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CD8 T cells play a critical role in immunity against intracellular pathogens and cancer. A primary objective of T cell-based vaccine strategies is the induction of durable and effective immune responses. Achieving this goal involves more than simply boosting the numbers of responding T cells. Of particular interest is the induction of CD8 T cells with polycytokine capability, specifically with the ability of CD8 T cells to co-produce IFNγ, TNFα and IL-2. The presence of these polycytokine-producing CD8 T cells correlates strongly with protection against foreign pathogens and cancer. Therefore, approaches capable of inducing such polyfunctional responses are needed. NKG2D engagement on CD8 T cells has been shown to result in increased effector response. However, the manner in which NKG2D engagement results in improved CD8 T cell effector response is unclear. Here we demonstrate in vitro and in vivo that NKG2D engagement by its natural ligand, Rae-1ε, shifts the balance from single cytokine to polycytokine (IL-2, IFNγ, and TFNα) production. These data define a previously unrecognized process in which NKG2D costimulation on CD8 T cells results in improved effector responses.

INTRODUCTION AND OBJECTIVE:Checkpoint inhibitors (CPI) have emerged as a way to activate the immune system’s antitumor properties by blocking inhibitory signals such as programmed cell death ligand...

Vitiligo is an acquired multifactorial disease that affects melanocytes and results in skin depigmentation. In this review, we examine the role of cells stress and self-reactive T cells responses. Given the canonical and non-canonical functions of NKG2D, such as authenticating stressed target and enhance TCR signaling, we examine how melanocyte stress leads to the expression of ligands that are recognized by the activating receptor NKG2D, and how its signaling results in the turning of T cells against self (melanocyte suicide by proxy). We also discuss how this initiation phase is followed by T cell perpetuation, as NKG2D signaling results in self-sustained long-lasting T cells, with improved cytolytic properties.

Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70i(Q435A)) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70i(Q435A) as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70i(Q435A) in mice affects anti-tumor responses. We found that HSP70i(Q435A) in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70i(Q435A)-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70i(Q435A)-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70i(Q435A) elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12192-021-01229-x.

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells in vitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

Natural killer group 2 member D (NKG2D) is a type II transmembrane receptor. NKG2D is present on NK cells in both mice and humans, whereas it is constitutively expressed on CD8(+) T cells in humans but only expressed upon T-cell activation in mice. NKG2D is a promiscuous receptor that recognizes stress-induced surface ligands. In NK cells, NKG2D signaling is sufficient to unleash the killing response; in CD8(+) T cells, this requires concurrent activation of the T-cell receptor (TCR). In this case, the function of NKG2D is to authenticate the recognition of a stressed target and enhance TCR signaling. CD28 has been established as an archetype provider of costimulation during T-cell priming. It has become apparent, however, that signals from other costimulatory receptors, such as NKG2D, are required for optimal T-cell function outside the priming phase. This review will focus on the similarities and differences between NKG2D and CD28; less well-described characteristics of NKG2D, such as the potential role of NKG2D in CD8(+) T-cell memory formation, cancer immunity and autoimmunity; and the opportunities for targeting NKG2D in immunotherapy.

In the study by Jacquemin et al., the authors reported that ligands for NKG2D are upregulated in vitiligo perilesional skin and especially in patients with active disease. The reasons for the elevated expression of NKG2D ligands are unknown. This study, however, provides a framework for understanding vitiligo: Skin resident CD8 T cells recognize and kill melanocytes through NKG2D signaling. This event results in the increased production and release of cyto/chemokines and the development of long-lasting CD8 T cells, which in turn causes the recruitment of new T cells, thus perpetuating and disseminating the disease.

Few costimulatory molecules have been shown to deeply impact the development and function of CD8 T cells. To our knowledge, only CD28, the archetype of TCR co-stimulation, has been reported to affect thymic T cell development. The role of NKG2D in peripheral T cells as a TCR costimulatory receptor is well established. However, its contribution to thymic education is unknown. We present data indicating noticeable differences in T cell development, TCR signaling, function and transcriptional program of T cells from NKG2D deficient mice compared to WT mice. We found changes in the Vβ chain usage and stagnation of the double-positive (DP) stage. Mechanistically, these changes were accompanied by TCR signaling strength reduction, shown by TCR Vβ5 chain, CD3, CD5 and CD45 reductions. Functionally, we found that peripheral T cells were unresponsive to peptide stimulation. Conversely, aCD3/CD28 antibodies mediated their phenotypic activation; however, generated functionally impotent cells. Finally, underpinning a defect in TCR signaling, we found that NKG2D deficient cells were unable to phosphorylate ERK or S6. From these data, we concluded that NKG2D shapes T cell thymic education and their functional imprint.

