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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.

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen–CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1−/− CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.

Background:A hypercoagulable state has been consistently reported in patients with severe Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), characterized by elevated D-dimer, prolonged PT, and mild thrombocytopenia, though the mechanism is unclear. We have previously shown that human immunodeficiency virus (HIV) infection causes depletion of the anticoagulant protein S and virus-mediated platelet activation. Based on early reports, we hypothesized that a similar process contributed to COVID-19-associated thrombosis. Aim:To probe platelet activation and coagulation factor activity in SARS-CoV-2-infected patients. Methods:Blood was collected from consenting patients with differing COVID-19 severity: outpatients (15), hospitalized inpatients (15), and healthy controls (8). Platelet-leukocyte aggregate (PLA) formation and monocyte profiling were measured by flow cytometry. Coagulation factors were assessed by enzymatic assays. PS, von Willebrand Factor (vWF), PC, cytokines, and anti-S-Protein (viral spike protein) IgG were measured by ELISAs. Results:Ninety percent of SARS-CoV-2+ out-patients and in-patients had circulating anti-S-Protein IgG, but plasma IL-6 and TNFα were only elevated in three in-patients, consistent with reports that systemic inflammation is relatively rare in this population. Immune response did not correlate with disease severity. Unlike in HIV1+/AIDS patients, total PS was not reduced in SARS-CoV-2+ patients. However, the anticoagulant pool of PS ("free PS") was reduced in plasma samples from in-patients compared to controls (47.2%±23.3% vs. 100.8±42.6%, p=0057), while out-patients had an intermediate concentration (73.1%±28.9%). Specific loss of free PS is likely mediated by an increase in C4-binding protein (C4bp), which binds PS. In-patients also had a trend toward elevated plasma tissue factor (TF) compared to controls (79.5±121.4 fM vs. 37.8±39.7 fM, p = 0.32). Endothelial cells and monocytes can express TF under inflammatory conditions. We evaluated endothelial damage and dysfunction by measuring E-Selectin, which was unchanged in either in-patients or out-patients, and von Willebrand Factor (vWF), which was elevated in in-patients compared to controls (143±29.8 ng/mL vs. 56.2±41.9 ng/mL, p=0.0023). Plasma from in-patients also had elevated myeloperoxidase (524±187 ng/mL vs. 127±35 ng/mL, p=0.0026) and had a trend toward increased platelet-leukocyte aggregates (14.6±11.7% vs. 5.2±3.7%, p=0.24), indicating platelet and leukocyte stimulation. Unlike in the HIV1+/AIDS patients, no virus was detectable in any of the SARS-CoV-2+ patient plasmas. Consistent with a lack of direct platelet-virus interaction, plasma PF4 and platelet Akt phosphorylation were unchanged in the patient samples. We also observed a trend toward increased TF on TF+/CD64+/CD11b+ monocytes from in-patients compared to controls (MFI = 3244±2340 vs. 1741±382, p=0.18). Two inpatients were followed until they were SARS-CoV-2-negative. In both, PLAs, IL-6, vWF, and plasma TF remained elevated out to 28 days and PS remained reduced, suggesting that hemostatic dysregulation persists after SARS-CoV-2 is undetectable. Conclusions:We propose that localized inflammation in SARS-CoV-2+ patients results in a decrease in anticoagulant PS, through a shift of the free and C4bp-bound forms. At the same time, this inflammation causes stimulation of endothelial cells, which secrete procoagulant vWF, monocytes, which express TF and release it into plasma on microvesicles, and platelets, which form platelet-leukocyte aggregates. These changes may not return to baseline post-infection, suggesting that long-term monitoring of thrombotic risk may be necessary for SARS-CoV-2+ patients. Disclosures No relevant conflicts of interest to declare.

