Secondary Cytotoxic T Lymphocytes Response Research Articles

Dynamics of an HIV model with cytotoxic T-lymphocyte memory

We consider a four-dimensional HIV model that includes healthy cells, infected cells, primary cytotoxic T-lymphocyte response (CTLp), and secondary cytotoxic T-lymphocyte response (CTLe). The CTL memory generation depends on CD4+ T-cell help, and infection of CD4+ T cells results in impaired T-cell help. We show that the system has up to five equilibria. By the Routh–Hurwitz theorem and central manifold theorem we obtain some sufficient conditions for the local stability, globally stability of the equilibria, and the bifurcations. We still discover the bistability case where in the system there may coexist two stable equilibria or a stable equilibrium together with a stable limit cycle. Several numerical analyses are carried out to illustrate the validity of our theoretical results.

Secondary heterosubtypic influenza infections result in radically different T cell immunodominance hierarchies and clinical outcomes (VIR5P.1137)

Abstract When human populations face a novel subtype influenza A virus (IAV) with pandemic potential, the role of CD8+ cytotoxic T lymphocyte (CTL) responses targeting epitopes conserved or cross-reactive between the novel and previously encountered IAVs can be crucial to limit disease severity. To discover immune correlates of protective efficacy, we used a model of H7N9 secondary infections in mice primed by prior H1N1or H9N2 infection. Each priming case significantly, but variably, reduced disease morbidity and mortality, virus load, and time to virus clearance after H7N9 challenge. The sizes of the respective memory CTL pools were the best predictors of protective efficacy. The secondary CTL responses were characterized by earlier, significantly greater airway infiltration of H7N9 virus-specific CTLs but with distinct epitope immunodominance hierarchies among the dominant KbPB1703, DbPA224, and DbNP366 epitopes for each priming case. The epitope conservation between the priming and challenge viruses clearly influenced but alone did not consistently predict the immunodominance. The receptor repertoire of CTLs targeting a crossreactive and non-crossreactive DbNP366 epitope variant were characterized to reveal the nature of epitope cross-reactivity. These findings will contribute to understanding human CTL responses and rational design of CTL-mediated vaccines, where protective efficacy and immunodominance hierarchies may be highly sensitive to the immunological history of hosts.

CD154+ CD4+ T-cell dependence for effective memory influenza virus-specific CD8+ T-cell responses.

CD40-CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8(+) cytotoxic T lymphocyte (CTL) responses. This is typically via CD4(+)CD154(+) T-cell-dependent 'licensing' of CD40(+) dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4(+) T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40-CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154(+), but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection.

Redundancy of the influenza A virus-specific cytotoxic T lymphocyte response in HLA-B*2705 transgenic mice limits the impact of a mutation in the immunodominant NP 383–391 epitope on influenza pathogenesis

During the 1993–1994 flu season, influenza A/H3N2 viruses emerged with an amino acid substitution (R384G) at the anchor residue of the HLA-B*2705 restricted NP 383–391 epitope located in the nucleoprotein (NP). The R384G substitution reached fixation rapidly and abrogated recognition of A/H3N2 viruses by NP 383–391-specific CD8+ T cytotoxic T lymphocytes (CTL) completely. To test the impact of the R384G substitution in the immunodominant NP 383–391 epitope in vivo, influenza A viruses that differ at position 384 of the NP only were generated by reverse genetics. These viruses with an arginin (384R) or a glycin (384G) at position 384 were used to inoculate HLA-B*2705-transgenic mice and C57Bl/6 mice. Infection of naïve C57Bl/6 and HLA-B*2705 mice with influenza virus containing the NP 383–391 epitope (384R) caused more weight loss compared to infection with the virus without the epitope (384G). In contrast, HLA-B*2705 transgenic mice primed for a secondary CTL response by infection with a heterosubtypic influenza A virus, did not display this difference in virulence and the outcome of infection with the 384R virus was somewhat reduced. This phenotype of the 384R-virus was not observed in primed C57Bl/6 mice lacking HLA-B*2705. The relative reduction of weight loss after infection of primed HLA-B*2705+ mice with the 384R virus correlated with the CTL response to the NP 383–391. However, no differences were observed in the kinetics of viral clearance between the two viruses in immune HLA-B*2705+ mice, which may be attributed at least partially to CTL responses to other HLA-B*2705 restricted epitopes that were similar in magnitude.

