Defining the role of IL-15 trans-presentation by distinct cell-types during the development and homeostasis of Natural Killer and invariant Natural Killer T cells

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK cells, I revealed the non-hematopoietic compartment provides IL-15 to NK cells in the early stages of development while hematopoietic cells were crucial for the generation and maintenance of mature NK cells. Regarding iNKT cells in the thymus, IL-15 trans-presentation by non-hematopoietic cells was crucial for the survival of mature iNKT cells. In the liver, both hematopoietic and non-hematopoietic compartments provided IL-15 to both immature and mature iNKT cells. This IL-15 signal helped mediate the survival and proliferation of both NK and iNKT cells as well as induce the functional maturation of mature iNKT cells via enhanced T-bet expression. In conclusion, my work illustrates an important notion that the immunological niche of NK and iNKT cells is tightly regulated and that this regulation is meticulously influenced by the tissue microenvironment.

Simultaneous induction of NKG2D and NKG2D ligand for promoting immune surveillance by IL-12 plus doxorubicin treatment

NKG2D (natural killer group 2, member D) and its ligands interaction in tumor microenvironment directs tumor infiltrating immune cells to recognize tumor cells, stimulate cytotoxic effector immune cells, and therefore eradicate tumor cells. IL-12, a cytokine produced by antigen presenting cells, has remarkable antitumor effect by activating innate and adaptive immunity. Doxorubicin, a commonly used chemotherapeutic agent also boosts the host antitumor immune response to cause tumor cell death. Our previous publication suggests that IL-12 plus doxorubicin enhances NKG2D function-dependent inhibition of tumor progression and promotes CD8+T cells infiltrating into tumors. The purpose of this study is to determine the underlying mechanism. Our study reveals a novel function of doxorubicin, which is to augment IL-12–induced NKG2D expression in CD8+T cells but not in NK or CD4+T cells. This observation was further validated by NK and CD8+T cell-depletion studies, in which only depletion of CD8+T cells abolished the expression of NKG2D in lymphocytes. The induced NKG2D expression in CD8+T cells is tightly associated with tumor-specific localization of CD8+T cells and improved antitumor efficacy. The IL-12 plus doxorubicin treatment-induced antitumor efficacy is also due to NKG2D ligand Rae-1 induction in tumors. Rae-1 induction in tumors is a long term effect in multiple tumor models, but not in normal tissues. A novel CD8+T cell direct contact dependent mechanism accounts for Rae-1 induction in vivo and in vitro, and CD80 is the receptor through which CD8+T cells interplay with tumor cells to upregulate Rae-1 on tumor cells. In summary, increased NKG2D expression in CD8+T cells in response to IL-12 plus doxorubicin was closely associated with tumor-specific localization of CD8+T cells and greater antitumor efficacy of the combined regimen than either agent alone. NKG2D ligand Rae-1 induction is triggered by the interaction of CD80 on tumor cells with tumor infiltrating CD+8 T cells.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

The role of plasmacytoid dendritic cells in the regulation of adaptive immunity

Plasmacytoid dendritic cells (pDCs) selectively express TLR7 which allows them to respond to RNA viruses and TLR9 which allows them to respond to DNA viruses and CpG oligonucleotides. Upon exposure to virus pDCs produce vast amounts of type I interferon (IFN) directly inhibiting viral replication and contributing to the activation of other immune cells. The ability of pDCs to promote B and T cell differentiation through type I IFN has been well documented although the role of additional factors including tumor necrosis factor (TNF) family members has not been thoroughly addressed. Here the expression of selected TNF family members in pDCs was examined and the role of TNF receptor-ligand interactions in the regulation of B and T lymphocyte growth and differentiation by pDCs was investigated. Upon stimulation with CpG-B, pDCs exhibit strong and stable expression of CD70, a TNF family ligand that binds to its receptor CD27 on memory B cells and promotes plasma cell differentiation and Ig secretion. Using an in vitro pDC/B cell co-culture system, it was determined that CpG-B-stimulated pDCs induce the proliferation of CD40L-activated human peripheral B cells and Ig secretion. This occurs independently of IFN and residual CpG, and requires physical contact between pDCs and B cells. CpG-stimulated pDCs induce the proliferation of both naive and memory B cells although Ig secretion is restricted to the memory subset. Blocking the interaction of CD70 with CD27 using an antagonist anti-CD70 antibody reduces the induction of B cell proliferation and IgG secretion by CpG-B-stimulated pDCs. Published studies have also indicated an important role for CD70 in promoting the expansion of CD4+ and CD8+ T cells and the development of effector function. CpG-B-stimulated pDCs induce naïve CD4+ T cell proliferation and production of multiple cytokines including IFN-γ, TNF-α, IL-10, IL-4, IL-5 and IL-13. Blocking the function of CD70 with an antagonist anti-CD70 antibody significantly reduced the induction of naïve CD4+ T cell proliferation by CpG-B-stimulated pDCs and the production of IL-4 and IL-13. Collectively these data indicate an important role for CD70 in the regulation of B and T lymphocyte growth and differentiation by pDCs. ^

CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT

Adjuvants are essential components of vaccine formulations that enhance adaptive immune responses to antigens, particularly for immunizations targeting the tolerogenic mucosal tissues, which are more biologically relevant for protective immunity against pathogens transmitted by the mucosal routes. Adjuvants possess the inherent capacity to bridge innate and adaptive immune responses through activating innate immune mediators. Here evidence is presented in support of the effectiveness of a synthetic glycolipid, alpha-Galactosylceramide (-GalCer), as an adjuvant for mucosal immunization with peptide and protein antigens, by oral and intranasal routes, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. The adjuvant activity of -GalCer delivered by the intranasal route was manifested in terms of potent activation of NKT cells, an important innate immunity mediator, along with the activation of dendritic cells (DC) which serve as the professional antigen-presenting cells. Data from this investigation provide the first evidence for mucosal delivery as an effective means to harness the adjuvant potential of α-GalCer for priming as well as boosting cellular immune responses to co-administered immunogens. Unlike systemic administration where a single dose of α-GalCer leads to anergy of responding NKT cells and thus hinders delivery of booster immunizations, we demonstrated that administration of multiple doses of α-GalCer by the intranasal route affords repeated activation of NKT cells and the induction of broad systemic and mucosal immunity. This is specifically advantageous, and may be even essential, for vaccination regimens against mucosal pathogens such as the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), where priming of durable protective immunity at the mucosal portals of pathogen entry would be highly desirable.

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

A retrospective cohort study of the duration of hepatitis B vaccine immunity and associated factors in pediatric dialysis patients

Dialysis patients are at high risk for hepatitis B infection, which is a serious but preventable disease. Prevention strategies include the administration of the hepatitis B vaccine. Dialysis patients have been noted to have a poor immune response to the vaccine and lose immunity more rapidly. The long term immunogenicity of the hepatitis B vaccine has not been well defined in pediatric dialysis patients especially if administered during infancy as a routine childhood immunization.^ Purpose. The aim of this study was to determine the median duration of hepatitis B immunity and to study the effect of vaccination timing and other cofactors on the duration of hepatitis B immunity in pediatric dialysis patients.^ Methods. Duration of hepatitis B immunity was determined by Kaplan-Meier survival analysis. Comparison of stratified survival analysis was performed using log-rank analysis. Multivariate analysis by Cox regression was used to estimate hazard ratios for the effect of timing of vaccine administration and other covariates on the duration of hepatitis B immunity.^ Results. 193 patients (163 incident patients) had complete data available for analysis. Mean age was 11.2±5.8 years and mean ESRD duration was 59.3±97.8 months. Kaplan-Meier analysis showed that the total median overall duration of immunity (since the time of the primary vaccine series) was 112.7 months (95% CI: 96.6, 124.4), whereas the median overall duration of immunity for incident patients was 106.3 months (95% CI: 93.93, 124.44). Incident patients had a median dialysis duration of hepatitis B immunity equal to 37.1 months (95% CI: 24.16, 72.26). Multivariate adjusted analysis showed that there was a significant difference between patients based on the timing of hepatitis B vaccination administration (p<0.001). Patients immunized after the start of dialysis had a hazard ratio of 6.13 (2.87, 13.08) for loss of hepatitis B immunity compared to patients immunized as infants (p<0.001).^ Conclusion. This study confirms that patients immunized after dialysis onset have an overall shorter duration of hepatitis B immunity as measured by hepatitis B antibody titers and after the start of dialysis, protective antibody titer levels in pediatric dialysis patients wane rapidly compared to healthy children.^

The Influence of Immunization Route on the Adjuvant Effect of alpha-Galactosylceramide