2628 Background: Preclinical models demonstrate that combined RT with immune checkpoint inhibitor (ICI) results in specific CD8+ T-cell phenotype associated with a tumor-reactive population resulting in significant tumor response. Sequential treatment could allow radiation to release tumor antigens from immune inaccessible areas and provide robust anti-tumor immune response with ICI. We report an interim analysis of the phase II clinical trial evaluating the efficacy and safety of the combination. Methods: Advanced NSCLC and HNSCC patients who had initiated on FDA approved single-agent ICI were eligible. Patients were given SBRT (BED>100Gy) or 30 Gy fractionated RT delivered as a 3-dimensional dose to a single metastatic site within 14 days of the first ICI dose. Primary objective was 6-month PFS and secondary objectives were safety and tolerability, 1Y PFS and OS. This interim analysis was done after enrollment of 43 patients. Results: Between 10/2017 to 1/2021, 43 patients were enrolled, and 38 included in this analysis. Median age was 62 years; 26 patients were male. 9 patients received ICI for NSCLC as first-line, 7 for NSCLC second-line and 22 for HNSCC second-line. 24 patients received pembrolizumab and 14 nivolumab; 21 had SBRT and 17 fractionated RT. Median follow up duration was 11.8m (range: 2.7 - 31.4m) for patients without progressive disease (PD). 10 patients were off-study, 7 continuing treatment. 15 died and 26 had PD. 14 patients died of malignancy and cause of death for one patient was unknown. 6-month PFS was 49.19% with median PFS of 5.5 months. (table) Fifty-two grade-3-5 adverse events (AEs) were reported among 21 subjects. Most common were transaminitis (n=15), lymphopenia (n=8), and GI side effects (n=4). Treatment related AEs included 19 grade-3 events, and none were grade 4/5. Two grade-5 AEs were from PD (oral bleeding and unspecified). There were 20 grade-1/2 and 3 grade-3 immune related adverse events (IRAEs). No grade-4/5 IRAEs were reported. Two patients discontinued treatment due to grade 3 transaminitis. Conclusions: Interim analysis shows that 6m PFS was acceptable with majority of patients being second-line metastatic HNSCC who historically had mPFS of 2.1-2.3 months and mOS 7.7-8.4 months in Checkmate-141/KEYNOTE-040 trials. Hence, the combination is of further interest and accrual will continue to reach the goal. The combination therapy was tolerable without unexpected AEs. Majority of deaths were from disease progression. No treatment related grade 4/5 adverse events were reported. Two patients discontinued treatment due to grade-3 IRAE. Clinical trial information: NCT03313804. [Table: see text]

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

Transfusion-related immunomodulation consists of both proinflammatory and anti-inflammatory responses after transfusion of blood products. Stored red blood cells (RBCs) suppress human T-cell proliferation in vitro, but the mechanism remains unknown. We hypothesized that cytokine synthesis by T cells may be inhibited when stored RBCs are present and that suppression between fresh and stored RBCs would be different.Purified human T cells were stimulated to proliferate with anti-CD3/anti-CD28 and then exposed to stored or fresh RBCs. Cells were placed in culture for 5 days. Cell culture supernatants were analyzed for the production of typical T-cell cytokines using multianalyte ELISArray kits.Stimulated T cells proliferated. RBC exposure markedly suppressed this proliferation. Interleukin 10, interleukin 17a, interferon γ, tumor necrosis factor α, and granulocyte macrophage colony-stimulating factor were increased in response to stimulation but depressed in the presence of stored RBCs. The use of fresh RBCs also resulted in depression of these cytokines when compared with stimulated T cells with no RBCs; however, this depression was less pronounced.T-cell activation is associated with both proinflammatory and anti-inflammatory cytokine release, comparable with patterns seen in trauma and acute injury. All of these responses are depressed by an exposure to stored RBCs. Decreased levels of these cytokines after RBC transfusion represents a potential contributor to the immunosuppressive complications seen in trauma patients after transfusion. This provides insight for future mechanistic studies to delineate the role of RBC transfusion in transfusion-related immunomodulation.

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of PGH2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis compared with wild-type (WT) mice associated with a marked reduction in Abs to type II collagen. In this study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum Abs in response to immunization with the T cell–dependent (TD) Ag DNP-keyhole limpet hemocyanin. Immunization with the T cell–independent type 1 Ag trinitrophenyl-LPS or the T cell–independent type 2 Ag DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleen of mPGES-1 null and WT mice were similar after immunization with DNP-keyhole limpet hemocyanin. To determine whether the effect of mPGES-1 and PGE2 was localized to hematopoietic or nonhematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in nonhematopoietic cells was the critical factor for reduced TD Ab production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of nonhematopoietic/mesenchymal stromal cells play a key role in TD humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation.

