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NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin β1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2β1 in NK cell biology. In this study, we show that in mice α2β1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2β1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)–infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2β1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2β1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell development and function. Numerous studies have shown the significant roles of miRNAs in regulating immune cells including natural killer (NK) cells. However, little is known about the role of miRNAs in NK cells with aging. We previously demonstrated that the aged C57BL/6 mice have significantly decreased proportion of mature (CD27−CD11b+) NK cells compared with young mice, indicating impaired maturation of NK cells with aging. Here, we performed deep sequencing of CD27+ NK cells from young and aged mice. Profiling of the miRNome (global miRNA expression levels) revealed that 49 miRNAs displayed a twofold or greater difference in expression between young and aged NK cells. Among these, 30 miRNAs were upregulated and 19 miRNAs were downregulated in the aged NK cells. We found that the expression level of miR‐l8la‐5p was increased with the maturation of NK cells, and significantly decreased in NK cells from the aged mice. Knockdown of miR‐181a‐5p inhibited NK cell development in vitro and in vivo. Furthermore, miR‐181a‐5p is highly conserved in mice and human. MiR‐181a‐5p promoted the production of IFN‐γ and cytotoxicity in stimulated NK cells from both mice and human. Importantly, miR‐181a‐5p level markedly decreased in NK cells from PBMC of elderly people. Thus, our results demonstrated that the miRNAs profiles in NK cells change with aging, the decreased level of miR‐181a‐5p contributes to the defective NK cell development and function with aging. This opens new strategies to preserve or restore NK cell function in the elderly., MicroRNAs (miRNAs) regulate gene expression and thereby influence cell development and function. Deep sequencing of R1 and R2 NK cells revealed that the miRNAs profiles in NK cells change with aging. MiR‐l8la‐5p expression decreases in aged NK cells, and the decreased level of miR‐181a‐5p contributes to the defective NK cell maturation and function with aging.

It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN‐γ) expression by group 1 innate lymphoid cells (G1‐ILCs), mostly NK in cells but also some ILC1. In response to the IFN‐γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN‐γ expression by G1‐ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged., Stotesbury et al. show that in aged mice, the trafficking of migratory dendritic cells (mDC) to the draining lymph node (dLN) in response to viral infection is decreased. Resulting in impaired expression of IFN‐γ by Group 1 Innate Lymphoid cells, reduced recruitment of and attenuated CXCL9 production by inflammatory monocytes (iMO). As a consequence, natural killer cell recruitment to the dLN is increased and susceptibility to lethal viral disease is increased.

CMV has been proposed to play a role in cancer progression and invasiveness. However, CMV has been increasingly studied as a cancer vaccine vector, and multiple groups, including ours, have reported that the virus can drive antitumor immunity in certain models. Our previous work revealed that intratumoral injections of wild-type murine CMV (MCMV) into B16-F0 melanomas caused tumor growth delay in part by using a viral chemokine to recruit macrophages that were subsequently infected. We now show that MCMV acts as a STING agonist in the tumor. MCMV infection of tumors in STING-deficient mice resulted in normal recruitment of macrophages to the tumor, but poor recruitment of CD8+ T cells, reduced production of inflammatory cytokines and chemokines, and no delay in tumor growth. In vitro, expression of type I IFN was dependent on both STING and the type I IFNR. Moreover, type I IFN alone was sufficient to induce cytokine and chemokine production by macrophages and B16 tumor cells, suggesting that the major role for STING activation was to produce type I IFN. Critically, viral infection of wild-type macrophages alone was sufficient to restore tumor growth delay in STING-deficient animals. Overall, these data show that MCMV infection and sensing in tumor-associated macrophages through STING signaling is sufficient to promote antitumor immune responses in the B16-F0 melanoma model.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

CD8 T lymphocytes are a major cell population of the adaptive immune system. A fundamental characteristic of the CD8 T lymphocyte pool is that it is composed of millions of clones; each with a unique T cell receptor capable of recognizing a limited number of peptides displayed at the cell surface bound to the grooves of major histocompatibility complex class I (MHC I) molecules. Naive CD8 T lymphocytes are normally resting and circulate between the blood and secondary lymphoid organs in search of their cognate peptide–MHC complexes. During viral infections, bone marrow–derived professional antigen-presenting cells (pAPCs) in secondary lymphoid organs display viral peptides on their MHC I molecules. Specific CD8 T lymphocytes that recognize these peptide–MHC adducts become activated (primed), proliferate extensively, and develop into effectors capable of killing infected cells, identified by the presence at their surface of the pertinent viral peptide–MHC complexes. This article describes how the process of priming naive CD8 T lymphocytes occurs.

