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Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.

e15211 Background: Immunotherapy approaches are limited in their ability to induce antigen-specific CD8+ T cells in vivo able to recognize and kill tumor cells. We developed a novel immunotherapy approach using enantiomerically pure, R-DOTAP cationic lipid nanoparticles and tumor-derived T cell antigens, and previously demonstrated that R-DOTAP formulations efficiently prime cytotoxic T cells through enhanced cross presentation and induction of type I interferons.[1] A phase I clinical trial of a R-DOTAP HPV16 peptide formulation confirmed induction of strong in vivo HPV-specific CD8+ cytolytic T-cells without associated systemic toxicities. In this study, we assessed R-DOTAP nanoparticle formulations containing whole protein (ovalbumin) or long multi-epitope peptides from the tumor antigen TARP (T-cell alternate reading frame protein): a 58-residue protein overexpressed in prostate and breast cancers, documented to be immunogenic in humans. Methods: R-DOTAP formulations were prepared containing ovalbumin (OVA) or TARP peptides. C57BL/6K mice were immunized with 10 μg/mouse of OVA plus R-DOTAP, CFA or sucrose on Days 0, 15 and 30. OVA-specific cellular and humoral responses following vaccination were assessed by measuring splenic CD4 and CD8 T cell IFN-γ production and circulating OVA-specific antibodies in serum. HLA-A2 transgenic mice (AAD mice) were vaccinated with long, multi-epitope TARP peptides delivered as an R-DOTAP admixture or with CFA or sucrose on Days 0 and 7. Antigen-specific T cell responses were measured by IFN-γ ELISpot assay. Results: OVA R-DOTAP formulations induced strong antigen-specific effector CD4 and CD8 immune and memory responses detected 7 and 30 days, respectively, following vaccination as well as OVA-specific antibody responses. In TARP peptide vaccinated mice, R-DOTAP formulations were able to present multiple CD8 T cell epitopes and stimulate responses that were superior to CFA. Conclusions: Our results suggest that R-DOTAP is a versatile immune activating therapy that can be formulated with long, multi-epitope tumor-derived peptides or whole proteins. R-DOTAP formulations induce quantitatively robust antigen-specific CD4 and CD8 T cells in vivo compared to well-established immune stimulants. Reference: 1.Gandhapudi SK, Ward M, Bush JP et al. Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response. J Immunol 2019;202:3524-3536

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11.10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high-affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs were 214.9 nm and -14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24h (5%). We further evaluated the antibody response of the Ni-NCs using His-Gag p41 as a model antigen. Formulations were administered subcutaneously to BALB/c mice at day 0 (prime) and 14 (boost) followed by serum collection on day 28. Serum His-Gag p41-specific antibody levels were found to be significantly higher at 1 and 0.5 μg doses of Gag p41-His-Ni-NCs (His-Gag p41 equivalent) compared with His-Gag p41 (1 μg) adjuvanted with aluminum hydroxide (AH). The serum IgG2a levels induced by Gag p41-His-Ni-NCs (1 μg) were significantly higher than AH adjuvanted His-Gag p41. The Ni-NCs alone did not result in the elevation of systemic IL-12/p40 and CCL5/RANTES inflammatory cytokine levels upon subcutaneous administration in BALB/c mice. In conclusion, the proposed Ni-NCs can bind His-tagged proteins and have the potential to be used as antigen delivery system capable of generating strong antigen-specific antibodies at doses much lower than with aluminum-based adjuvant and causing no significant elevation of systemic pro-inflammatory IL-12/p40 and CCL5/RANTES cytokines.

The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system.

