Naoto Hirano

Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells.

During a search for genes that maintain T cell quiescence, we determined that Tob, a member of an anti-proliferative gene family, was highly expressed in anergic T cell clones. Tob was also expressed in unstimulated peripheral blood T lymphocytes and down-regulated during activation. Forced expression of Tob inhibited T cell proliferation and transcription of cytokines and cyclins. In contrast, suppression of Tob with an antisense oligonucleotide augmented CD3-mediated responses and abrogated the requirement of costimulation for maximal proliferation and cytokine secretion. Tob associated with Smad2 and Smad4 and enhanced Smad DNA-binding. The inhibitory effect of Tob on interleukin 2 (IL-2) transcription was not mediated by blockade of NFAT, AP-1 or NF-κB transactivation but by enhancement of Smad binding on the −105 negative regulatory element of the IL-2 promoter. Thus, T cell quiescence is an actively maintained phenotype that must be suppressed for T cell activation to occur.

CIZ, a Zinc Finger Protein That Interacts with p130 cas and Activates the Expression of Matrix Metalloproteinases

ABSTRACT p130 cas (Cas) is a docking protein that contains an SH3 domain and multiple tyrosine residues. p130 cas is located at focal adhesions, is tyrosine phosphorylated in response to integrin stimulation, and is thought to transmit signals, via c-Crk and other proteins, for the remodeling of actin stress fibers and cell movement. In a search for the ligands of the SH3 domain of p130 cas by far-Western screening, we cloned a novel protein named CIZ (for Cas-interacting zinc finger protein). CIZ consists of the following: a putative leucine zipper; a serine/threonine-rich region; a proline-rich sequence; five, six, or eight Krüppel-type C2H2 zinc fingers; and the glutamine-alanine repeat. CIZ binds Cas in cells and is located in the nucleus and at focal adhesions. We showed that CIZ is a nucleocytoplasmic shuttling protein, by using the transient interspecies heterokaryon formation assay. In order to search for the targets of CIZ in nucleus, we determined the DNA binding consensus of CIZ as (G/C)AAAAA(A) by cyclic amplification and selection of targets analysis. The consensus-like sequences are found in several promoters of matrix metalloproteinases (MMPs), which are the enzymes used to degrade the extracellular matrix proteins. CIZ binds to a consensus-like sequence in the MMP-1 (collagenase) promoter. Overexpression of CIZ upregulates the transcriptions from MMP-1, MMP-3 (stromelysin), and MMP-7 (matrilysin) promoters, and this transactivation was enhanced in the presence of Cas. Furthermore, the stable overexpression of CIZ promoted the production of MMP-7 in culture medium. In summary, CIZ, a novel zinc finger protein, binds Cas, is a nucleocytoplasmic shuttling protein, and regulates the expression of MMPs.

Long-Lived Antitumor CD8+ Lymphocytes for Adoptive Therapy Generated Using an Artificial Antigen-Presenting Cell

Purpose: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL). Experimental Design: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined. Results: Stimulation of CD8+ T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC33, produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined. Conclusions: With clinical grade aAPC33 in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.

Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.

Antitumor CD8+ T cells educated in vitro can persist as memory T cells and induce antitumor responses in humans without prior conditioning or cytokine treatment. Memory that Keeps Going and Going Senior scientists consoling their trainees about failed experiments tout the value of persistence. Yet, persistence is not only important for the scientist tirelessly pipetting at two in the morning, it is key for immunological memory as well. Melanoma is a malignant tumor of melanocytes, the pigment cells found in the skin. Advanced-stage melanoma has a poor prognosis, with patients on average surviving less than a year. A new promising therapy for advanced-stage melanoma patients harnesses the immune system to attack the tumor cells. In adoptive T cell therapy, cytotoxic cells specific for the tumor are transferred into patients, where they then traffic to and destroy the tumor cells. However, one limitation of this therapy is keeping the tumor-specific T cells alive in the patient, which has been largely unsuccessful in the absence of extensive treatment of the patient. Butler et al. now report a means to expand these T cells that allows them to persist in the absence of extra patient manipulation. The authors use artificial antigen-presenting cells to turn antitumor T cells into memory T cells, which survive longer and respond more quickly than normal effector T cells. These artificial antigen-presenting cells express molecules that “costimulate” the T cells, resulting in T cells that both look and act like memory T cells in the culture dish. These educated tumor-specific cells were then introduced into patients with advanced-stage melanoma. These cells functioned as memory cells in the patients as well: persisting for long periods of time, homing to the tumor site, and demonstrating tumor-specific activation and function, all in the absence of further patient manipulation. Although this is an early clinical trial with a small number of patients, there is some indication that this treatment may have clinical benefit for patients with this devastating disease. Persistence, both of the T cells and the scientists running the study, has finally paid off. Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8+ T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.