Direct presentation (DP) and cross presentation (CP) on MHC I by professional APCs are defined by the internal or external source of the Ag, respectively. Although some Ags are substrates for both DP and CP, others are only substrates for DP. The reasons for this difference remain largely unknown. In this study, we studied in tissue culture and also in vivo, the effects of altering the length and sequence of the amino acid chains flanking an MHC class I restricted determinant (the chicken OVA OVA258–265, SIINFEKL) that is normally a good substrate for both DP and CP. We demonstrate that CP but not DP strictly requires flanking N and C-terminal extensions of minimal length. Furthermore, we show that removal but not replacement of just one amino acid 22 residues downstream from the determinant is sufficient to strongly affect CP without affecting either protein stability or DP. Thus, our work shows that the flanking residues of an antigenic determinant can differentially affect CP and DP, and that features of the Ag other than half-life can have a major impact in CP. Our studies may have implications for understanding CP in viral infections and possibly for the design of new vaccines.

In B lymphocytes, the B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) facilitates signaling from the antigen receptor. Mice lacking BCAP have a predominantly immature pool of B cells with impaired immune function and increased susceptibility to apoptosis. Unexpectedly, we have found that natural killer (NK) cells from BCAP-deficient mice are more mature, more long-lived, more resistant to apoptosis, and exhibit enhanced functional activity compared with NK cells from wild-type mice. Surprisingly, these effects are evident despite a severe impairment of the immunoreceptor tyrosine-based activation motif-mediated Akt signaling pathway. The seemingly paradoxical phenotype reveals inherent differences in the signals controlling the final maturation of B cells and NK cells, which depend on positive and negative signals, respectively. Both enhanced interferon-γ responses and augmented maturation of NK cells in BCAP-deficient mice are independent of available MHC class I ligands. Our data support a model in which blunting of BCAP-mediated activation signaling in developing NK cells promotes functionality, terminal maturation, and long-term survival.

The immune system detects viral infections and mutations in parenchymal cells when antigens from these cells are crosspresented on MHC class I molecules of professional antigen-presenting cells (APC). Exogenous antigens are crosspresented through TAP-dependent (cytosolic) or poorly understood TAP-independent (vacuolar) pathways. The TAP-independent pathway is blocked by the cysteine protease inhibitor, leupeptin, but not by proteasome inhibitors, which is opposite to the effects of these agents on the TAP-dependent pathway. Dendritic cells lacking the cysteine protease cathepsin S lack the TAP-independent pathway. Mice whose APC lack cathepsin S have reduced crosspriming to particulate and cell-associated antigens, as well as to influenza virus. Cathepsin S-deficient phagosomes generate a class I-presented peptide poorly. In contrast, cathepsin S-sufficient phagosomes and recombinant cathepsin S produce the mature epitope. Therefore, cathepsin S plays a major role in generating presented peptides for the vacuolar pathway of crosspresentation, and this mechanism is active in vivo.

Priming of CD8+ T cells requires presentation of short peptides bound to MHC class I molecules of professional APCs. Cross-presentation is a mechanism whereby professional APC present on their own MHC class I molecules peptides derived from degradation of Ags synthesized by other Ag “donor cells.” The mechanism of cross-presentation is poorly understood, and the nature of the transferred Ag is unknown. In this report, we demonstrate that the bulk of a cross-presented Ag transferred from donor cells recently infected with vaccinia virus are proteasomal products that are susceptible to peptidases within the donor cell cytosol and not full-length proteins or mature epitopes either free or bound to chaperones.

The factors that determine CD4 + T cell (T CD4 + ) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the T CD4 + repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV- and VACV-specific T CD4 + at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from T CD4 + following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific T CD4 + as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on T CD4 + responses and memory. IMPORTANCE Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted T CD4 + repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific T CD4 + , comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of T CD4 + responses at both acute and memory time points, with VACV-specific T CD4 + memory being notably compromised. These data offer insight into the impact of epitope-extrinsic factors on the resulting T CD4 + responses.

Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8+ T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8+ T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8 + T cells recruited to the response. Thus, during lethal virus challenge, memory CD8+ T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ. © 2013 Elsevier Inc., R01AI048849, R01AI065544, 5U19AI083008; P30CA006927; NIH T32 CA-009035036

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the non-anchor positions of the Db-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for Db was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for Db binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical Db-binding motif but unable to bind to the class I molecule with high affinity could be made to bind Db, by changing unfavorable AAs to favourable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind Db. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs, were present at secondary anchor positions.

CD4 + T cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4 + T cells have been described, whether those generated during the course of a viral infection play a role in virus control remains unknown. Here we show that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4 + T cells in the draining lymph node and liver of resistant mice have a cytotoxic phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4 + T cells have impaired virus control. Thus, perforin-dependent CD4 + T-cell killing of infected cells is an important mechanism of antiviral defense.

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy., National Institute of Allergy and Infectious Diseases grants (U19AI083008, R01AI065544); National Cancer Institute; Instituto de Salud Carlos III; Spanish Ministry of Science and Innovation

Summary It is well established that natural killer (NK) cells confer resistance to many viral diseases, but in only a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virus-infected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1 b are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.

The percentages and absolute numbers of mononuclear peripheral blood cells (MNC) were studied in vaccinated (Vac) and non-vaccinated (control) cattle, challenged with foot-and-mouth disease virus (FMDV). All Vac cattle but none of the controls resisted challenge. Cell populations were studied immediately before and one week after challenge, by direct and indirect immunofluorescence, using polyclonal and monoclonal antibodies against different bovine markers. Total B-lymphocytes, as assessed with polyclonal anti-IgG (H + L) antisera, as well as total mononuclear cells, were normal before and after infection, and did not change in Vac or control groups. Before challenge Vac cattle had higher numbers of ILA-29+ (a putative marker for null cells or, alternatively, for gamma delta T-cells) than control cattle. After challenge, in control cattle, the number of total T-cells, BoT4-bearing (helper) T-cells and BoT8-bearing (cytotoxic/suppressor) T-cells were decreased, while IgM-bearing B-lymphocytes, as well as monocyte/macrophage cells were increased. The number of IL-A29-bearing T-cells did not change after infection in either group. After challenge, Vac cattle also showed increased numbers of IgM-bearing B-lymphocytes and monocyte/macrophage cells, whereas T-subpopulations did not change significantly.

It is uncertain how immunity protects against systemic viral diseases. Here, we demonstrate that in the absence of persistent virus, not only antibodies but also recall responses by long-lived memory CD8 + T cells prevent mousepox, a disease caused by ectromelia virus, a close relative of the virus of human smallpox. Moreover, we show that to protect, recall CD8 + T cells directly kill targets in the lymph node draining the primary site of infection thus curbing systemic viral spread. Therefore, our work provides the basis for a model where lymph nodes are not just organs where lymphocytes become activated and proliferate but also the sites where a major fight against virus spread takes place.

It is believed that CD8+ T lymphocytes or Abs can independently clear many primary viral infections, including those caused by Orthopoxviruses (OPV), a genus that includes the human pathogens variola and monkeypox and the vaccine species vaccinia virus. However, most experiments addressing the role of Abs and CD8+ T cells in protection have used viruses that are not specific for the host. In the present study, we used the mouse-specific OPV ectromelia virus and mice deficient in CD40, B cells, or CD8+ T cells and adoptive transfers of CD8+ T or B lymphocytes to show that the protection afforded by CD8+ T cells is incomplete. Despite sustained CD8+ T cell responses, in the absence of Ab responses ectromelia virus persists. This results in delayed disease and inexorably leads to death. Therefore, CD8+ T lymphocytes and Abs are not redundant but complementary and essential to survive infections with a highly pathogenic viruses in the natural host.

Cross-presentation of phagocytosed antigens on major histocompatibility (MHC) class I molecules is known to occur via two alternative routes; one involving processing and loading of the antigenic peptides inside a vacuolar compartment, the other requiring processing inside the cytosol and loading to MHC-I in the endoplasmic reticulum. A new pathway has been described recently in which antigens are transported to the cytosol, processed by phagosome-associated proteasomes and returned back to the phagosome for MHC-I loading.

