Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/ melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA ؊ and CD45RO ؉ after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA ؉ and CD45RO ؊ phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss.

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways1. Wnt/β-catenin is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation2,3, but its role in the generation and maintenance of memory T cells is unknown. We found that the induction of Wnt/β-catenin signaling using inhibitors of glycogen-sythase-kinase-3β or the Wnt protein family member, Wnt3a, arrested CD8+ T cell development into effector cells. By blocking T-cell differentiation, Wnt signaling enabled the generation of CD44low, CD62Lhigh, Sca-1high, CD122high, Bcl-2high self-renewing, multipotent CD8+ memory stem cells with proliferative and anti-tumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of stemness in mature memory CD8+ T cells and have important implications for the design of novel vaccination strategies and adoptive immunotherapies.

IntroductionThe role of CD4 ϩ cells in antitumor immunity remains controversial and poorly understood. 1,2 They are known to mediate potent therapeutic effect in the setting of hematopoietic stem cell allotransplantation and donor lymphocyte infusion in hematologic malignancy, 3,4 but antigen-specific T helper (Th) cells have been studied to much lesser extent. A lack of clarity regarding CD4 ϩ cells is due, in no small part, to the complexity of their biology. CD4 ϩ T cells can differentiate into diverse subsets with specific phenotypes that can have self-reinforcing and opposing functions, but these T-cell subsets have not been comprehensively studied in tumor-bearing mice.Historically, CD4 ϩ T lymphocytes have been thought of as mere providers of stimuli to help the putatively more important CD8 ϩ effectors, which eliminate cancer by direct cytotoxicity. [5][6][7] There are several studies showing that CD4 ϩ T helper (Th) cells are capable of protecting the host against tumor challenge and even of mediating tumor regression on their own in the setting of either solid or hematopoietic disease. [8][9][10][11][12][13] Furthermore, protection was maintained against MHC class II-negative multiple myeloma model and involved cross-presentation by professional antigenpresenting cells (APCs) and activation of tumoricidal activity mediated by macrophages secreting IFN-␥. 14 A similar IFN-␥-dependent mechanism was involved in the rejection of MHC class II-negative tumor in severe combined immunodeficient (SCID) mice. 15 In some cases, the ability to reject antigen-expressing tumor by specific naive Th cells was thought to be substantially better than the ability of CD8 ϩ cells. 16 Classically, effector CD4 ϩ T cells have been categorized into T helper 1 (Th1) and T helper 2 (Th2) subsets. 17,18 Limited studies indicate that both subtypes elicit antitumor effects, 19-21 but the Th1-polarized cells, secreting IFN-␥ and capable of enhancing activity of cytotoxic CD8 ϩ lymphocytes, have traditionally been regarded as more efficient. [22][23][24][25] However, it is also clear that CD4 ϩ T regulatory cells (T regs ) can efficiently suppress the function of antitumor CD8 ϩ T cells. 5,[26][27][28] Recently, the novel Th17 lineage, generated in the presence of TGF-␤ and IL-6 and expanded under the influence of IL-23, [29][30][31] has been associated with responses against certain infections and implicated in the development of autoimmunity in animal models that had been previously linked to Th1-type responses (experimental autoimmune encephalitis, collagen-induced arthritis). 32,33 They also seem to play an important role in the pathogenesis of graft-versus-host disease (GVHD). 34,35 Th17 cells have been found in various tumors, including mycosis fungoides, Sézary syndrome, and prostate cancer. 36,37 Kryczek et al reported the presence of naturally occurring Th17 cells and T regs in the tumor microenvironment and tumor-draining lymph nodes in both human and mice tumors. 38 Proinflammatory cytokines including IL-17A, IL-6, and I...

Baseline gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with beneficial clinical response to ipilimumab and more frequent occurrence of ipilimumab-induced colitis.

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8 + T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8 + T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8 + T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.

Effector cells derived from central memory CD8 ؉ T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1 ؊ phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8 ؉ T cells may allow superior efficacy upon adoptive transfer.

