Timothy Bullock

Route of Immunization with Peptide-pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control

We have established that the route of immunization with peptide-pulsed, activated DC leads to memory CD8+ T cells with distinct distributions in lymphoid tissues, which determines the ability to control tumors growing in different body sites. Both intravenous (i.v.) and subcutaneous (s.c.) immunization induced memory T cells in spleen and control of metastatic-like lung tumors. s.c. immunization also induced memory T cells in lymph nodes (LNs), imparting protection against subcutaneously growing tumors. In contrast, i.v. immunization-induced memory was restricted to spleen and failed to impart protective immunity against subcutaneously growing tumors. Memory cell distribution and tumor control were both linked to injection route–dependent localization of DCs in lymphoid compartments. Using peripheral LN–ablated mice, these LNs were shown to be essential for control of subcutaneously growing tumors but not lung metastases; in contrast, using immunized asplenic mice, we found that the spleen is necessary and sufficient for control of lung tumors, but unnecessary for control of subcutaneously growing tumors. These data demonstrate the existence of a previously undescribed population of splenic-resident memory CD8 T cells that are essential for the control of lung metastases. Thus, regional immunity based on memory T cell residence patterns is an important factor in DC-based tumor immunotherapy.

Induction of CD70 on Dendritic Cells through CD40 or TLR Stimulation Contributes to the Development of CD8+ T Cell Responses in the Absence of CD4+ T Cells

The expansion of CD8+ T cells in response to Ag can be characterized as either dependent or independent of CD4+ T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4+ T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4+ T cell help, the expansion of primary CD8+ T cells after immunization with TNF-α- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8+ T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8+ T cells without the aid of CD4+ T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4+ T cell-independent expansion of primary CD8+ T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8+ T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8+ T cell responses in the absence of CD4+ T cells.

Antigens derived from melanocyte differentiation proteins: self-tolerance, autoimmunity, and use for cancer immunotherapy

Summary: A large set of peptide antigens presented by class I major histocompatibility complex (MHC) molecules on human and murine melanomas and recognized by CD8+ T cells have been defined. These peptides represent attractive candidates for the development of therapeutic and/or prophylactic approaches to treat this cancer. However, the majority of the peptides that are presented by multiple tumors and recognized by T cells from multiple patients arise from proteins that are also expressed in normal melanocytes. It is expected that immune responses to such peptides will be compromised by self‐tolerance or, alternatively, that stimulation of effective immune responses will be accompanied by autoimmune vitiligo. In this review, we describe a preclinical model to evaluate these issues and recent data to suggest that tolerance can be overcome to generate effective antitumor responses. This model also allows the rapid and systematic examination of parameters for the effective use of synthetic peptide vaccines.

Antigen Density Presented By Dendritic Cells In Vivo Differentially Affects the Number and Avidity of Primary, Memory, and Recall CD8 + T Cells

We studied the size and avidity of primary and recall CD8+ T cell responses in vivo in mice immunized with dendritic cells presenting different densities of a MHC class I-restricted peptide. Increasing the epitope density on a fixed number of dendritic cells increased the size of the primary response, yet had no influence on the avidity of the effector cells. However, epitope density-based selection of cells with different avidities was evident in the subsequent memory population, and in recall responses. Additionally, mice primed with different peptide densities had similarly sized quiescent memory and recall responses. Our findings provide evidence for an important role for epitope density in the selection of T cells in vivo.

The Density of Peptides Displayed by Dendritic Cells Affects Immune Responses to Human Tyrosinase and gp100 in HLA-A2 Transgenic Mice

Several HLA-A*0201-restricted peptide epitopes that can be used as targets for active immunotherapy have been identified within melanocyte differentiation proteins. However, uncertainty exists as to the most effective way to elicit CD8+ T cells with these epitopes in vivo. We report the use of transgenic mice expressing a derivative of HLA-A*0201, and dendritic cells, to enhance the activation of CD8+ T cells that recognize peptide epitopes derived from human tyrosinase and glycoprotein 100. We find that by altering the cell surface density of the immunizing peptide on the dendritic cells, either by pulsing with higher concentrations of peptide, or by changing the MHC-peptide-binding affinity by generating variants of the parent peptides, the size of the activated CD8+ T cell populations can be modulated in vivo. Significantly, the density of peptide that produced the largest response was less than the maximum density achievable through short-term peptide pulsing. We have also found, however, that while some variant peptides are effective at eliciting both primary and recall CD8+ T cell responses that can recognize the parental epitope, other variant epitopes lead to the outgrowth of CD8+ T cells that only recognize the variant. HLA-A*0201 transgenic mice provide an important model to define which peptide variants are most likely to stimulate CD8+ T cell populations that recognize the parental, melanoma-specific peptide.