Inhibitory checkpoint blockade therapy is an immunomodulatory strategy that results in the restoration of T cell functions, and its efficacy depends on the recognition of tumor cells for destruction. Considering the factors at play, one could propose that anti-tumor responses will not occur if tumor cells are immunologically invisible to T cells. In this study, we tested a strategy based on the modulation of cancer cell's immunovisibility through HDAC inhibition. In a model (heterotopic and orthotopic) of mouse urothelial bladder cancer, we demonstrated that the use of intratumoral or intravesical HDACi in combination with systemic anti-PD-1 was effective at inducing curative responses with durable anti-tumor immunity capable of preventing tumor growth at a distal site. Mechanistically, we determined that protective responses were dependent on CD8 cells, but not NK cells. Of significance, in an in vitro human model, we found that fully activated T cells fail at killing bladder cancer cells unless tumor cells were pretreated with HDACi. Complementary to this observation, we found that HDACi cause gene deregulation, that results in the upregulation of genes responsible for mediating immunorecognition, NKG2D ligands and HSP70. Taken together, these data indicate that HDAC inhibition results in the elimination of the tumor cell's "invisibility cloak" that prevents T cells from recognizing and killing them. Finally, as checkpoint blockade therapy moves into the adjuvant setting, its combined use with locally administrated HDACi represents a new approach to be included in our current therapeutic treatment toolbox.

SummaryIn light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.

Background The development of memory responses is an evolutionary function of the adaptive immune system. We propose that for the immune system to populate the memory compartment with the best-suited CD8 T cells it utilizes a process of certification or molecular accreditation mediated through Natural Killer Group 2D (NKG2D). This process of certification assures that the memory compartment is filled with CD8 T cells that have demonstrated their ability to kill their cognate targets through a two-step process that utilizes T cell receptor (TCR) and NKG2D signaling. Methods One week after immunization with peptide-pulsed dendritic cells, NKG2D signaling was transiently blocked in vivo with a single injection of neutralizing antibodies. Under such conditions, we determined the importance of NKG2D signaling during the effector phase for memory formation without compromising NKG2D signaling at the memory phase. Both open (polyclonal) and closed (monoclonal) CD8 T cell repertoires were studied. Results We show that signaling through NKG2D mediated this certification. Temporary blockade of NKG2D signaling during the effector phase resulted in the formation of highly defective memory CD8 T cells characterized by altered expression of the ribosomal protein S6 and epigenetic modifiers, suggesting modifications in the T cell translational machinery and epigenetic programming. Finally, these uncertified memory cells were not protective against a B16 tumor challenge. Conclusion Signaling through NKG2D during the effector phase (certification) favors the development of functional memory CD8 T cells, a previously undescribed role for NKG2D. Temporary blockade of NKG2D signaling during the effector phase results in the formation of highly defective memory CD8 T cells potentially by affecting the expression of the ribosomal protein S6 and epigenetic modifiers, suggesting alterations in T cell translational machinery and epigenetic programming. Electronic supplementary material The online version of this article (10.1186/s40425-019-0531-2) contains supplementary material, which is available to authorized users.

CD8+ T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4+ T cells, leading to the tenet that CD8+ T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8+ T cell priming, we demonstrate that CD8+ T cells, themselves, actively limit their own memory potential through CD8+ T cell-derived IFN-γ–dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8+ T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8+ T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.

CD4-unhelped CD8(+) T cells are functionally defective T cells primed in the absence of CD4(+) T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8(+) T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8(+) T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8(+) T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4(+) T cells in a CD4-dependent influenza model and rescues HIV-specific CD8(+) T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8(+) T cells from their pathophysiological fate and may provide therapeutic benefits.

