Douglas C. Palmer

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The γ C cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of γ C cytokine–responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.

Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8 + T cells

T cell differentiation is a progressive process characterized by phenotypic and functional changes. By transferring tumor-specific CD8+ T cells into tumor-bearing mice at various stages of differentiation, we evaluated their efficacy for adoptive immunotherapy. We found that administration of naive and early effector T cells, in combination with active immunization and IL-2, resulted in the eradication of large, established tumors. Despite enhanced in vitro antitumor properties, more-differentiated effector T cells were less effective for in vivo tumor treatment. Several events may underlie this paradoxical phenomenon: (a) downregulation of lymphoid-homing and costimulatory molecules; (b) inability to produce IL-2 and access homeostatic cytokines; and (c) entry into a proapoptotic and replicative senescent state. While the progressive acquisition of terminal effector properties is characterized by pronounced in vitro tumor killing, in vivo T cell activation, proliferation, and survival are progressively impaired. These findings suggest that the current methodology for selecting T cells for transfer is inadequate and provide new criteria for the generation and the screening of optimal lymphocyte populations for adoptive immunotherapy.

CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells

CD4+ T cells control the effector function, memory, and maintenance of CD8+ T cells. Paradoxically, we found that absence of CD4+ T cells enhanced adoptive immunotherapy of cancer when using CD8+ T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4+CD25− Th cells (Th cells) with tumor/self-reactive CD8+ T cells and vaccination into CD4+ T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4+ T cells that contained a mixture of Th and CD4+CD25+ T regulatory cells (Treg cells) or Treg cells alone prevented effective adoptive immunotherapy. Maintenance of CD8+ T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2−/− mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring Treg cells to be effective.

Tumor-specific Th17-polarized cells eradicate large established melanoma

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells

IL-15 and IL-2 possess similar properties, including the ability to induce T cell proliferation. However, whereas IL-2 can promote apoptosis and limit CD8+ memory T cell survival and proliferation, IL-15 helps maintain a memory CD8+ T cell population and can inhibit apoptosis. We sought to determine whether IL-15 could enhance the in vivo function of tumor/self-reactive CD8+ T cells by using a T cell receptor transgenic mouse (pmel-1) whose CD8+ T cells recognize an epitope derived from the self/melanoma antigen gp100. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the in vivo antitumor activity of adoptively transferred CD8+ T cells. These results provide several avenues for improving adoptive immunotherapy of cancer in patients.

IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy

IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8(+) T cells, but their distinct effects on antigen-driven differentiation of naive CD8(+) T cells into effector CD8(+) T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8(+) T cells into granzyme B- and CD44-expressing effector CD8(+) T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2-mediated acquisition of a cytolytic CD8(+) T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8(+) T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.

Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function

Naive CD8+ T cells rely upon oxidation of fatty acids as a primary source of energy. After antigen encounter, T cells shift to a glycolytic metabolism to sustain effector function. It is unclear, however, whether changes in glucose metabolism ultimately influence the ability of activated T cells to become long-lived memory cells. We used a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells. We found that cells exhibiting limited glucose incorporation had a molecular profile characteristic of memory precursor cells and an increased capacity to enter the memory pool compared with cells taking up high amounts of glucose. Accordingly, enforcing glycolytic metabolism by overexpressing the glycolytic enzyme phosphoglycerate mutase-1 severely impaired the ability of CD8+ T cells to form long-term memory. Conversely, activation of CD8+ T cells in the presence of an inhibitor of glycolysis, 2-deoxyglucose, enhanced the generation of memory cells and antitumor functionality. Our data indicate that augmenting glycolytic flux drives CD8+ T cells toward a terminally differentiated state, while its inhibition preserves the formation of long-lived memory CD8+ T cells. These results have important implications for improving the efficacy of T cell-based therapies against chronic infectious diseases and cancer.

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8 + T cells via TLR4 signaling

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.

Adoptively transferred effector cells derived from naïve rather than central memory CD8+ T cells mediate superior antitumor immunity

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.