Background Red blood cells (RBCs) suppress T-cell responsiveness through a mechanism requiring cell–cell contact. Questions remain as to whether this effect is an allogeneic response, related to cell death, or dependent on particular components of the RBCs. Study Design and Methods Peripheral T cells were isolated from healthy donors and exposed to stored allogeneic RBCs or autologous RBCs after processing. RBCs were lysed by hypotonic solvent to produce cellular ghosts. Tritiated thymidine proliferation assays were utilized. Cultures were saturated with interleukin (IL)-2 to determine whether impaired IL-2 synthesis played a role. Results T-cell proliferation was suppressed by both autologous and allogeneic RBCs. RBC membrane integrity does enhance T-cell suppression. T-cell death is not responsible for the suppressive changes. IL-2 synthesis is suppressed in RBC-exposed T cells but addition of exogenous IL-2 does not rescue proliferative capabilities. Proliferation of T cells was inhibited with RBC exposure but mitigated with the addition of fresh RBCs. Conclusions T-cell suppression is enhanced by intact RBCs but this effect is unrelated solely to alloantigens. Neither apoptosis nor necrosis of T cells contributes to this phenomenon. IL-2 synthesis is suppressed after RBC exposure as a consequence of T-cell inhibition, but is not the primary cause of suppression. Fresh RBCs do not mediate T-cell suppression, indicating that changes in the RBC and development of the storage lesion may occur during initial blood bank processing.

Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1+/+ and HSF1−/− gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1−/− T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B–induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1−/− T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1−/− mice, but not HSF1+/+ mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.

Transfusion of packed red blood cells (RBCs) produces a myriad of immunologic derangements, from suppressive to stimulatory. Proliferation of human T cells is suppressed in vitro after exposure to processed red blood cells (PRBCs). We hypothesized that this effect would be mitigated by using fresh RBCs. We also hypothesized that this suppressive effect was a generalized effect on lymphocyte proliferation and would be observed in both CD4+ and CD8+ T-cell subpopulations as well as B cells.We isolated human T cells from donor peripheral blood mononuclear cells and exposed them to either blood bank PRBCs or fresh RBCs from volunteer donors and stimulated them with anti-CD3/anti-CD28. Human B cells were stimulated with lipopolysaccharide and exposed to PRBCs or fresh RBCs. We measured proliferation of B cells by thymidine incorporation assays. We also treated RBCs with citrate-phosphate-dextrose (CPD) at different time points before culture them with stimulated T cells to determine the role of this common RBC storage solution in lymphocyte proliferation.In vitro proliferation of CD4+ and CD8+ T cells was suppressed by blood bank RBCs. This suppression is eliminated when fresh RBCs were used. The B cells showed inhibition of proliferation when exposed to similar conditions, which appeared to be consistent over serial dilutions. Fresh RBCs exposed to CPD did not appear suppressive in the first 6 h after exposure.T-cell and B-cell proliferation inhibition by blood banked RBCs suggests a generalized effect of RBCs on cellular proliferation. The lack of suppression by fresh RBCs further suggests that something involved in blood banking alters RBC properties such that they attain a suppressive phenotype. One such blood banking component, CPD, does not appear to affect this suppressive phenotype within the first 6 h.

Packed red blood cell (PRBC) transfusion suppresses immunity and increases morbidity and mortality. Leukocyte reduction has failed to abrogate these effects, thus implicating red blood cells themselves or their components. PRBC impair proliferation of immortal (Jurkat) T cells by depleting arginine from the extracellular environment. The effect of PRBC on isolated ex vivo T-cell proliferation has not been reported. We hypothesize that PRBCs depress mitogen-stimulated proliferation in isolated human and mouse T cells.Human peripheral T cells were isolated by Ficoll-Hypaque gradient, purified by magnetic separation, and stimulated with anti-CD3 or anti-CD28. DO11.10 transgenic mouse splenic T cells were stimulated with ovalbumin. Cells were cultured at 1 x 10(6)/mL in 96-well plates or in 24-transwell plates in the presence of PRBC (0.015-5% by volume, stored for 4-6 weeks). In culture media, arginine and citrulline were varied. Proliferation was measured at 72 hours by thymidine incorporation. T-cell viability, apoptosis, and receptor zeta chain were measured by flow cytometry.PRBC significantly depressed human peripheral and mouse splenic T-cell proliferation in a dose-dependent manner. PRBC arginase blockade by N-omega-hydroxy-nor-l-arginine only partly restored proliferation. Cell contact was required in both cell types for maximal effect. Depressed zeta chain in human peripheral T cells was partly restored by arginase blockade. Salvage by high-dose arginine and citrulline was unsuccessful. Decreased proliferation was not related to cell death.PRBC suppresses mitogen-stimulated human and antigen-stimulated mouse T-cell proliferation by mechanisms independent of arginine depletion. This is a novel mechanism for transfusion-associated immune suppression.

