CD122 has been implicated to suppress autoimmune type 1 diabetes (T1D) development in animal models. However, the mechanisms remain poorly understood. We find that in vivo administration of an anti-CD122 mAb (CD122 blockade) restores immune tolerance in nonobese diabetic (NOD) mice via multiple mechanisms. First, CD122 blockade selectively ablates pathogenic NK cells and memory phenotype CD8+ T cells from pancreatic islets. In contrast, islet CD4+Foxp3+ Tregs are only mildly affected. Second, CD122 blockade suppresses IFN-γ production in islet immune cells. Third, CD122 blockade inhibits the conversion of islet Th17 cells into diabetogenic Th1 cells. Furthermore, a combination of anti-CD122 mAb and Treg-trophic cytokines (IL-2 or IL-33) enhances the abundance and function of islet Tregs. In summary, these data provide crucial mechanistic insights into CD122 blockade-mediated immunoregulation and support therapeutic benefits of this combinational treatment in T1D.

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies.

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.

Engineered human IgG antibodies with longer serum half-lives in primates.

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.

Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells.

The 1D10 antigen is the target for Hu1D10 (apolizumab), a humanized HLA-DR beta-chain-specific antibody that is currently in clinical trials for hematologic malignancies. We demonstrate that Hu1D10 induces caspase-independent apoptosis following secondary cross-linking in primary chronic lymphocytic leukemia (CLL) cells. Generation of reactive oxygen species (ROS) and signal transduction, as evidenced by phosphorylation of Syk and AKT, were noted. The source of the Hu1D10-induced ROS was examined using the Raji lymphoblastic cell line with engineered defects in the mitochondrial respiratory chain. Hu1D10 treatment of clones with deficient mitochondrial respiration produced ROS suggesting a cytoplasmic source. Administration of ROS scavengers to primary CLL cells prior to Hu1D10 treatment diminished AKT activation. Treatment with Hu1D10 and the phosphatidylinositol 3-kinase inhibitor LY294002 demonstrated in vitro synergy with enhanced apoptosis. In conjunction with an ongoing clinical trial, blood samples were collected following intravenous infusion of Hu1D10 and analyzed for phosphorylation of AKT. Two of 3 patient samples showed a sustained increase in AKT phosphorylation following Hu1D10 administration. These data suggest that Hu1D10 ligation in CLL cells induces death and survival signals for which combination therapies may be designed to greatly enhance efficiency of both Hu1D10 and other class II antibodies in development.