Weili Yan1, Anekant Jain1, Ronan O’Carra2, JeroldG Woodward3, Wenxue Li4, Guanhan Li4, Avindra Nath4, Russell J Mumper11Division of Molecular Pharmaceutics and the Center for Nanotechnology in Drug Delivery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky; 3Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY, USA; 4Department of Neurology, Johns Hopkins University, Baltimore, MD, USAAbstract: Lipid-based nanoparticles (NPs) with a small amount of surface-chelated nickel (Ni-NPs) were developed to easily formulate the human immunodeficiency virus (HIV) his-tagged Tat (his-Tat) protein, as well as to formulate and co-deliver two HIV antigens (his-p24 and his-Nef) on one particle. Female BALB/c mice were immunized by subcutaneous injection with his-Tat/Ni-NP formulation (1.5 µg his-Tat/mouse) and control formulations on day 0 and 14. The day 28 anti-Tat specific immunoglobulin G titer with his-Tat/Ni-NPs was significantly greater than that with Alum/his-Tat. Furthermore, splenocytes from his-Tat/Ni-NP-immunized mice secreted significantly higher IFN-γ than those from mice immunized with Alum/his-Tat. Although Ni-NPs did not show better adjuvant activity than Tat-coated anionic NPs made with sodium dodecyl sulfate (SDS/NPs), they were less toxic than SDS/NPs. The initial results indicated that co-immunization of mice using his-p24/his-Nef/Ni-NP induced greater antibody response compared to using Alum/his-p24/his-Nef. Co-delivery of two antigens using Ni-NPs also increased the immunogenicity of individual antigens compared to delivery of a single antigen by Ni-NPs. In conclusion, Ni-NPs are an efficient delivery system for HIV vaccines including both single antigen delivery and multiple antigen co-delivery.Keywords: nanoparticle, nickel, HIV, antigen co-delivery, vaccine

The purpose of these studies was to prepare nanoparticles (NPs) with a small amount of surface-chelated nickel for obtaining enhanced binding of histidine-tagged (his-tag) proteins compared to non-histidine-tagged protein binding to charged nanoparticles.NPs were prepared from oil-in-water microemulsion precursors using emulsifying wax, 3 mM Brij 78 and 0.1 mM DOGS-NTA-Ni lipid (referred to as Ni-NPs). The amount of lipid entrapped in the NPs was quantitated by atomic emission spectroscopy (AES). The Ni-NPs were investigated for binding to two his-tag proteins, green fluorescent protein (GFP) and his-tag HIV-1 Gag p24. In vivo studies in mice were carried out to evaluate the immune responses obtained to his-tag Gag p24 bound to Ni-NPs.AES studies demonstrated that approximately 5% of the DOGS-NTA-Ni lipid used was entrapped in the NPs. The optimal binding ratio his-tag GFP and his-tag Gag p24 to Ni-NPs was found to be 1:33.7 and 1:35.4 w/w, respectively. This interaction was stable at 37 degrees C in PBS, pH 7.4 over 4 h and the interaction of his-tag GFP with the Ni-NPs was enhanced compared to control NPs prepared with no Ni on the surface (NTA-NPs). The in vivo studies demonstrated enhanced serum IgG and IgG2a responses to his-tag Gag p24 bound to Ni-NPs compared to protein adjuvanted with Alum or adsorbed on the surface of control NTA-NPs.Ni-NPs can be used to bind strongly to his-tag proteins. This system was demonstrated to have potential applications in vaccine delivery for enhancing immune responses to protein-based vaccines.

A significant emphasis has been placed on the development of adjuvants and/or delivery systems to improve both antibody production and cell-mediated immune responses. We previously reported on a novel anionic nanoparticle, which led to enhanced humoral and T helper type-1 (Th1) biased immune responses in mice when coated with cationized model antigen. Tat (1-72) is a conserved regulatory HIV-1 protein. It was hypothesized that HIV vaccine strategies employing Tat (1-72) may be a promising approach. Although previous reports have suggested that Tat (1-86) may be immunosuppressive, it was demonstrated in this present study that Tat (1-72) was not immunosuppressive when co-administered to mice with ovalbumin (OVA). Tat (1-72) was coated on novel anionic nanoparticles. BALB/c mice were immunized with Tat (5 microg)-coated nanoparticles (15 microg) by subcutaneous injection on days 0 and 14. Antibody and cytokine release were determined on day 28 and compared to Tat (5 microg) adjuvanted with Alum (15 microg) as a Th2 control, Tat (5 microg) adjuvanted with Lipid A (50 microg) as a Th1 control. Immunization of BALB/c mice with Tat-coated nanoparticles resulted in antibody levels (IgG and IgM) comparable to those elicited from Tat and Alum. However, Tat-coated nanoparticles led to a Th1 biased immune response. The IFN-gamma release from splenocytes with Tat-coated nanoparticles was comparable to that from mice immunized with Tat and Lipid A, and 3.3-fold greater than that from mice immunized with Tat and Alum. These studies warrant further investigation of these nanoparticles to enhance both antibody and cellular-based immune responses.