Summary HSP70i and other stress proteins have been used in anti-tumor vaccines. This begs the question whether HSP70i plays a unique role in immune activation. We vaccinated inducible HSP70i (Hsp70-1) knockout mice and wild-type animals with optimized TRP-1, a highly immunogenic melanosomal target molecule. We were unable to induce robust and lasting depigmentation in the Hsp70-1 knockout mice, and in vivo cytolytic assays revealed a lack of cytotoxic T-lymphocyte activity. Absence of T-cell infiltration to the skin and maintenance of hair follicle melanocytes were observed. By contrast, depigmentation proceeded without interruption in mice lacking a tissue-specific constitutive isoform of HSP70 (Hsp70-2) vaccinated with TRP-2. Next, we demonstrated that HSP70i was necessary and sufficient to accelerate depigmentation in vitiligo-prone Pmel-1 mice, accompanied by lasting phenotypic changes in dendritic cell subpopulations. In summary, these studies assign a unique function to HSP70i in vitiligo and identify HSP70i as a targetable entity for treatment.

While the effects of TCR affinity and TGFβ on CD8(+) T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8(+) T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8(+) T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFβ in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8(+) T-cell-mediated cancer immunotherapeutic strategies.

HER2/neu is an oncogene amplified and over-expressed in 20-30% of breast adenocarcinomas. Treatment with the humanized monoclonal antibody trastuzumab has shown efficacy in combination with cytotoxic agents, although resistance occurs over time. Novel approaches are needed to further increase antibody efficacy. In this study, we provide evidence in a mouse breast cancer therapeutic tumor model that the combination of active immunization with a modified HER2/neu DNA vaccine and passive infusion of an anti-HER2/neu monoclonal antibody leads to significant regression of established tumors. Our data indicate that combination therapy with a HER2/neu DNA vaccine and trastuzumab may have clinical activity in breast cancer patients.

CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-β have been studied individually, the influence of CD8 co-receptor on TGF-β function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-β-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αβCD8 molecule, we demonstrate that cells expressing full-length αβCD8 were highly susceptible, αβCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-β. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-β suppression. Resistance was not associated with TGF-β receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-β. Based on the important role which TGF-β-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies.

Recombinant lentivector immunization has been demonstrated to induce potent CD8 T cell responses in vivo. In this study, we investigated whether lentivector delivering a self/tumor Ag, tyrosinase related protein 1 (TRP1), could stimulate effective antitumor T cell responses. We found that immunization with lentivector expressing mutated TRP1 Ag elicited potent CD8 T cell responses against multiple TRP1 epitopes. Importantly, the activated CD8 T cells effectively recognize wild-type TRP1 epitopes. At peak times, as many as 10% of CD8 T cells were effector cells against TRP1 Ag. These cells killed wild-type TRP1 peptide-pulsed target cells in vivo and produced IFN-γ after ex vivo stimulation. The CD8 T cell responses were long-lasting (3–4 wk). Immunized mice were protected from B16 tumor cell challenge. In a therapeutic setting, lentivector immunization induced potent CD8 T cell responses in tumor bearing mice. The number of infiltrating T cells and the ratio of CD8/CD4 were dramatically increased in the tumors of immunized mice. The tumor-infiltrating CD8 T cells were functional and produced IFN-γ. The potent CD8 T cell responses stimulated by lentivector immunization eliminated small 3-day s.c. B16 tumors and strongly inhibited the growth of more established 5-day tumors. These studies demonstrate that genetic immunization with lentivector expressing mutated self/tumor Ag can generate potent CD8 T cell immune responses and antitumor immunity that prevent and inhibit B16 tumor growth, suggesting that lentivector immunization has the potential for tumor immunotherapy and immune prevention.

Vitiligo is a T-cell-mediated autoimmune disease of the skin. Progressive depigmentation accelerates in response to stress. Personal trauma, contact with bleaching phenols, overexposure to UV, and mechanical injury can lead to progressive loss of melanocytes. This study was focused on the role of stress protein heat shock protein (HSP)70 for translating stress into an autoimmune disease to melanocytes. Intracellular HSP70 can act as a cytoprotectant, preventing apoptosis in cells under stress. Isoform HSP70i can be secreted by live cells, and in prior in vitro studies, HSP70 has been shown to activate dendritic cells and elicit an immune response to chaperoned proteins and peptides. Here, the role of HSP70 in precipitating and perpetuating vitiligo was assessed in vivo in a mouse model of autoimmune vitiligo. In this model, depigmentation was introduced by gene gun vaccination with eukaryotic expression plasmids encoding melanocyte differentiation antigens. Inclusion of human and mouse-derived inducible HSP70 in the vaccination protocol significantly increased and accelerated depigmentation in this model, accompanied by the induction of prolonged humoral responses to HSP70. Cytotoxicity toward targets loaded with a K(b)-restricted tyrosinase-related protein 2-derived peptide correlated with depigmentation. The data presented strongly support a role for HSP70i in progressive depigmentation in vivo.