IntroductionInfusion of tumor-reactive T cells is emerging as an effective treatment for Epstein-Barr virus-associated malignancies and for melanoma. 1-3 T cells with reactivity against tumor antigens can be generated through T-cell receptor (TCR) gene engineering, 4 expanded in vitro, and administered to patients. 5 This approach permits not only targeting of any antigen for which a TCR can be isolated but also selection and induction of T-cell subsets with optimal therapeutic potential. Identification of these subsets is a critical challenge in the advance of cancer immunotherapy. 6,7 Th17-lineage CD4 ϩ T cells, which express the transcription factor ROR-␥t and produce the proinflammatory cytokine interleukin-17 (IL-17), demonstrate enhanced antitumor immunity. 8,9 IL-17-producing CD8 ϩ T cells have been identified in mice and in humans, 10-13 and they can be generated through in vitro priming with Th17-polarizing cytokines. 14 However, a role for these cells in antitumor immunity has not been described. CD4 ϩ T cells can differentiate into lineages with diverse effector functions. In contrast, CD8 ϩ T cells, through the redundant expression of Eomes and T-bet, are fated to develop into cytolytic effector cells that produce IFN-␥ and express granzyme B and perforin. 11,15 It is not known if induction of IL-17 production in this cytolytic lineage would enhance its ability to mediate tumor destruction. Interestingly, the improved antitumor immunity observed with Th17 cells appears to be IL-17-independent, suggesting that it is not IL-17 secretion per se, but rather another characteristic conferred by Th17-polarization, that improves their function. 8 To study tumor therapy with type 17-skewed CD8 ϩ T cells, we used the pmel-1 model of adoptive immunotherapy of B16 melanoma. 16 This model reproduces the clinical challenge of targeting the shared tumor/self-antigen, gp100, in poorly immunogenic, vascularized B16 melanoma tumors. Pmel-1, gp100-specific, TCR-transgenic CD8 ϩ T cells that were primed and expanded before infusion were used to parallel human peripheral blood lymphocytes that are TCR transduced and expanded for clinical therapy. 5 Methods Mice and tumor linesPmel-1/Thy1.1 TCR-transgenic mice have been described previously. 16,17 They were housed in the National Institutes of Health (NIH) Clinical Research Center vivarium and maintained in compliance with the NIH Animal Care and Use Committee. C57BL/6 mice were purchased from The Jackson Laboratory. The B16F10 and MCA205 tumor lines were obtained from the National Cancer Institute (NCI) tumor repository. All animal studies were approved by the NCI Animal Care and Use Committee. In vitro assaysType 17 skewing was accomplished by priming pmel-1/Thy1.1 splenocytes with 1 M hgp100 25-33 in rmIL-6 (5 ng/mL) and recombinant human transforming growth factor-␤ (rhTGF-␤; 10 ng/mL; R&D Systems), and anti-IFN-␥ (10 g/mL; eBioscience). Beginning 2 days after priming, cell cultures were expanded with rhIL-2 (30 IU/mL; Novartis). Control cells were primed and expand...

Gut microbiota composition influences the clinical benefit of immune checkpoints in patients with advanced cancer but mechanisms underlying this relationship remain unclear. Molecular mechanism whereby gut microbiota influences immune responses is mainly assigned to gut microbial metabolites. Short-chain fatty acids (SCFA) are produced in large amounts in the colon through bacterial fermentation of dietary fiber. We evaluate in mice and in patients treated with anti-CTLA-4 blocking mAbs whether SCFA levels is related to clinical outcome. High blood butyrate and propionate levels are associated with resistance to CTLA-4 blockade and higher proportion of Treg cells. In mice, butyrate restrains anti-CTLA-4-induced upregulation of CD80/CD86 on dendritic cells and ICOS on T cells, accumulation of tumorspecific T cells and memory T cells. In patients, high blood butyrate levels moderate ipilimumab-induced accumulation of memory and ICOS + CD4 + T cells and IL-2 impregnation. Altogether, these results suggest that SCFA limits anti-CTLA-4 activity.