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen, including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.

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.

Chronic infection with Toxoplasma gondii induces a potent resistance against reinfection, and IFN-γ production by CD8+ T cells is crucial for the protective immunity. However, the molecular mechanisms that regulate the secondary response remain to be elucidated. In the current study, we examined the role of IL-2 in IFN-γ production by CD8+ immune T cells in their secondary responses using T. gondii–specific CD8+ T cell hybridomas and splenic CD8+ immune T cells from chronically infected mice. The majority (92%) of CD8+ T cell hybridomas produced large amounts of IFN-γ only when a low amount (0.5 ng/ml) of exogenous IL-2 was provided in combination with T. gondii Ags. Inhibition of cell proliferation by mitomycin C did not affect the enhancing effect of IL-2 on the IFN-γ production, and significant increases in transcription factor T-bet expression were associated with the IL-2–mediated IFN-γ amplification. Splenic CD8+ immune T cells produced similar low levels of IL-2 in the secondary response to T. gondii, and a blocking of IL-2 signaling by anti–IL-2Rα Ab or inhibitors of JAK1 and JAK3 significantly reduced IFN-γ production of the T cells. This IL-2–mediated upregulation of IFN-γ production was observed in mitomycin C–treated CD8+ immune T cells, thus independent from their cell division. Therefore, endogenous IL-2 produced by CD8+ immune T cells can play an important autocrine-enhancing role on their IFN-γ production in the secondary responses to T. gondii, suggesting an importance of induction of CD8+ immune T cells with an appropriate IL-2 production for vaccine development.

Background: Transfusion of packed red blood cells (PRBCs) is associated with morbidity and mortality. The mechanisms are not fully understood. Packed red blood cells deplete extracellular arginine and possess transporters for arginine, an amino acid essential for normal immunity. We hypothesize that the membrane yþ amino acid transporter contributes to arginine depletion in PRBCs. Materials and methods: We titrated PRBCs to a 10% hematocrit with phosphate-buffered saline, blocked PRBC yþ transporters using n-ethylmaleimide (0.2 mM), and measured arginine and ornithine levels using liquid chromatographyemass spectroscopy. We added radiolabeled L-arginine [4,5- 3 H] (10 mmol/L) added to similar culture conditions and measured arginine uptake in counts per minute (CPM). We examined storage periods of 6e 9d , 1e4 wk, and 6 wk, and correlated donor demographics with arginine uptake. Results: n-Ethylmaleimide blockade of yþ transporters impaired PRBC arginine depletion from culture media (117.6 � 8.6 mM versus 76.9 � 5.8 mM; P < 0.001) and reduced intracellular L-arginine (7,574 � 955 CPM versus 18,192 � 1,376 CPM; P < 0.01). Arginine depletion increased with storage duration (1 wk versus 6 wk; P < 0.002). With n-ethylmaleimide treatment, 6-wk-old PRBCs preserved more culture arginine (P < 0.008) than at shorter durations. Nine-day storage duration increased L-arginine uptake compared with 6- to 8-day storage (n ¼ 77, R ¼ 0.225, P < 0.05). Extracellular arginine depletion and extracellular ornithine synthesis varied among donors and correlated inversely (R ¼� 0.5, P ¼ 0.01). Conclusions: Membrane yþ transporters are responsible for arginine depletion by PRBCs. Membrane yþ activity increases with storage duration. Arginine uptake varies among donors. Membrane biology of RBCs may have a role in the negative clinical effects associated with PRBC transfusion.