Professional APCs (pAPC) can process and present on their own MHC class I molecules Ags acquired from Ag donor cells (ADC). This phenomenon of cross-presentation is essential in the induction of CD8+ T cell responses to viruses that do not infect pAPC and possibly contributes to the induction of CD8+ responses to many other viruses. However, little is known about the mechanisms underlying this process. In this study, we show that dendritic cells and macrophages cross-present a model Ag supplied by vaccinia virus-infected ADC via the cytosolic route. Strikingly, we also found that cross-presentation of Ags provided by vaccinia-infected cells occurs within a couple of hours of pAPC/ADC interaction, that the duration of cross-presentation lasts for only 16 h, and that cross-presentation can occur at early times of infection when the ADC are still alive.

Ovarian cancer is the fifth leading cause of death and the most lethal gynecological cancer among women in the United States. Persistent activation of signal transducer and activator of transcription 3 (STAT3), a cytoplasmic transcription factor, is frequently detected in EOC. STAT3 transduces signals from cytokines such as interleukin 6 (IL-6) via interactions with the IL-6 receptor and Janus kinases (JAK). JAK2 activates STAT3 by phosphorylation, leading to dimerization and translocation of STAT3 to the nucleus where it activates transcription of target genes regulating proliferation, survival and motility. Importantly, in addition to tumor cells, STAT3 signaling is also critical for immune cell activity and, in particular, inflammatory response. The inflammatory tumor microenvironment is important for ovarian cancer progression; therefore, we hypothesized that disruption of the STAT3 pathway would block ovarian tumor progression by: 1) directly inhibiting the growth of tumor cells; and 2) reducing a pro-tumorigenic inflammatory microenvironment. To target JAK2-mediated activation of STAT3 we used AZD1480, a JAK2-selective small molecule inhibitor. The effects of AZD1480 treatment on cell proliferation, apoptosis, adhesion and motility were evaluated in cultured human ovarian carcinoma cells. To study the effects of AZD1480 in vivo, we used MISIIR-TAg mice, a transgenic mouse model of ovarian carcinoma. Tumor growth in MISIIR-TAg mice was monitored and quantified in mice by weekly magnetic resonance imaging (MRI). Drug treatment-mediated alterations in gene expression were evaluated by microarray analysis and changes in the inflammatory response were evaluated by flow cytometry analysis of cells extracted from ovarian tumors, spleens and peritoneal washes. AZD1480 treatment significantly reduced primary ovarian tumor growth in transgenic mice. Microarray analysis showed changes in expression of genes involved in the acute immune response, such as Gbp6, Ifi44, Irgm, Igtp, Gzmb and Cd69. Analysis of immune cell populations by flow cytometry showed a significant decrease in the number and percent of T helper and T regulatory cells present in the peritoneal cavity of drug-treated mice compared to controls. As T regulatory cells are associated with a poor prognosis in ovarian cancer patients, the decrease of this subpopulation in drug-treated mice suggests a change in the tumor microenvironment that may contribute to reduced tumor growth. Taken together, these results indicate that targeting JAK2/STAT3 impedes ovarian tumor growth through complex mechanisms, including the reduction of primary tumor growth and inflammation in the tumor microenvironment. These findings highlight the potential utility of targeting the JAK2/STAT3 pathway for the treatment of ovarian cancer patients. Citation Format: Galina Gritsina, Fang Xiao, Shane W. O'Brien, Marisa A. Maglaty, Ren-Huan Xu, Luis J. Sigal, Samuel Litwin, Denise C. Connolly. Targeting the JAK2/STAT3 pathway in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1113. doi:10.1158/1538-7445.AM2014-1113

Cytotoxic T lymphocytes (CTLs) are thought to detect viral infections by monitoring the surface of all cells for the presence of viral peptides bound to major histocompatibility complex (MHC) class I molecules. In most cells, peptides presented by MHC class I molecules are derived exclusively from proteins synthesized by the antigen-bearing cells. Macrophages and dendritic cells also have an alternative MHC class I pathway that can present peptides derived from extracellular antigens; however, the physiological role of this process is unclear. Here we show that virally infected non-haematopoietic cells are unable to stimulate primary CTL-mediated immunity directly. Instead, bone-marrow-derived cells are required as antigen-presenting cells (APCs) to initiate anti-viral CTL responses. In these APCs, the alternative (exogenous) MHC class I pathway is the obligatory mechanism for the initiation of CTL responses to viruses that infect only non-haematopoietic cells.