Manipulation of Avidity to Improve Effectiveness of Adoptively Transferred CD8+ T Cells for Melanoma Immunotherapy in Human MHC Class I-Transgenic Mice

The adoptive transfer of tumor-reactive CD8+ T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8+ T cell populations that recognize either tyrosinase369–376 or gp100209–217 from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-γ accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8+ T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

Analysis of MHC Class II Antigen Processing by Quantitation of Peptides that Constitute Nested Sets

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3–36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.

Control of established melanoma by CD27 stimulation is associated with enhanced effector function and persistence, and reduced PD-1 expression, of tumor infiltrating CD8+ T cells

The immune response to the tumor can be enhanced by targeting costimulatory molecules on T cells. As the CD70-CD27 costimulatory axis plays an important role in the activation, survival, and differentiation of lymphocytes, we have examined the efficacy of agonistic anti-CD27 antibodies as monotherapies for established melanoma in a murine model. We show that this approach leads to a substantial reduction in the outgrowth of both experimental lung metastases and subcutaneous tumors. Anti-CD27 treatment supports the maintenance of tumor-specific CD8+ T cells within the tumor, reduces the frequency of FoxP3-expressing CD4+ T cells within tumors, and potentiates the ability of NK1.1+ and CD8+ tumor infiltrating cells to secrete IFNγ upon coculture with tumor cells. The enhanced effector function correlated with lower levels of PD-1 expression on CD8+ T cells from anti-CD27–treated mice. Despite the modulating effect of anti-CD27 on multiple cell types, only CD8+ T cells were absolutely required for tumor control. The CD4+ T cells were dispensable, whereas NK1.1+ cells were needed during early stages of tumor growth but not for the effectiveness of anti-CD27. Thus, CD27-mediated costimulation provides a potent boost to multiple aspects of the endogenous responses to tumor, and may be exploited to enhance tumor immunity.

Immunity to Melanoma Antigens: From Self‐Tolerance to Immunotherapy

The development of effective immune therapy for cancer is a central goal of immunologists in the 21st century. Our laboratories have been deeply involved in characterization of the immune response to melanoma and translation of laboratory discoveries into clinical trials. We have identified a cohort of peptide antigens presented by Major Histocompatibility Complex (MHC) molecules on melanoma cells and widely recognized by T cells from melanoma patients. These have been incorporated into peptide-based vaccines that induce CD8(+) and CD4(+) T-cell responses in 80-100% of patients. Major objective clinical tumor regressions have been observed in some patients, and overall survival in vaccinated patients exceeds expected stage-specific survival. New clinical trials will determine the value of combination of melanoma helper peptides (MHP) into multipeptide vaccines targeting CD8 cells. New trials will also evaluate new approaches to modulating the host-tumor relationship and will develop new combination therapies. Parallel investigations in murine models are elucidating the immunobiology of the melanoma-host relationship and addressing issues that are not feasible to approach in human trials. Based on the fact that the largest cohort of melanoma antigens are derived from normal proteins concerned with pigment production, we have evaluated the mechanisms of self-tolerance to tyrosinase (Tyr) and have determined how T cells in an environment of self-tolerance are impacted by immunization. Using peptide-pulsed dendritic cells as immunogens, we have also used the mouse model to establish strategies for quantitative and qualitative enhancement of antitumor immunity. This information creates opportunities for a new generation of therapeutic interventions using cancer vaccines.

Immune Responses to the HLA-A*0201-Restricted Epitopes of Tyrosinase and Glycoprotein 100 Enable Control of Melanoma Outgrowth in HLA-A*0201-Transgenic Mice

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.