Background The prevalence of peanut allergies is increasing. Peanuts and many other allergen sources contain significant amounts of triglycerides, which affect absorption of antigens but have unknown effects on sensitization and anaphylaxis. We recently reported that dietary medium-chain triglycerides (MCTs), which bypass mesenteric lymph and directly enter portal blood, reduce intestinal antigen absorption into blood compared with long-chain triglycerides (LCTs), which stimulate mesenteric lymph flow and are absorbed in chylomicrons through mesenteric lymph. Objective We sought to test how dietary MCTs affect food allergy. Methods C3H/HeJ mice were fed peanut butter protein in MCT, LCT (peanut oil), or LCT plus an inhibitor of chylomicron formation (Pluronic L81). Peanut-specific antibodies in plasma, responses of the mice to antigen challenges, and intestinal epithelial cytokine expression were subsequently measured. Results MCT suppressed antigen absorption into blood but stimulated absorption into Peyer patches. A single gavage of peanut protein with MCT, as well as prolonged feeding in MCT-based diets, caused spontaneous allergic sensitization. MCT-sensitized mice experienced IgG-dependent anaphylaxis on systemic challenge and IgE-dependent anaphylaxis on oral challenge. MCT feeding stimulated jejunal-epithelial thymic stromal lymphopoietin, Il25 , and Il33 expression compared with that seen after LCT feeding and promoted T H 2 cytokine responses in splenocytes. Moreover, oral challenges of sensitized mice with antigen in MCT significantly aggravated anaphylaxis compared with challenges with the LCT. Importantly, the effects of MCTs could be mimicked by adding Pluronic L81 to LCTs, and in vitro assays indicated that chylomicrons prevent basophil activation. Conclusion Dietary MCTs promote allergic sensitization and anaphylaxis by affecting antigen absorption and availability and by stimulating T H 2 responses.

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.

Heat shock factor 1 (HSF1) is a highly conserved transcriptional regulator that controls the expression of a variety of different gene products during heat shock and other environmental stresses (24, 33). Previous studies examining either the resistance of Caenorhabditis elegans to bacterial infection or the ability of mice to produce IgG have suggested that HSF1 may be important for generating protective immune responses (18, 38). In most cell types, the heat shock response that leads to trimerization and nuclear translocation of HSF1 occurs only during severe thermal stress, at temperatures at or above 42°C (32). However, in T cells, HSF1 can be activated at 39.5°C, a temperature that is within the range of a typical fever response (11). Fever is induced during infection by Toll-like receptor (TLR) ligands that trigger the release of proinflammatory cytokines such as interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) and prostaglandin E2, which acts on the hypothalamus to temporarily increase the thermoregulatory set point of the body (7). In mice, fever has been characterized as an elevation of 1.3 to 2.1°C above the basal core body temperature (13). Although raising the body temperature to produce a fever is an energy-costly process, the evolutionary conservation of febrile responses suggests that fever is beneficial for the host and that the immune mechanisms designed to clear infections must be well adapted to functioning at elevated temperatures (15). Since HSF1 is activated at much lower temperatures in T cells than in other cell types, we hypothesized that it might be needed to protect T cell function during infections that induce a fever response. In a previous in vitro study, we showed that T cells isolated from HSF1-deficient mice were unable to proliferate efficiently at physiologic fever temperature (39.5°C) (26). To further address this question in vivo, we chose to study the role of HSF1 during Listeria monocytogenes infection, since clearance of this intracellular bacterial pathogen is strictly dependent on protective T cell responses. Early resistance to listeriosis is mediated primarily by gamma interferon (IFN-γ) and TNF-α, and mice that lack either of these cytokines are highly susceptible to infection (3, 16, 28, 30). However, the activation and proliferation of CD8+ T cells that can lyse infected cells and release the bacteria from their protective intracellular niche are required for sterilizing immunity against L. monocytogenes. Although fever is a commonly described symptom associated with human listeriosis (22, 40), the ability of L. monocytogenes to induce fever in small animal models has not been well characterized. One early study showed that the rectal temperature of rabbits increased by 2°C following injection with a partially purified toxin derived from L. monocytogenes and that this response peaked at 24 h postinjection (35). However, there are no reports in the literature describing the fever response in mice following L. monocytogenes infection. In this paper, we monitored the core body temperature of mice infected with L. monocytogenes and found that both HSF1-deficient and wild-type mice maintain a fever response during the first 2 to 3 days postinfection. Surprisingly, we showed that CD8+ T cell responses were not impaired in HSF1-deficient mice during L. monocytogenes infection. However, HSF1−/− mice infected with at least 1 × 104 CFU of L. monocytogenes displayed increased lethality that appeared to be caused by misregulated production of proinflammatory cytokines such as TNF-α.

