Cytotoxic T-lymphocyte Precursor Frequencies Research Articles

Vaccination against persistent viral infection exacerbates CD4+ T-cell-mediated immunopathological disease.

Lymphocytic choriomeningitis virus (LCMV) infection of normal mice results in a fatal immunopathologic meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL). We have previously shown that female beta2-microglobulin-deficient (beta2m-/-) mice, which are also deficient in CD8+ T cells, are susceptible to LCMV-induced immune-mediated meningitis, characterized by significant weight loss and mortality. This LCMV disease in beta2m-/- mice is mediated by CD4+ T lymphocytes. Our previous studies have also demonstrated that male beta2m-/- mice are less susceptible than female beta2m-/- mice to LCMV-induced, immune-mediated mortality and weight loss. In this report, we show that vaccination of male beta2m-/- mice enhances immunopathology following intracranial infection with LCMV. We observed increased production of gamma interferon (IFN-gamma), an increase in CD4+ CTL precursor frequency, and an increased frequency of IFN-gamma-producing cells from spleen cells of vaccinated male beta2m-/- mice. Vaccinated male beta2m-/- mice also had significantly increased inflammation in the cerebrospinal fluid (CSF), characterized by a large CD4+ T-cell infiltrate. CSF cells from vaccinated mice showed increased production of IFN-gamma on day 7 postchallenge. Neither vaccinated nor control beta2m-/- mice were able to clear virus, and the two groups had similarly high levels of virus early after infection. These results suggest that the magnitude of the early immune response is more important than the level of virus in the brain in determining the outcome of immunopathology in beta2m-/- mice. We show here that vaccination can increase CD4+ T-cell-dependent immunopathology to a persistent viral infection.

Dominant cytotoxic T lymphocyte response to the immediate-early trans-activator protein, BZLF1, in persistent type A or B Epstein-Barr virus infection.

Five healthy human leukocyte antigen-B8 (HLA-B8)-positive virus carriers were studied to investigate the CD8+ cytotoxic T lymphocyte (CTL) response to an HLA-B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early trans-activator protein, BZLF1. Of the 5 virus carriers, 4 were infected with type A and 1 with type B EBV. Using limiting-dilution analysis of peripheral blood mononuclear cells, a high RAKFKQLLQ-specific CTL precursor frequency was demonstrated after specific peptide or autologous lymphoblastoid cell line stimulation in both type A and type B EBV carriers. The RAKFKQLLQ-specific CTL precursor frequencies in all 5 persons were at least as dominant as those observed with two other EBV-associated, HLA-B8-restricted latent epitopes, FLRGRAYGL and QAKWRLQTL. These findings show that healthy virus carriers maintain a high frequency of BZLF1-specific memory T cells, potentially to control virus spread from lytically infected cells.

Rapid effector function in CD8+ memory T cells.

The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon gamma (IFN-gamma) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-gamma release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I-restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.

Bone Marrow Contains Virus-Specific Cytotoxic T Lymphocytes

AbstractImmunizing bone marrow donors prior to bone marrow transplant (BMT) has the potential for adoptively transferring specific immunity against opportunistic pathogens. Studies have shown that long-term antibody production occurs in the bone marrow and that specific humoral immunity may be transferred from donor to recipient following BMT. However, the magnitude and duration of T-cell memory in the bone marrow compartment has not been adequately investigated. In this study, virus-specific CD8+ T-cell responses in the bone marrow were compared with those observed in the spleen of mice acutely infected with lymphocytic choriomeningitis virus (LCMV). During the acute stages of infection, most CD8+ T cells in the spleen and bone marrow showed upregulated surface expression of the activation/memory marker, LFA-1 (LFA-1hi). After clearing LCMV infection, the antiviral immune response subsided to homeostatic levels and the ratio of CD8+/LFA-1hi to CD8+/LFA-1lo T cells in the spleen and bone marrow of LCMV immune mice returned to the value observed in naive mice. Virus-specific ex vivo effector cytotoxic T-lymphocyte (CTL) responses could be identified in both spleen and bone marrow compartments at 8 days postinfection. LCMV-specific CTL precursor (CTLp) frequencies peaked in the bone marrow at 8 days postinfection and averaged one in 200 to one in 650 CD8+ T cells, a frequency similar to that observed in the spleen. After clearing the acute infection, potent LCMV-specific CTL memory responses could be demonstrated in the bone marrow for at least 325 days postinfection, indicating long-term persistence of antiviral T cells at this site. Adoptive transfer of LCMV-immune bone marrow into severe combined immunodeficiency (SCID) mice provided protection against viral challenge, whereas SCID mice that received naive bone marrow became chronically infected upon challenge with LCMV. These results indicate that after acute viral infection, virus-specific memory T cells can be found in the bone marrow compartment and are maintained for an extended period, and when adoptively transferred into an immunodeficient host, they are capable of conferring protection against chronic viral infection.