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

One strategy to generate T-cell responses to tumors is to alter subdominant epitopes through substitution of amino acids that are optimal anchors for specific MHC molecules, termed heteroclitic epitopes. This approach is manually error-prone and time-consuming. In here, we describe a computer-based algorithm (EpitOptimizer) for the streamlined design of heteroclitic epitopes. Analysis of two cancer-related proteins showed that EpitOptimizer-generated peptides have enhanced MHC-I binding compared with their wild-type counterparts; and were able to induce stronger CD8+ T-cell responses against their native epitope. These data demonstrate that this approach can serve as the basis of epitope-engineering against cancer and intracellular pathogens.

Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.

Purpose: Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen.Experimental Design: Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection.Results: T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone.Conclusions: Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.

Xenogeneic DNA vaccination can elicit tumor immunity through T cell and antibody-dependent effector mechanisms. Blockade of CTLA-4 engagement with B7 expressed on APCs has been shown to enhance T cell-dependent immunity. We investigated whether CTLA-4 blockade could increase T-cell responses and tumor immunity elicited by DNA vaccines. CTLA-4 blockade enhanced B16 tumor rejection in mice immunized against the melanoma differentiation antigens tyrosinase-related protein 2 and gp100, and this effect was stronger when anti-CTLA-4 was administered with booster vaccinations. CTLA-4 blockade also increased the T-cell responses to prostate-specific membrane antigen (PSMA) when given with the second or third vaccination. Based on these pre-clinical studies, we suggest that anti-CTLA-4 should be tested with xenogeneic DNA vaccines against cancer and that special attention should be given to sequence and schedule of administration.

Major histocompatibility complex (mhc)–encoded molecules govern immune responses by presenting antigenic peptides to T cells. The extensive polymorphism of genes encoding these molecules is believed to enhance immune defense by broadening the array of antigenic peptides available for T cell recognition, but direct evidence supporting the importance of this mechanism in combating pathogens is limited. Here we linkmhcpolymorphism-driven diversification of the cytotoxic T lymphocyte (CTL) repertoire to the generation of high-avidity, protective antiviral T cells and to superior antiviral defense. Thus, much of the beneficial effect of themhcpolymorphism in immune defense may be due to its critical influence on the properties of the selected CTL repertoire.

Summary: The immune repertoire contains T cells and B cells that can recognize autologous cancer cells. This repertoire is directed against self, and in some cases altered self (mutations). Priming immune responses against self antigens can be difficult. Strategies are presented using altered self to elicit immunity against self in poorly immunogenic tumor models. Mechanisms underlying immunity to self antigens on cancer cells show that the immune system can use diverse strategies for cancer immunity, in both the immunization and the effector phases. CD4+ T cells are typically, but not always, required for immunization. The effector phase of tumor immunity can involve cytotoxic T cells, macrophages with activating Fc receptors, and/or killer domain molecules. This diversity in the effector phase is observed even when immunizing with conserved paralogs. A consequence of tumor immunity is potentially autoimmunity, which may be undesirable. Autoimmunity uses similar mechanisms as tumor immunity, but tumor immunity and autoimmunity can uncouple. These studies open up strategies for active immunization against cancer.