Antiviral protection following immunization correlates with humoral but not cell‐mediated immunity

Smallpox was a deadly disease when it was rife yet despite its eradication more than 30 years ago, the possibility of accidental or intentional release has driven research in search of better definitions of correlates of protective immunity. Mousepox, a disease caused by ectromelia virus (ECTV), is arguably one of the best surrogate small animal models for smallpox. Correlates of protection in mousepox are well defined during primary infection, whereas those in a secondary infection, which have definite relevance to vaccination strategies, are less well understood. We previously established that neutralizing antibody (Ab), which is generated far more rapidly during a secondary infection compared with a primary infection, has a key role during a secondary virus challenge. In this study, we show that the route of immunization or the use of homologous or heterologous virus vaccines for immunization does not influence the ability of mice to control high-dose virulent ECTV challenge or to mount a substantial secondary neutralizing Ab response. In contrast, the recall cytotoxic T lymphocyte (CTL) responses generated under these regimes of immunization were varied and did not correlate with virus control. Furthermore, unlike the recall Ab response that was generated rapidly, the kinetics of the secondary antiviral CTL response was no different to a primary infection and peaked only at day 8 post-challenge. This finding further underscores the importance of Ab in conferring protection during secondary poxvirus infection. This information could potentially prove useful in the design of safer and more efficacious vaccines against poxviruses or other diseases using poxvirus vectors.

Maintenance or Emergence of Chronic Phase Secondary Cytotoxic T Lymphocyte Responses after Loss of Acute Phase Immunodominant Responses Does Not Protect SIV-Infected Rhesus Macaques from Disease Progression

The simian immunodeficiency virus- (SIV-) infected rhesus macaque is the preferred animal model for vaccine development, but the correlates of protection in this model are not completely understood. In this paper, we document the cytotoxic T lymphocyte (CTL) response to SIV and its effects on viral evolution in an effort to identify events associated with disease progression regardless of MHC allele expression. We observed the evolution of epitopes targeted by CTLs in a group of macaques that included long-term nonprogressing (LTNP), slowly progressing (SP), normally progressing (NP), and rapidly progressing (RP) animals. Collectively, our data (1) identify novel CTL epitopes from an SP animal that are not restricted by known protective alleles, (2) illustrate that, in this small study, RP and NP animals accrue more mutations in CTL epitopes than in SP or LTNP macaques, and (3) demonstrate that the loss of CTL responses to immunodominant epitopes is associated with viral replication increases, which are not controlled by secondary CTL responses. These findings provide further evidence for the critical role of the primary cell-mediated immune responses in the control of retroviral infections.

Clonal restriction of the expansion of antigen-specific CD8+ memory T cells by transforming growth factor-β

Recent evidence showed that transforming growth factor-beta (TGF-beta) regulates the global expansion of CD8+ T cells, which are CD44hi, a marker for memory cells. However, it is not clear whether this regulatory mechanism also applies to the antigen-specific CD8+ memory cells. By using a murine mixed lymphocyte culture (MLC) model, we examined the effect of TGF-beta on antigen-specific CD8+ memory cells [cytotoxic T lymphocyte (CTL)]. We found that the secondary CTL response in CD8+ memory cells from untreated MLC was not affected by TGF-beta but augmented by interleukin (IL)-2, whereas the CD8+ memory cells from TGF-beta-pretreated MLC (MLC-TGF-beta) failed to mount a significant, secondary CTL response, even when IL-2 was added. In exploring this dichotomy, in combination with flow cytometry analysis, we found that prolonged exposure to TGF-beta reduces the CTL activity in CD8+ memory cells. The increase by IL-2 and the reduction by TGF-beta of the CTL responses were clonal-specific. TGF-beta did not affect the CTL response to a third-party antigen or polyclonal T cell activation. Experiments performed with transgenic 2C cells gave similar results. Cell-cycle study performed with adoptive transfer of the cell tracker-labeled MLC cells revealed that the in vivo expansion of CD8+ memory cells from MLC-TGF-beta was restricted severely, and the restriction was clonal-specific, thus offering direct evidence to show that TGF-beta induces clonal restriction of CD8+ memory cell expansion.

Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen

The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNγ), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNγ-producing CTLs and their reaccumulation in the spleen. The data suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present.

Prior vaccination increases the epitopic breadth of the cytotoxic T-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection.

Although recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Simian immunodeficiency virus (SIV) gag DNA-vaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection.

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.

Enhancement of primary and secondary cellular immune responses against human immunodeficiency virus type 1 gag by using DNA expression vectors that target Gag antigen to the secretory pathway.

In this study, we have investigated the influence of antigen targeting after DNA vaccination upon the induction of cellular immune responses against human immunodeficiency virus type 1 (HIV-1) Gag. In addition to the standard version of HIV-1 Gag, we constructed Gag expression vectors that encode a secreted (Sc-Gag) and a cytoplasmic (Cy-Gag) Gag molecule. Although all three HIV-1 Gag expression vectors induced detectable humoral and cellular immune responses, after intramuscular injection the DNA vector encoding the Sc-Gag generated the highest primary cytotoxic T-lymphocyte (CTL) and T-helper responses. Mice immunized with one of the HIV-1 Gag DNA vectors (but not with the control vector pcDNA3. 1) developed a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag, and this response persisted for 125 days. The magnitude of the protection correlated with the levels of Gag-specific ex vivo CTL activity and the number of CD8(+) T cells producing gamma interferon. The DNA vector encoding the Sc-Gag induced higher levels of protection and greater secondary CTL responses than did the DNA vector encoding Cy-Gag.

Gamma interferon is not required for mucosal cytotoxic T-lymphocyte responses or heterosubtypic immunity to influenza A virus infection in mice.

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.

Loss of resistance to a highly immunogenic tumor with age corresponds to the decline of CD8 T cell activity.

A number of ultraviolet light-induced tumors that do not grow progressively in 3- or 9-month-old animals grow progressively in animals starting at approximately 15 months of age. We investigated the immune response to one such tumor, 6130, in young and old mice to determine in nature of this breakdown in immune protection with age. The 6130 tumor cells that grow progressively in 22-month-old animals still retain sensitivity of cytotoxic T lymphocytes (CTLs) generated against the highly immunogenic "6130-A" antigen and consequently are still immunologically rejected when transplanted into 3- and 9-month-old animals, but not in mice older than 15 months. Investigation of the CD8+ CTL response found that in animals 3-12 months old, primary and secondary tumor-specific CTL responses to 6130 can be generated in vivo and in vitro. On the other hand, the CD8+ CTL responses to 6130 seen in animals 15 months of age or older were significantly depressed or absent. These results indicate that a decline in antigen-specific CD8+ CTL is the major cause of increased susceptibility to 6130 tumor growth in older animals.

Antiviral cytotoxic T-cell memory by vaccination with recombinant Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterium that is able to escape phagocytic vesicles and replicate in the cytoplasm of infected cells. As with viral vectors, this intracytoplasmic life cycle provides a means for introducing foreign proteins into the major histocompatibility complex class I pathway of antigen presentation. Using recombinant L. monocytogenes (rLM) strains expressing the full-length nucleoprotein (NP) or a single cytotoxic T-lymphocyte (CTL) epitope from lymphocytic choriomeningitis virus (LCMV), we analyzed antiviral CTL responses induced by rLM vaccination. After vaccination, rLM was cleared from the host within 7 days while inducing an LCMV-specific ex vivo CD8+ effector CTL response. Virus-specific CTL memory was maintained for 6 months postvaccination, as demonstrated by vigorous secondary CTL responses after in vitro stimulation. A single immunization with rLM that expressed either the full-length NP gene or the CTL epitope alone resulted in LCMV NP-specific CTL precursor frequencies of approximately 1/10(4) CD8+ T cells. A second rLM vaccination resulted in enhanced virus-specific CTL activity and in vitro proliferation. rLM-vaccinated mice were protected against chronic viral infection by an accelerated virus-specific memory CTL response. These mice cleared infectious virus as well as viral antigen, suggesting that sterilizing immunity was achieved. In contrast to mice that received wild-type LM, rLM-vaccinated mice were protected from virally induced immunosuppression and splenic atrophy associated with chronic LCMV infection. The ability to elicit long-term cell-mediated immunity is fundamental in designing vaccines against intracellular pathogens, and these results demonstrate the efficacy of recombinant LM vaccination for inducing protective antiviral CTL memory.