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

Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42°C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39°C vs 42°C), within the physiological range of fever. In this study, we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h, whereas HSF1−/− T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1−/− mice at fever temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle progression under the stressful conditions encountered during fever.

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.

The induction of heat shock protein gene expression in response to stress is critical for the ability of organisms to cope with and survive exposure to these stresses. However, most studies on HSF1-mediated induction of hsp70 gene expression have utilized immortalized cell lines and temperatures above the physiologically relevant range. For these reasons much less is known about the heat shock response as it occurs in mammalian cells within tissues in the intact organism. To gain insight into this area we determined the temperature thresholds for activation of HSF1 DNA binding in different mouse tissues. We have found that HSF1 DNA binding activity and hsp70 synthesis are induced in spleen cells at significantly lower temperatures relative to cells of other tissues, with a temperature threshold for activation (39 degrees C) that is within the physiological range for fever. Furthermore, we found that the lowered temperature set point for induction of the stress response in spleen is specific to T-lymphocytes residing within this tissue and is not exhibited by B-lymphocytes. This lowered threshold is also observed in T-lymphocytes isolated from lymph nodes, suggesting that it is a general property of T-lymphocytes, and is seen in different mouse strains. Fever is an early event in the immune response to infection, and thus activation of the cellular stress response in T-lymphocytes by fever temperatures could serve as a way to give these cells enough time to express hsps in anticipation of their function in the coming immune response. The induced hsps likely protect these cells from the stressful conditions that can exist during the immune response, for example increasing their protection against stress-induced apoptosis.

Studies of amyloid precursor protein transgenic mice suggest that immune responses to amyloid beta peptide (Abeta) may be instrumental in the removal of plaques from the brain, but the initial clinical trial of an Abeta vaccine in patients with Alzheimer s disease (AD) was halted as the result of serious neurological complications in some patients. We now provide evidence that AD patients exhibit an enhanced immune response to Abeta and that, contrary to expectations, Abeta antibodies enhance the neurotoxic activity of the peptide. Serum titers to Abeta were significantly elevated in AD patients and Abeta antibodies were found in association with amyloid plaques in their brains, but there was no evidence of cell-mediated immune responses to Abeta in the patients. Abeta antibodies were detected in the serum of old APP mutant transgenic mice with plaque-like Abeta deposits, but not in the serum of younger transgenic or nontransgenic mice. Serum from APP mutant mice potentiated the neurotoxicity of Abeta. Our data suggest that a humoral immune response to Abeta in AD patients may promote neuronal degeneration, a process with important implications for the future of vaccine-based therapies for AD.

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.

This chapter focuses on modulation of expression of major histocompatibility complex (MHC) molecules by mycoplasmas. The mycoplasmas have diverse and multiple effects on immunocompetent cells, including T and B lymphocytes, natural killer cells, and macrophages. At least six different mycoplasma species have the ability to produce a factor that are termed M1aF, that induces MHC class I and class II expression on a variety of cell types. These cells include primary murine bone marrow macrophage cultures, cultures of several different murine and human macrophage cell lines, and at least one murine glioma cell line, G26-20. The ability of mycoplasmas to produce M1aF is independent on the type of media that they are grown in. Mycoplasmas grow in both SP4 and Hayflick's media and both support the production of this factor. M1aF is detectable under reduced serum concentration in the culture medium to as low as 5%. Two very important considerations need to be kept in mind concerning the indicator cell lines used. The first regards the inherent instability of the cell lines in terms of their ability to respond to either M1aF or IFN-γ. Another important consideration is to make sure that the indicator line does not become contaminated with mycoplasmas.

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.

