Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes.

Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmAxgzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.

High peptide affinity for MHC class I does not correlate with immunodominance.

Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147-155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147-155. Kdm-expressing target cells saturated with synthetic NPP147-155 (10-5 M) were lysed similarly to Kdw-expressing targets by NPP147-155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm-peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147-155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.

Spontaneous mutation at position 114 in H-2Kd affects cytotoxic T cell responses to influenza virus infection.

Vaccinia virus (VV)-encoded MHC class I Kd molecules which differ by a single amino acid change from glutamine (Kdw, wild type) to histidine (Kdm, mutant) at position 114 located in the floor of the peptide binding groove were compared in terms of peptide binding and cytotoxic T (Tc) cell recognition. Most anti-viral Tc cells were not affected or only marginally affected. However, the Kdm molecule did not detectably present the immunodominant peptide (NPP147-155) of influenza virus nucleoprotein (NP), encoded by the full-length NP gene either in influenza A virus or recombinant VV. This defect could be overcome by using exogenous synthetic NPP147-155 or translation from a minigene encoding NPP147-155 in VV. Kdw presented NPP147-155 encoded by the full-length NP gene, but Kdw-NPP147-155 complexes were at least 100-fold less abundant than after translation from a minigene.

In vivo administration of major histocompatibility complex class I-specific peptides from influenza virus induces specific cytotoxic T cell hyporesponsiveness.

We have been investigating the immunogenicity of two class I major histocompatibility complex-specific peptides with a sequence derived from influenza virus nucleoprotein specific for Kd and one for Db. Peptide-modified splenocytes are unable to immunize for a primary cytotoxic T (Tc) cell response in vivo, or secondary response in vitro. Peptide-modified stimulator cells can boost virus-primed splenocytes for a strong secondary response in vitro. Animals primed with syngeneic peptide-modified splenocytes upon challenge with virus in vivo do not generate strong secondary Tc cell responses on day 3 after challenge in contrast to virus primed animals. Day 6 responses of virus-challenged, peptide-primed animals are reduced as compared to unprimed mice. This hyporesponsiveness is independent of CD8+ T cells in the priming population and can be elicited with tumor cell lines. The data are discussed in the framework of the two-signal model of immune induction.

Cellular immune responses in the murine lung to local immunization with influenza A virus glycoproteins in micelles and immunostimulatory complexes (iscoms).

Primary immunization with a single inoculum of either micelles or iscoms containing influenza A virus glycoproteins failed to induce either B or cytotoxic T (Tc) cell responses. In contrast, immunization with two inocula of iscoms, but not micelles, resulted in the appearance of influenza virus-specific antibody-secreting cells (ASC) but not Tc cells in the lung. There was a 10-fold increase in Tc cell precursor frequency and an increase in ASC generated by secondary in vitro stimulation of lung cell cultures obtained from mice primed with iscoms but not micelles. In mice primed with infectious virus, secondary immunization with either micelles or iscoms increased the number of ASC in the lung and elicited virus-specific Tc cell responses. In contrast homologous virus challenge failed to induce detectable secondary B or Tc cell responses.

Cytotoxic T cells recognize very early, minor changes in ectromelia virus-infected target cells.

Target cells (P-815 mastocytoma cells) infected with ectromelia virus became susceptible to lysis by H-2 compatible specific effector T cells within one hour of exposure of the cells to virus. This is long before viral progeny are produced and shed from the cell. Polyacrylamide gel electrophoresis (PAGE) profiles of the plasma membranes from infected and uninfected P-815 cells pulsed with 35S-methionine for one or a few hours after infection with virus were very complex and showed no consistent differences. P-815 cells, infected with ectromelia virus in the presence of an inhibitor of protein synthesis, pactamycin, slowly became susceptible to cell mediate lysis when the pactamycin was removed. The number of polypeptide species synthesized under these conditions was reduced to only three, of molecular weights between 10,000-50,000 daltons. Specific, newly synthesized membrane components recognized by mouse convalescent sera were isolated by immune complexing and examined by PAGE. Six polypeptide bands were seen, the major one correlating with one observed in the pactamycin experiment. The results suggested that the convalescent serum recognized both viral and host cell coded antigens. The significance of these findings is discussed.