We reported that antigenic preparations from Yersinia enterocolitica stimulate murine T cells in a manner consistent with that of superantigens. As a consequence we examined whether Y. enterocolitica antigenic preparations stimulate human T-cell cultures. Human T cells, enriched from peripheral blood lymphocytes, were stimulated to proliferate in the presence of Y. enterocolitica cytoplasmic and membrane preparations. This activity has also been shown to be sensitive to protease treatment, indicating the presence of a protein, and when separated by ion-exchange chromatography a single peak of activity is resolved. Furthermore, this proliferation was inhibited, in a dose-dependent manner, by the presence of antibodies directed against MHC class II antigens, indicating a requirement for these molecules. When these cells were stained with a panel of V beta-specific antibodies to determine if there was an enrichment of a particular V beta-bearing T-cell subset after stimulation, results indicate a significant enrichment of T cells bearing V beta 3, V beta 12, V beta 14, and V beta 17 over controls. Taken together, these data are consistent with a Y. enterocolitica product acting as a superantigen for human T cells.

Mycoplasma fermentans (incognitus strain) is a recently identified new human pathogen and suspected cofactor in acquired immune deficiency syndrome. Because this organism appears to exert strong immunosuppressive properties of its own, we decided to investigate whether it was capable of inducing MHC class II expression, as we have observed for other species of mycoplasma. In this report we demonstrate that M. fermentans (incognitus strain) is capable of producing factors that increase MHC class II expression as well as MHC class I expression on the myelomonocytic cell line, WEHI-3 cells, We also present data showing that these mycoplasmal factors induce small, although significant, increases in MHC class I and II antigens on a mouse glioma cell line, G26-20, and MHC class II expression on the human monocyte cell lines, U-937 and HL-60. Using nuclear run-on analysis, we show that the mycoplasma-induced increase in MHC expression is at least partially due to an increase in transcription of the MHC genes. Furthermore, we show that the factor that mediates this activity is sensitive to protease treatment, indicating that it is, at least in part, protein. These results demonstrate that M. fermentans (incognitus strain) is capable of modulating the expression of immunologically important MHC genes in both murine and human cell lines, which may prove to be an important factor in the pathogenesis of this organism.

Background: A small fraction of dietary protein survives enzymatic degradation and is absorbed in potentially antigenic form. This can trigger inflammatory responses in patients with celiac disease or food allergies, but typically induces systemic immunological tolerance (oral tolerance). At present it is not clear how dietary antigens are absorbed. Most food staples, including those with common antigens such as peanuts, eggs, and milk, contain long-chain triglycerides (LCT), which stimulate mesenteric lymph flux and postprandial transport of chylomicrons through mesenteric lymph nodes (MLN) and blood. Most dietary antigens, like ovalbumin (OVA), are emulsifiers, predicting affinity for chylomicrons. We hypothesized that chylomicron formation promotes intestinal absorption and systemic dissemination of dietary antigens. Methodology/Principal Findings: Absorption of OVA into MLN and blood was significantly enhanced when OVA was gavaged into fasted mice together with LCT compared with medium-chain triglycerides (MCT), which do not stimulate chylomicron formation. The effect of LCT was blocked by the addition of an inhibitor of chylomicron secretion, Pluronic L81. Adoptively transferred OVA-specific DO11.10 T-cells proliferated more extensively in peripheral lymph nodes when OVA was gavaged with LCT than with MCT or LCT plus Pluronic L-81, suggesting that dietary OVA is systemically disseminated. Most dietary OVA in plasma was associated with chylomicrons, suggesting that these particles mediate systemic antigen dissemination. Intestinal-epithelial CaCo-2 cells secreted more cell-associated, exogenous OVA when stimulated with oleicacid than with butyric acid, and the secreted OVA appeared to be associated with chylomicrons. Conclusions/Significance: Postprandial chylomicron formation profoundly affects absorption and systemic dissemination of dietary antigens. The fat content of a meal may affect immune responses to dietary antigens by modulating antigen absorption and transport.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill 51 Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia − . The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.

Peritoneal exudate cells from strains of mice both resistant and susceptible to challenge with mouse hepatitis virus strain JHM were examined for extrinsic and intrinsic antiviral activity. Thioglycolate-elicited and resident peritoneal cells from uninfected mice were able to suppress viral growth in a permissive cell. The active cell in both populations is an adherent, radiation-resistant, Thy-1.2 antigen- and Ia antigen-negative cell. The suppression of virus replication was not related to nonspecific cellular cytotoxicity directed against the permissive host cell, and no interferon was detected. The expression of extrinsic antiviral activity was not related to the ability of the host to resist mouse hepatitis virus infection by virtue of either age or genetic background. The expression of intrinsic antiviral activity, on the other hand, correlated with the ability of the host to resist virus challenge, indicating a characteristic distinction between these two in vitro mechanisms of macrophage-mediated antiviral activity with regard to host resistance to viral infection. Further, the ability of a macrophage to support viral replication itself was independent of the ability of the macrophage to suppress virus growth in another cell.

Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conserved Qa/Tla genes seen in other species. The remaining 10-19 bands displayed significant polymorphism; no two animals had identical band patterns when studied with all three enzymes. The polymorphism was markedly decreased between animals of the same ELA serotypes. Unique bands were identified in both A1 horses and all four A6 animals. Pvu II digestions of lymphocyte DNA were hybridized with mouse MHC class II probes. A cDNA probe for the E alpha gene revealed only a single nonpolymorphic band. In contrast, a cDNA probe for the H-2 A alpha locus displayed three to five strong bands in each animal with polymorphism that was most pronounced between horses of different ELA serotypes. Genomic DNA probes for A beta and E beta genes both revealed multiple polymorphic bands. However, cross-hybridization between these two probes prevented distinction between A beta and E beta equivalent loci. The reduced polymorphism evident within ELA specificities is consistent with the concept that the equine lymphocyte antigen system includes two families of closely linked MHC genes.

By producing long-term, stable, cytolytic T lymphocyte clones and utilizing targets expressing only a single gene product derived from the stimulator mouse strain, we have been able to directly demonstrate that T cells recognize distinct epitopes expressed on a single H-2 molecule. These multiple determinants are distinguishable by inhibition patterns with monoclonal antibodies (mAb). When two T cell clones, P-2.14 and P-2.17, are tested on an L cell transfected with the Dp gene (λ12a), the T cells kill the transfected targets as well as blasts derived from Dp mouse strains. mAb 7-16.10 inhibits recognition and killing of Dp targets by both P-2.14 and P-2.17. This mAb 7-16.10, however, competees with H-2.m22. Interestingly mAb 11-20.3 which also recognizes the H-2.m22 specificity inhibits clone P-2.14 but not P-2.17. The mAb 7-16.10, however, competes with 11-20.3 for binding to the surface of L cells expressing the Dp gene. Thus the two T cells must recognize an overlapping specificity. Other mAb which bind to the H-2Dp molecule are unable to inhibit either of these two cytolytic T cell clones. Paradoxically, any of the mAb when tested individually are sufficient to inhibit the polyclonal response derived from in vitro mixed lymphocyte culture. Therefore, by using targets expressing only a single H-2 molecule derived by DNA-mediated gene transfer and cytolytic T cell clones we have been able to directly demonstrate the presence of multiple epitopes on a single molecule and define their inhibition with mAb directed to that same molecule.

Mouse L cells transfected with a genomic clone containing the H-2Ld gene (8-5 cells) were shown to function as targets for H-2Ld-specific cytotoxic T lymphocytes (CTL). The CTL-mediated lysis of 8-5 cells was shown to be H-2Ld specific by the use of (i) CTL with restricted reactivity, (ii) unlabeled target inhibiton, and (iii) monoclonal antibody inhibiton. We also demonstrated that 8-5 cells could function as targets for antibody-plus-complement-mediated cell lysis. Specificity was confirmed by using H-2Ld-specific monoclonal antibodies. These experiments demonstrate that the gene products of a major histocompatibility complex genomic clone can be functionally expressed in a foreign cell and can mediate immunologically specific cellular interactions.

A λ clone containing the Dp gene was used to transform L cells. The Dp product expressed was identified by two-dimensional gel electrophoreis and flow cytometry. The Dp product expressed by the L cells was recognized by DP-specific flow cytometry. The Dp product expressed by the L cells was recognized by Dp-specific but not Kp-specific killer T cells. This killing was inhibited by monoclonal antibodies specific for Dp but not Kp or Kk antigens. Similarly, lymphocytic choriomeningitis virus (LCMV) killer T cells from B 10.P mice were able to kill LCMV-infected L12a cells, but not LCMV-infected Ltk+. Again only Dp monoclonal antibodies could inhibit this killing.