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Merozoite surface protein–1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti–MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti–MSP-119 antibodies. Our results suggest that an effective MSP-119–based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Immunization with effective cancer vaccines can offer a much needed adjuvant therapy to fill the treatment gap after liver resection to prevent relapse of hepatocellular carcinoma (HCC). However, current HCC cancer vaccines are mostly based on native shared-self/tumor antigens that are only able to induce weak immune responses. In this study we investigated whether the HCC-associated self/tumor antigen of alpha-fetoprotein (AFP) could be engineered to create an effective vaccine to break immune tolerance and potently activate CD8 T cells to prevent clinically relevant carcinogen-induced autochthonous HCC in mice. We found that the approach of computer-guided methodical epitope-optimization created a highly immunogenic AFP and that immunization with lentivector expressing the epitope-optimized AFP, but not wild-type AFP, potently activated CD8 T cells. Critically, the activated CD8 T cells not only cross-recognized short synthetic wild-type AFP peptides, but also recognized and killed tumor cells expressing wild-type AFP protein. Immunization with lentivector expressing optimized AFP, but not native AFP, completely protected mice from tumor challenge and reduced the incidence of carcinogen-induced autochthonous HCC. In addition, prime-boost immunization with the optimized AFP significantly increased the frequency of AFP-specific memory CD8 T cells in the liver that were highly effective against emerging HCC tumor cells, further enhancing the tumor prevention of carcinogen-induced autochthonous HCC. Conclusions: Epitope-optimization is required to break immune tolerance and potently activate AFP-specific CD8 T cells, generating effective antitumor effect to prevent clinically relevant carcinogen-induced autochthonous HCC in mice. Our study provides a practical roadmap to develop effective human HCC vaccines that may result in an improved outcome compared to the current HCC vaccines based on wild-type AFP. (Hepatology 2014;59:1448-1458)

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8(+) T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8(+) T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.

The ability to form memory is a major characteristic of the adaptive immune system. NKG2D, as a costimulatory molecule for CD8+ T cells, was shown to rescue memory formation in absence of CD4+ help. However, what exactly is the function of NKG2D on CD8+ T cells and how important is NKG2D for memory formation is not known. To recapitulate a full course of CD8+ T cell activation, from priming to memory recall, we immunized C57BL/6 mice with peptide-pulsed dendritic cells and followed the CD8+ T cell responses at priming, effector and memory phases. Using NKG2D deficient (NKG2D-KO) CD8+ T cells, we found that NKG2D signaling was not necessary to achieve CD8+ T cell priming. However, NKG2D-KO CD8+ T cells showed lower effector functions (reduced in vivo kill and reduced cytokine secretion upon in vitro restimulation) compared to wildtype CD8+ T cells. Blocking NKG2D signaling at the effector phase with a single injection of anti-NKG2D antibody (non-depleting) recapitulated the results observed with NKG2D-KO CD8+ T cells. Importantly, the CD8+ T cells that lacked NKG2D signaling at effector phase failed to differentiate into protective memory cells. These results indicate that NKG2D signaling certifies CD8+ T cells to become memory cells and that this certification occurs during the effector phase.

In this study, we demonstrate that engagement of two different natural killer receptors (NKRs) can lead to contrasting effects in the development of self-reactive CD8+T cells and autoimmune vitiligo. Specifically, using a mouse model, we show that CD8+T-cell targeting of a melanocyte antigen, tyrosinase-related protein-1 (TRP-1) in combination with delivery of the NKG2D ligands (Rae-1ϵ or H60), results in strong CD8+T-cell responses against TRP-1 and in the development of autoimmune vitiligo. In contrast, targeting of TRP-1 in combination with delivery of CD48, the natural ligand for the NKR 2B4, leads to reduced formation of TRP-1-reactive CD8+T-cell responses and decreased development of vitiligo. These data indicate that autoimmune vitiligo is limited by insufficient signals, despite plentiful self-reactive T cells in the peripheral immune system. To our knowledge, this is the first experimental evidence supporting the role of NKRs in modulating CD8+T-cell autoimmune vitiligo. This study supports the utilization of NKR signaling as a therapeutic avenue toward prevention of vitiligo and other autoimmune diseases.

A main goal of cancer immunology research is the formation of Ag-specific memory T cell immunity capable of activation upon tumor re-encounter. The requirements necessary to overcome the inhibitory signals present in the tumor microenvironment and form such memory T cell responses are unknown. In contrast to previous studies targeting tumors expressing highly immunogenic model Ags, we demonstrate that alleviating tumor-induced suppression along with vaccination against authentic Ags during the perioperative period provides long-lasting protection against a highly suppressive and poorly immunogenic melanoma. In this study, we employed DNA vaccination with an immunologically optimized mouse melanoma-shared Ag, Trp1ee/ng, combined with systemic TGF-β blockade during the perioperative period of primary tumor resection, to confer protection against B16 melanoma, and against JBRH, an independently derived melanoma unrelated to B16. Importantly, we demonstrate that correlative to memory responses, perioperative immunotherapy increases the formation of tumor-infiltrating and tumor-reactive CD8+ T cells expressing low levels of the transcription factor T-bet, defined as memory precursor effector cells. We show that conditions for an immunologically fertile environment are met when TGF-β blockade and vaccination are applied during the perioperative period of primary tumor resection. These findings address limitations of current CD8+ T cell immunotherapies against cancer by generating effective CD8+ T cell memory recall responses.