We have studied the ability of three different Mycoplasma species to induce proliferation of bone marrow-derived macrophages (BMM). We observed a significant mitogenic effect when BMM cells from BALB/c, DBA/2J, SJL, and C57BL/6 mice were incubated with membranes derived from Mycoplasma arginini or M. arthritidis but not when they were incubated with an equivalent amount of M. pulmonis membrane. We also determined that pretreatment of mycoplasma membrane preparations with papain eliminated the ability of these preparations to induce BMM proliferation. To determine whether these membrane fractions acted indirectly by stimulating the production of soluble factors known to stimulate proliferation of BMM cells, we performed blocking studies with antibodies directed against colony-stimulating factor 1 (CSF-1), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor. Our results indicate that antibodies directed against either CSF-1 or IL-3 failed to block mycoplasma-initiated proliferation of BMM cells. However, when anti-GM-CSF was added to proliferative cultures at the time of initiation, we saw a dose-dependent reduction of mycoplasma-initiated proliferation. We conclude that the ability of mycoplasma membranes to initiate the proliferation of BMM is not shared by all species of mycoplasma and that it involves the production of GM-CSF by an as yet undetermined cell.

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.

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.

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.

Numerous studies have implicated trans -acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II genes while others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans -acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role trans -acting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk − cell line and normal B cells to determine if the existence of positive trans -acting factors from the B cell would lead to the expression of Ltk − class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk − class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggest that Ltk − cells possess negative trans -acting factors that appear to predominate over the positive trans -acting factors possessed by B cells. We have further extended these studies to test the MHC-inducing activity of IFN-γ and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN-γ with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II genes were induced by IL-4. Taken together, these results indicate that Ltk − cells possess negative trans -acting factors that not only maintain the Ia − phenotype of these cells, but also block the action of positive trans -acting factors from B cells.

We have cloned six different class I genes from a B10.P sperm library. After cotransfection with the herpes simplex tk gene, one L-cell line was found to react with six H-2Dp-specific monoclonal antibodies. The cell line L12a did not react with Kp-specific monoclonal antibodies. This identification was confirmed by mapping a 2.5 kb Bam H1 restriction fragment present in the lambda 12a DNA clone to the D-TL region of H-2p. Only a single 8.8 kb Bam H1 fragment can be assigned to Kp by restriction fragment length polymorphism, while many others map to the D-TL interval. A restriction map of lambda 12a is presented.

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.

We have applied purification strategies similar to those used to purify murine dendritic cells to human peripheral blood in an attempt to enrich for stimulators of the human mixed lymphocyte reaction (MLR). Equilibrium density centrifugation of peripheral blood mononuclear cells yields a population of low density cells that are potent stimulators of a human MLR. The stimulator cells are Dr+, nonlymphocytic, and weakly adherent. Strongly adherent monocytes, also present in the low-density cell population, do not stimulate a human MLR. This contrasts with other human MLR studies that ascribe stimulatory activity to adherent monocytes, and it indicates functional and morphological heterogeneity among monocytes.

We have examined the capacity of non-bone marrow-derived (NBMD) cells to express endogenous Ia genes by evaluating E alpha mRNA expression in various tissues derived from long term bone marrow chimeras. These chimeras were produced in the combination BIO.A(4R) into B10.A and allowed to rest for greater than 100 days before use. Since B10.A(4R) is incapable of transcribing the E gene, E mRNA expression would most likely represent Ia expression by the host B10.A NBMD tissues in these long term chimeras. Our results demonstrate that host E alpha mRNA expression was readily detectable in kidney, lung and thymus, but not in the spleen of chimeric animals. During graft-vs-host disease (GVHD) E alpha expression was enhanced up to 4-fold in kidney and was also detectable in heart extracts as well. These experiments demonstrate that resident host cells, most likely NBMD, in a variety of organs are capable of la gene expression. This expression appears to be both constituitive and inducible. Furthermore, different tissues show a marked quantitative difference in the magnitude of NBMD la expression with kidney showing the highest level.

Mouse hepatitis viruses are members of the coronavirus group of animal viruses. Although named for their propensity to induce acute hepatitis in animals stressed by a variety of conditions, it has become clear that as a group they possess the ability to cause a diverse group of diseases in their natural host (Wege, et al, 1982). One strain of MHV, named JHM virus (JHMV), was isolated from mice found to have demyelinated lesions of the central nervous system (Bailey, et al, 1949). More recently it has been found that JHMV is not only capable of causing acute encephalomyelitis with demyelination but also chronic demyelination probably due to the establishment of a latent infection of oligodendroglia, the cells of myelin within the central nervous system (Herndon, et al, 1975; Stohlman and Weiner, 1981).