Induction of peptide-specific primary cytotoxic T lymphocyte responses from human peripheral blood.

Various protocols were developed and compared for eliciting specific cytotoxic T lymphocyte (CTL) cell lines from the unselected human peripheral blood mononuclear cells of naive donors. Interleukin-7 and CD4+ T cells primed in vitro by keyhole limpet hemocyanin were shown to act together in the generation of these responses. Primary responses were consistently induced with a variety of different HLA class I-binding malarial peptides. Primary CTL responses could be induced from unselected CD8+ and from CD45RA+CD8+ T cells. The CTL lines derived from these naive donors were CD8+ and demonstrated a high level of HLA class I-restricted killing for > 3 months after priming in vitro. They were also able to recognize and kill targets infected with a recombinant vaccinia virus containing the full-length antigen. In addition, this same protocol enhanced up to fourfold the levels of secondary CTL responses induced. The optimal method presented for naive cytotoxic T cell stimulation is simple, rapid and generally applicable and should provide a useful tool for both basic research and human therapy.

Conditioning of the secondary cytotoxic T-lymphocyte response to YC8 tumor

Studies from our laboratory demonstrated that conditioned resistance to the syngeneic YC8 lymphoma was established by multiple conditioned stimulus (CS)/unconditioned stimulus (US) associations. The conditioned stimulus used was exposure to the odor of camphor for 1 h and the unconditioned stimulus was an injection of DBA/2 spleen cell alloantigen that shares minor histocompatibility determinants with the YC8 lymphoma. To demonstrate a cellular basis for immune resistance to the YC8 tumor, BALB/c mice primed with DBA/2 spleen cell alloantigen were conditioned using a single trial CS/US association paradigm. Conditioned animals showed a measurable conditioned elevation of the cytotoxic T-lymphocyte (CTL) response to the YC8 tumor. Control groups in which the CS and US were not given in the proper sequence were unable to mount a conditioned response. These studies show that a secondary CTL response can be upregulated by the central nervous system (CNS).

Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule.

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2Kd to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2Kd. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2Kd-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2Kd; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2Kd. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2Kd to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2Kd class I molecule, and only a fraction of these serve as CTL epitopes.

In vitro human cytotoxic T cell responses against influenza A virus can be induced and selected by synthetic peptides

Studies on human anti-influenza cytolytic activities have demonstrated that cytotoxic T lymphocytes (CTL) from HLA-B37 individuals react preferentially with the peptide corresponding to residues 335-349 of the nucleoprotein, whereas CTL from HLA-A2 donors recognize peptide 57-68 from the viral matrix as a dominant epitope. We studied the secondary CTL response, obtained from peripheral blood mononuclear cells, of an HLA-A2+,B37+ individual stimulated either by infectious virus or by synthetic peptides. Only an HLA-B37-restricted response was detected after stimulation by the whole virus, showing an immunodominance of this activity over that restricted by HLA-A2. Moreover, human cytotoxic cell lines were successfully obtained after stimulation of peripheral blood mononuclear cells with synthetic peptides. Under these conditions, it was possible to selectively reveal the existence of an HLA-A2-restricted activity directed against the matrix peptide. These results demonstrate that, at least in vitro, it is possible to stimulate a latent repertoire by using synthetic peptides. Nevertheless, we could not induce a response against the matrix or the nucleoprotein peptides in HLA-A2- or B37- individuals, suggesting that a finer selection of synthetic peptides would be necessary for their possible utilization to induce CTL during vaccination.

Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to stimulate the secondary anti-PrV CTL response. Cultures of both murine or swine lymphocytes that had been stimulated with gIII- virus contained only approximately half as many lytic units as did those stimulated with either wild-type virus, a gX- virus mutant, or a gI-/gp63- virus mutant. Thus, a large proportion of the PrV-specific CTLs that are induced by immunization with PrV of both mice and pigs are directed against gIII. Furthermore, glycoproteins gI, gp63, and gX play at most a minor role in the CTL response of these animals to PrV.