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4+CD25+ regulatory T (Treg) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to Treg cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by Treg cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.

Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1 19 ) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1 19 can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab′) 2 fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human γ1 and γ3 constant regions, retain the ability to bind to both parasites and recombinant MSP1 19 , and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcγ receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.

Objective clinical responses can be achieved in melanoma patients by infusion of T cell receptor (TCR) gene transduced T cells. Although promising, the therapy is still largely ineffective, as most patients did not benefit from treatment. That only a minority of the infused T cells were genetically modified and that these were extensively expanded ex vivo may have prevented their efficacy. We developed novel and generally applicable retroviral vectors that allow rapid and efficient selection of T cells transduced with human TCRs. These vectors encode two TCR chains and a truncated CD34 molecule (CD34t) in a single mRNA transcript. Transduced T cells were characterized and the effects of CD34-based enrichment of redirected T cells were evaluated. Both CD8(+) and CD4(+) T cells could be transduced and efficiently co-expressed all introduced transgenes on their surface. Importantly, more than fivefold enrichment of both the frequency of transduced cells and the specific anti-tumor reactivity of the effector population could be achieved by magnetic beads-based enrichment procedures readily available for clinical grade hematopoietic stem cell isolation. This CD34-based enrichment technology will improve the feasibility of adoptive transfer of clinically relevant effectors. In addition to their enhanced tumor recognition, the enriched redirected T cells may also show superior reactivity and persistence in vivo due to the high purity of transduced cells and the shortened ex vivo culture.

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8 + T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53–61, was able to activate high levels of PSA-specific CD8 + T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53–61 was also the only peptide that induced human T cells to produce IFNγ after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNγ producing CD8 + T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP → B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.

T cells recognizing self antigens expressed by cancer cells are prevalent in the immune repertoire. However, activation of these autoreactive T cells is limited by weak signals that are incapable of fully priming naive T cells, creating a state of tolerance or ignorance. Even if T cell activation occurs, immunity can be further restricted by a dominant response directed at only a single epitope. Enhanced antigen presentation of multiple epitopes was investigated as a strategy to overcome these barriers. Specific point mutations that create altered peptide ligands were introduced into the gene encoding a nonimmunogenic tissue self antigen expressed by melanoma, tyrosinase-related protein-1 (Tyrp1). Deficient asparagine-linked glycosylation, which was caused by additional mutations, produced altered protein trafficking and fate that increased antigen processing. Immunization of mice with mutated Tyrp1 DNA elicited cross-reactive CD8(+) T cell responses against multiple nonmutated epitopes of syngeneic Tyrp1 and against melanoma cells. These multi-specific anti-Tyrp1 CD8(+) T cell responses led to rejection of poorly immunogenic melanoma and prolonged survival when immunization was started after tumor challenge. These studies demonstrate how rationally designed DNA vaccines directed against self antigens for enhanced antigen processing and presentation reveal novel self epitopes and elicit multi-specific T cell responses to nonimmunogenic, nonmutated self antigens, enhancing immunity against cancer self antigens.

Peripheral T cell diversity is virtually constant in the young, but is invariably reduced in aged mice and humans. CD8+ T cell clonal expansions (TCE) are the most drastic manifestation of, and possible contributors to, this reduced diversity. We show that the presence of TCE results in reduced CD8+, but not CD4+, T cell diversity, and in functional inability to mobilize parts of the CD8+ T cell repertoire affected by TCE. In the model of herpes simplex virus (HSV)-1 infection of B6 mice, >90% of the responding CD8+ T cells use Vβ10 or Vβ8 and are directed against a single glycoprotein B (gB498-505) epitope, gB-8p. We found that old animals bearing CD8+ TCE within Vβ10 or Vβ8 families failed to mount an effective immune response against HSV-1, as judged by reduced numbers of peptide-major histocompatibility complex tetramer+ CD8 T cells and an absence of antiviral lytic function. Furthermore, Vβ8 TCE experimentally introduced into young mice resulted in lower resistance to viral challenge, whereas Vβ5+ TCE induced in a similar fashion did not impact viral resistance. These results demonstrate that age-related TCE functionally impair the efficacy of antiviral CD8+ T cell immunity in an antigen-specific manner, strongly suggesting that TCE are not the mere manifestation of, but are also a contributing factor to, the immunodeficiency of senescence.