Clinical impact of HLA-DR15, a minor population of paroxysmal nocturnal haemoglobinuria-type cells, and an aplastic anaemia-associated autoantibody in children with acquired aplastic anaemia

Aplastic anaemia (AA) is defined as a pancytopenia caused by bone marrow failure, and its pathogenesis is thought to involve autoimmune processes. Several predictive markers of the response to immunosuppressive therapy (IST) have been proposed, which appear to reflect the immune pathophysiology. We prospectively investigated the presence of human leucocyte antigen (HLA)‐DR15, a minor population of paroxysmal nocturnal haemoglobinuria (PNH)‐type cells, and antibodies to the recently identified autoantigen postmeiotic segregation increased 1 (PMS1) in 103 children with AA enrolled in a multicentre study. In contrast to adults, children with AA did not show an increased frequency of HLA‐DR15. In addition, a sensitive flow cytometric assay revealed that children with AA have a much lower prevalence of PNH‐type cells (21·4%) than reported for adults with this disease. An immunoblotting assay detected anti‐PMS1 antibody in 15 of 103 (14·6%) of the children. Finally, the response rate to IST was not significantly different between patients with and without DR15 (45·5% vs. 54·0%), PNH‐type cells (68·2% vs. 53·1%) or anti‐PMS1 antibody (40·0% vs. 59·1%). The current study did not confirm a correlation between these markers and the response to IST, suggesting that there is a difference in the pathophysiologies of adult and paediatric AA.

Efficient Presentation of Naturally Processed HLA Class I Peptides by Artificial Antigen-Presenting Cells for the Generation of Effective Antitumor Responses

Appropriate presentation of tumor-associated antigens (TAA) by antigen-presenting cells (APC) is required for the development of clinically relevant antitumor T-cell responses. One common approach, which uses APC pulsed with synthetic peptides, can sometimes generate ineffective immune responses. This failure may, in part, be attributed to the formation of HLA/synthetic pulsed peptide complexes that possess different conformations compared with those of endogenously presented peptides. In addition, endogenous peptides may undergo post-translational modifications, which do not occur with synthetic peptides. Because our goal is to induce immunity that can recognize TAA that are endogenously presented by tumors, we designed an APC that would not only express the required immunoaccessory molecules but also naturally process and present target antigenic peptides. In this study, we generated an artificial APC (aAPC) that can endogenously present any chosen HLA-A*0201 (A2)–restricted peptide by processing a fusion protein that contains a unique “LTK” sequence linked to the antigenic peptide. Proteasome-dependent processing is so effective that the presented peptide can be directly eluted from the cell surface and identified by biochemical methods. Furthermore, we found that aAPC, engineered to endogenously present peptide derived from the melanoma antigen MART1, can be used to prime and expand antitumor CTL that target MART1-expressing tumor cells in a HLA-A2-restricted manner. Our engineered aAPC could serve as an “off-the-shelf” APC designed to constitutively express class I–restricted TAA peptides and could be used to generate effective T-cell responses to treat human disease.