Potent vaccine formulations ideally include adjuvants to activate innate immune responses and enhance antigen-specific adaptive immunity. The synthetic glycolipid alpha-Galactosylceramide (α-GalCer) effectively activates the innate immune mediating NKT cells to produce cytokines and activate downstream immune cells, resulting in development of humoral and cell mediated immune responses to co-administered antigens. While a single intravenous immunization of α-GalCer strongly activates NKT cells, multiple doses by this route are well documented to induce anergy in NKT cells. Anergy is defined as the deficiency in NKT proliferation and cytokine production, including IL-4 and IFNγ. However, our studies have shown that two doses of α-GalCer administered intranasally by the intranasal route leads to reactivation of NKT cells and improved adaptive immune responses after each subsequent dose. I therefore investigated the role of multiple routes of immunization in activation of NKT cells, i.e. anergy versus repeated activation. Specifically, I hypothesized that the differential capacity of NKT cells to produce IFNγ, as a result of route of immunization with α-GalCer, influences the induction of adaptive immune responses to co-administered antigen. Our experimental design utilizes the observation that intranasal immunization primarily induces immune responses in the lungs while intravenous immunization induces responses in the liver. Using intracellular cytokine staining for IFNγ production and Elispot analyses for determining NKT and T cell activation, respectively, it was determined that administering two consecutive intravenous doses resulted in anergy to NKT cells (no IFNγ production) in the liver and lack of adaptive immunity while second immunization by the intranasal route overcame anergy in the lung. The outcome in the other tissues analyzed was mixed and could be the result of tissue microenvironment among others possible reasons. When intranasal dosing preceded systemic, NKT cells were reactivated to produce IFNγ and induced positive adaptive immune responses in the responding lung tissue. These results indicate that the mechanism by which mucosal and systemic immunization routes activate NKT cells may differ in that there is a differential tissue-specific effect induced by each route. Future studies are necessary to determine the reason for these tissue-specific effects and how they relate to NKT cell activation.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^

Immunologic characteristics of immunogenic variants generated by {\it in vitro\/} treatment of a methylcholanthrene-induced murine fibrosarcoma MCA-F with 1 methyl-3-nitro-1-nitrosoguanidine, 5 -aza-2$\sp\prime$-deoxycytidine, or ultraviolet radiation

This study addresses the questions of whether the frequency of generation and in vivo cross-reactivity of highly immunogenic tumor clones induced in a single parental murine fibrosarcoma cell line MCA-F is more closely related to the agent used to induce the Imm$\sp{+}$ clone or whether these characteristics are independent of the agents used. These questions were addressed by treating the parental tumor cell line MCA-F with UV-B radiation (UV-B), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), or 5-aza-2$\sp\prime$-deoxycytidine (5-azaCdR). The frequency of Imm$\sp{+}$ variant generation was similarly high for the three different agents, suggesting that the frequency of Imm$\sp{+}$ generation was related more closely to the cell line than to the inducing agent used. Cross-reactivity was tested with two Imm$\sp{+}$ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental immunity. Under these conditions each clone, except one, immunized against itself. The MNNG-induced clones engendered stronger antivariant immunity but a weaker variant cross-reactive immunity could also be detected.^ This study also characterized the lymphocyte populations responsible for antivariant and antiparental immunity in vivo. Using the local adoptive transfer assay (LATA) and antibody plus complement depletion of T-cell subsets, we showed that immunity induced by the Imm$\sp{+}$ variants against the parent MCA-F was transferred by the Thy1.2$\sp{+}$, L3T4a$\sp{+}$, Lyt2.1$\sp{-}$ (CD4$\sp{+}$) population, without an apparent contribution by Thy1.2$\sp{+}$, L3T4a$\sp{-}$, Lyt2.1$\sp{+}$ (CD8$\sp{+}$) cells. A role for Lyt2.1$\sp{+}$T lymphocytes in antivariant, but not antiparent immunity was supported by the results of LATA and CTL assays. Immunization with low numbers of viable Imm$\sp{+}$ cells, or with high numbers of non viable Imm$\sp{+}$ cells engendered only antivariant immunity without parental cross-protection. The associative recognition of parental antigens and variant neoantigens resulting in strong antiparent immunity was investigated using somatic cells hybrids of Imm$\sp{+}$ variants of MCA-F and an antigenically distinct tumor MCA-D. An unexpected result of these latter experiments was the expression of a unique tumor-specific antigen by the hybrid cells. These studies demonstrate that the parental tumor-specific antigen and the variant neoantigen must be coexpressed on the cell surface to engender parental cross-protective immunity. (Abstract shortened with permission of author.) ^