The major histocompatibility complex (MHC) of the mouse, H–2 on chromosome 17, contains several families of genes encoding cell-surface molecules which have a role in mediating immune responses1,2. The class I genes encode a family of homologous membrane proteins including the transplantation antigens K, D and L. These antigens reflect extensive genetic polymorphism which is apparent in the many different class I gene constellations or haplotypes found in mice (for example, BALB/c mice exhibit the H–2d haplotype and their class I molecules are denoted Kd, DD and Ld). Transplantation antigens serve as targets for T-cell killing in allogeneic immune responses such as in vivo graft rejection3 and destruction of allogeneic cells by cytotoxic T cells in vitro4. However, the physiological role of transplantation antigens may be to serve as restricting elements in virus-mediated T-cell killing of infected self cells. Virus infection of mice generates killer T cells whose receptors must interact with the foreign viral antigen and a class I molecule or restricting element for the cytotoxic effector function to be activated5,6. Thus the T-cell receptor recognizes the viral antigen in the context of a class I molecule. To study the interaction between the T-cell receptor and the class I restricting element, we have used the mouse L-cell transformant 8–5 which expresses Ld molecules7 and the K7-65 transformant expressing K molecules (R.S.G. et al., in preparation). Mouse L cells are fibroblasts derived from C3H mice of H–2k haplotype and monoclonal antibodies can be used to distinguish H–2d molecules from the endogenous H–2k products. Recently, we have demonstrated that both Ld (ref. 8) and Kd molecules (unpublished data) expressed on transformed L cells can act as target antigens for alloreactive cytotoxic T lymphocytes. Here we show that the Ld molecule on transformed mouse L cells can serve as a restricting element in lymphocytic choriomeningiris virus (LCMV) infection, whereas its Kd counterpart cannot.

Mouse L cells ( H-2 k ) transfected with a genomic clone containing the gene for transplantation antigen H-2L express the H-2L d molecule on the cell surface in a form indistinguishable from the native molecules. To determine the applicability of this system to the study of major histocompatibility complex (MHC) gene function, several biological properties of a cloned H-2L d transformant, 8-5, were evaluated. When 8-5 cells were used as an immunogen in C3H ( H-2 k ) mice, an antiserum was produced with activity predominantly directed against H-2L d specificities. This illustrates the utility of this approach for defining previously uncharacterized class I gene products expressed in transformed cells. The H-2L d molecules expressed on 8-5 cells also function as direct target antigens and restricting elements for H-2L d specific and lymphocytic choriomeningitis virus specific cytotoxic T lymphocytes respectively. In both instances, recognition of 8-5 cells by effector T cells was blocked by addition of H-2L d specific antisera or monoclonal antibodies.

Publisher Summary This chapter discusses the relationship between ultraviolet (UV) light exposure and skin-cancer formation in experimental animals. A detailed kinetic analysis into the phenomenon of UV induced tumor susceptibility has yielded many observations. Animals exposed to UV light gain tumor susceptibility after approximately 2 weeks of intermittent exposure. This is far less than the dosage of UV needed at the same expose rate to elicit tumor formation. In a study described in the chapter to determine whether any testable immunologic functions are altered by UV exposure, several in vivo and in vitro assays were performed. The immunologic impairment of UV exposed mice may be limited to the recognition and immunologic responses generated in response to subtle antigenic differences rather than to strong alloantigenic and mitogenic influences.

C3Hf/HeN mice support the progressive growth of most transplanted syngeneic ultraviolet (uv) light-induced tumors following short-term uv exposure whereas nonexposed mice reject these tumors. Because an immunosuppressive role in uv-mediated tumor susceptibility might be suggested by these observations, a comparison of several immunological parameters has been conducted. These include antibody production, proliferation in response to antigenic and mitogenic stimulation, and the generation of cytotoxic effector cells by normal or short-term uv-exposed mice. The results indicate that short-term uv irradiation of mice does not result in any readily detectable alterations of the host's immune system other than the consistent loss of the antitumor response mediating rejection of the transplanted uv-induced tumor.

The majority of skin tumors induced in mice by ultraviolet (UV) light are rejected when implanted into normal syngeneic recipients. Subcarcinogenic levels of UV light exposure render the normally resistant mice susceptible to tumor challenge. The immunoregulatory effect of UV light appears to be additive, since the growth rate of a tumor transplant is dependent upon the length of UV exposure administered prior to implantation. This suppressive influence does not appear to be directly mediated by the UV light, because the amputation of UV-irradiated tail skin allows for a retention of tumor resistance in otherwise tumor-susceptible hosts. UV-irradiated mice could also be immunized against UV tumors, which suggests that immune recognition of tumor-specific transplantation antigens has not been inhibited. The ability of UV exposure to alter normal immunological reactivity to UV-induced tumors is possibly an integral factor in the mechanism underlying UV carcinogenesis.