Presence of anti‐kinectin and anti‐PMS1 antibodies in Japanese aplastic anaemia patients

While anti‐kinectin antibodies are frequently found in aplastic anaemia (AA) patients from the US (39%), we detected antibodies in only three of 30 (10%) of Japanese AA cases. Additionally, population differences in anti‐postmeiotic segregation increased 1 (PMS1) responses were seen, with detectable antibody in 10% Japanese and 0% US AA patients. In one of the Japanese AA patients with detectible anti‐kinectin antibodies, the autoantibody disappeared coincidentally with the partial resolution of pancytopenia. These results support the hypothesis that the epidemiology of AA is heterogeneous and suggest that anti‐kinectin autoantibody titre may serve as a surrogate maker for the disease activity of AA.

Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

Background Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. Methods/Principal Findings We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. Conclusions We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro.

A panel of human cell-based artificial APC enables the expansion of long-lived antigen-specific CD4+ T cells restricted by prevalent HLA-DR alleles

Many preclinical experiments have attested to the critical role of CD4(+) T cell help in CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. Recent clinical trials have demonstrated that reinfusion of CD4(+) T cells can induce responses in infectious diseases and cancer. However, few standardized and versatile systems exist to expand antigen-specific CD4(+) T(h) for clinical use. K562 is a human erythroleukemic cell line, which lacks expression of HLA class I and class II, invariant chain and HLA-DM but expresses adhesion molecules such as intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. With this unique immunologic phenotype, K562 has been tested in clinical trials of cancer immunotherapy. Previously, we created a K562-based artificial antigen-presenting cell (aAPC) that generates ex vivo long-lived HLA-A2-restricted CD8(+) CTL with a central/effector memory phenotype armed with potent effector function. We successfully generated a clinical version of this aAPC and conducted a clinical trial where large numbers of anti-tumor CTL are reinfused to cancer patients. In this article, we shifted focus to CD4(+) T cells and developed a panel of novel K562-derived aAPC, where each expresses a different single HLA-DR allele, invariant chain, HLA-DM, CD80, CD83 and CD64; takes up soluble protein by endocytosis and processes and presents CD4(+) T-cell peptides. Using this aAPC, we were able to determine novel DR-restricted CD4(+) T-cell epitopes and expand long-lived CD4(+) T-cells specific for multiple antigens without growing bystander Foxp3(+) regulatory T cells. Our results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD8(+) CTL and CD4(+) T(h).

Dissociation of its opposing immunologic effects is critical for the optimization of antitumor CD8+ T-cell responses induced by interleukin 21.

Purpose: Interleukin 21 (IL-21) is a promising new cytokine, which is undergoing clinical testing as an anticancer agent. Although IL-21 provides potent stimulation of CD8+ T cells, it has also been suggested that IL-21 is immunosuppressive by counteracting the maturation of dendritic cells. The dissociation of these two opposing effects may enhance the utility of IL-21 as an immunotherapeutic. In this study, we used a cell-based artificial antigen-presenting cell (aAPC) lacking a functional IL-21 receptor (IL-21R) to investigate the immunostimulatory properties of IL-21. Experimental Design: The immunosuppressive activity of IL-21 was studied using human IL-21R+ dendritic cells. Antigen-specific CD8+ T cells stimulated with human cell–based IL-21R-aAPC were used to isolate the T-cell immunostimulatory effects of IL-21. The functional outcomes, including phenotype, cytokine production, proliferation, and cytotoxicity were evaluated. Results: IL-21 limits the immune response by maintaining immunologically immature dendritic cells. However, stimulation of CD8+ T cells with IL-21R- aAPC, which secrete IL-21, results in significant expansion. Although priming in the presence of IL-21 temporarily modulated the T-cell phenotype, chronic stimulation abrogated these differences. Importantly, exposure to IL-21 during restimulation promoted the enrichment and expansion of antigen-specific CD8+ T cells that maintained IL-2 secretion and gained enhanced IFN-γ secretion. Tumor antigen-specific CTL generated in the presence of IL-21 recognized tumor cells efficiently, demonstrating potent effector functions. Conclusions: IL-21 induces opposing effects on antigen-presenting cells and CD8+ T cells. Strategic application of IL-21 is required to induce optimal clinical effects and may enable the generation of large numbers of highly avid tumor-specific CTL for adoptive immunotherapy.