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

The adaptive immune system is capable of recognizing cancer through T- and B-cell receptors. However, priming adaptive immunity against self antigens is potentially a difficult task. Presentation of altered self to the immune system is a strategy to elicit immunity against poorly immunogenic antigens. We have shown that immunization with conserved paralogues of tumor antigens can induce adaptive immunity against self antigens expressed by cancer. Remarkably, cancer immunity elicited by closely related paralogues can generate distinct adaptive immune responses, either antibody or T-cell dependent. Cancer immunity induced by xenogeneic immunization follows multiple and alternative pathways. The effector phase of tumor immunity can be mediated by cytotoxic T cells or macrophages and perhaps natural killer cells for antibody-dependent immunity. Helper CD4+ T cells are typically, but not always, required to generate immunity. Autoimmunity is frequently observed following immunization. Cancer immunity and autoimmunity use overlapping mechanisms, and therefore they are difficult to uncouple, but distinct pathways can be discerned that open the eventual possibility of uncoupling tumor immunity from autoimmunity. Studies examining the molecular basis for immunogenicity of conserved paralogues are facilitating the development of new strategies to rationally design vaccines that trigger adaptive immune responses to cancer.

Cancer cells express self-antigens that are weakly recognized by the immune system. Even though responses against autologous cells are difficult to induce, the immune system is still able to mount a response against cancer. The discovery of the molecular identity of antigens that are recognized by the immune system of melanoma patients has led to the elucidation of tumor immunity at a cellular and molecular level. Multiple pathways in both the priming and effector phases of melanoma rejection have been described. Animal models' active immunotherapies for melanoma show a requirement for the cellular compartment of the immune system in the priming phase, primarily CD4+T cells. More diverse elements are required for the effector phase, including components from the innate immune system (macrophages, complement and/or natural killer cells) and from the adaptive immune system (CD8+T cells and B cells). Minor differences in amino-acid sequences of antigens must determine the particular mechanisms involved in tumor rejection. Since the immune system contains T and B cells that recognize and reject autologous cells, a consequence of tumor immunity is potential autoimmunity. There are distinct pathways for tumor immunity and autoimmunity. The requirements for autoimmunity at the priming phase seem to be CD4+/IFN-gamma dependent while the effector mechanisms are alternative and redundant. Vitiligo (autoimmune hypopigmentation) can be mediated by T cells, FcgammaR+macrophages and/or complement.

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 Db-restricted peptide, hgp10025–33, were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp10025–27 epitope was substituted with the weaker Db-binding epitope from mgp100 (mgp10025–27) or a mutated epitope unable to bind Db did not reject B16 melanoma. Mice immunized with a minigene construct of hgp10025–33 rejected B16 melanoma, whereas mice immunized with the mgp10025–33 minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2b mice with melanoma.

The adaptive immune system is capable of recognizing cancer through T- and B-cell receptors. However, priming adaptive immunity against self antigens is potentially a difficult task. Presentation of altered self to the immune system is a strategy to elicit immunity against poorly immunogenic antigens. We have shown that immunization with conserved paralogues of tumor antigens can induce adaptive immunity against self antigens expressed by cancer. Remarkably, cancer immunity elicited by closely related paralogues can generate distinct adaptive immune responses, either antibody or T-cell dependent. Cancer immunity induced by xenogeneic immunization follows multiple and alternative pathways. The effector phase of tumor immunity can be mediated by cytotoxic T cells or macrophages and perhaps natural killer cells for antibody-dependent immunity. Helper CD4+ T cells are typically, but not always, required to generate immunity. Autoimmunity is frequently observed following immunization. Cancer immunity and autoimmunity use overlapping mechanisms, and therefore they are difficult to uncouple, but distinct pathways can be discerned that open the eventual possibility of uncoupling tumor immunity from autoimmunity. Studies examining the molecular basis for immunogenicity of conserved paralogues are facilitating the development of new strategies to rationally design vaccines that trigger adaptive immune responses to cancer.