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector system to induce protective immunity against diverse pathogens. Replication-competent chimeric viruses can be constructed by inserting foreign antigenic sequences within the poliovirus polyprotein. When inserted sequences are flanked by poliovirus protease recognition sites the recombinant polyprotein is processed to mature and functional viral proteins plus the exogenous antigen. It previously has been shown that poliovirus recombinants can induce antibody responses against the inserted sequences but it is not known whether poliovirus or vaccine vectors derived from it can elicit effective cytotoxic T lymphocyte (CTL) responses. To examine the ability of the recombinant poliovirus to induce CTL responses, a segment of the chicken ovalbumin gene, which includes the H2-Kb-restricted CTL epitope SIINFEKL, was cloned at the junction of the P1 and P2 regions. This recombinant virus replicated with near wild-type efficiency in culture and stably expressed high levels of the ovalbumin antigen. Murine and primate cells infected with the recombinant virus appropriately processed the SIINFEKL epitope and presented it within major histocompatibility complex class I molecules. Inoculation of mice with recombinant poliovirus that expresses ovalbumin elicits an effective specific CTL response. Furthermore, vaccination with these recombinant poliovirus induced protective immunity against challenge with lethal doses of a malignant melanoma cell line expressing ovalbumin.

Relative affinities were determined for the interaction of H-2D b with all the peptides from the A/PR/8/34 strain of influenza virus that contained the D b -binding motif. The results indicated that, even though 23 peptides with the appropriate motif were identified and analysed, binding of only five of them could be detected at peptide concentrations lower than 10 −7 M. Of these five, only one, TGICNQNII, bound with better affinity than the nucleoprotein-derived natural epitope, ASNENMETM. The origin of the higher binding peptide was the influenza neuraminidase, a protein for which little cytosolic processing would be expected since it is a surface glycoprotein. To establish why many of the influenza-derived peptides did not bind, the role of non-anchor residues on D b -peptide interactions was analysed, using a scheme where QDIENEEKI, a non-binding peptide from the influenza virus polymerase 1, was sequentially converted to ASNENMETI, which binds to D b with an affinity similar to that of ASNENMETM. Although all positions examined influenced peptide binding, peptide residue no. 2 (P2) was of particular importance. Therefore, each of the 20 naturally occurring amino acids were inserted at this position to investigate their effects on peptide-MHC interaction. The results indicated that amino acids having side chains with charged or ring structures were deleterious, while non-polar and polar residues were either neutral or facilitated binding to different degrees. Our data also indicated that every residue of the peptide contributes to the stability of the MHC-peptide complex, and the final affinity is dependent on the nature of the amino acids at each position, not just on those at a small number of anchor positions. The results also suggested that increased stability, as indicated by the half-life of the peptide-MHC class I complex, might play an important role in selecting the immunodominant epitope.

A lactate dehydrogenase (LDH)-based immunoassay, referred to as CPEIA (cell panning enzyme immunoassay), has been developed for the detection of cell-surface antigens. CPEIA is similar to a panning assay, in that it is based on the capture of cells bearing an antigen of interest by means of an antibody immobilized to a 96-well microtiter plate. Attachment of the cells is then measured by addition of a substrate for the intracellular enzyme lactate dehydrogenase. The substrate solution also contains the nonionic detergent Triton X-100 to lyse the cells and release LDH, which converts the substrate p-iodonitrotetrazolium violet (INT) from yellow to red. The intensity of the color resulting from the LDH-catalyzed reaction is proportional to the number of cells bound to the plate. The procedure does not require fixation of the cells, centrifugation, and blocking steps, resulting in a more convenient assay. CPEIA has been used for the detection of MHC class I antigens and other molecules on the surfaces of mouse cell lines and concanavalin A (ConA)-stimulated T lymphocytes. In addition, the assay has been used to detect peptide binding to Db and Kb MHC class I molecules on the surface of the mutant cell line RMA-S. The half-maximal responses for peptide-MHC class I interactions at different peptide concentrations can be determined with the assay, allowing the apparent dissociation constants to be calculated.