A single [3H]thymidine-based limiting dilution analysis to determine HTLp and CTLp frequencies for bone marrow donor selection.

Histocompatibility between recipient and donor is a critical factor in allogeneic BMT which, to a large extent, determines the incidence of GVHD after BMT. Functional histocompatibility assays, such as the helper T lymphocyte precursor frequency assay (HTLp) and the cytotoxic T lymphocyte precursor frequency assay (CTLp), have proved to be helpful tools in facilitating donor selection procedures. However, a major drawback of these assays is that they are laborious and require large numbers of cells. We therefore adapted a [3H]thymidine-based assay, the 'JAM' test, as a read-out for CTLp frequencies, to replace the more cumbersome 51Cr-release assay. Furthermore, we applied an experimental setup that enables the assessment of HTLp and CTLp frequencies from a single limiting dilution assay to reduce the number of cells needed. The newly developed assay is relatively easy to perform and has the advantage that different subsets of T cells can be quantified in a single ongoing alloreaction. When the combined assay was applied in unrelated donor selection it proved to be a sensitive method that enables differentiation in suitability of distinct donors for a single patient. Therefore, the combined HTLp/CTLp assay appears to be a practical and sensitive method for identifying functional histocompatibility in related and unrelated donor/recipient combinations.

Direct MHC class I complementary DNA transfer to thymus induces donor-specific unresponsiveness, which involves multiple immunologic mechanisms.

Our purposes were 1) to determine whether direct transfer of cDNA encoding allogeneic MHC class I Ag to the rat thymus would be capable of inducing donor-specific unresponsiveness and 2) to study the immunologic mechanism of this effect. Plasmid DNA encoding donor strain (ACI-RT1.Aa) MHC class I Ag was directly injected into Lewis (RT1(l)) rat recipient thymus 7 to 10 days before ACI liver transplantation. A single dose of anti-lymphocyte serum was given i.p. on the day of thymic injection. Rats injected intrathymically with plasmid DNA and treated with anti-lymphocyte serum demonstrated prolonged survival in 9 of 13 rats (>100 days). PCR was used to demonstrate that RT1.Aa cDNA was expressed in thymus transiently and later appeared in spleen. CTL limiting dilution assays showed that CTL precursor frequency was decreased in tolerant liver recipients. To test the hypothesis of clonal deletion vs anergy, CTL limiting dilution assays cultures were restimulated with donor cells and IL-2 to reverse anergy. Restimulation caused CTL precursor frequency to return to near normal in only one of five tolerant rats, suggesting clonal deletion or a dense anergic state. Passive transfer of splenocytes from tolerant rats to naive recipients prolonged cardiac allograft survival, suggesting that suppressor-type cells may also contribute to thymic tolerance in our model. In summary, our data suggest that donor MHC class I Ag expressed in thymus by direct DNA injection, followed by liver allografting, results in donor-specific unresponsiveness. The mechanism of this effect is complex, involving multiple immunologic mechanisms.

Molecular characterization of HLA-C incompatibilities in HLA-ABDR-matched unrelated bone marrow donor-recipient pairs. Sequence of two new Cw alleles (Cw*02023 and Cw*0707) and recognition by cytotoxic T lymphocytes.

While the influence of HLA-AB and -DRB1 matching on the outcome of bone marrow transplantation (BMT) with unrelated donors is clear, the evaluation of HLA-C has been hampered by its poor serological definition. Because the low resolution of standard HLA-C typing could explain the significant number of positive cytotoxic T lymphocyte precursor frequency (CTLpf) tests found among HLA-AB-subtype, DRB1/B3/B5-subtype matched patient/donor pairs, we have identified by sequencing the incompatibilities recognized by CD8+ CTL clones obtained from such positive CTLpf tests. In most cases the target molecules were HLA-C antigens that had escaped detection by serology (e.g. Cw*1601, 1502 or 0702). Direct recognition of HLA-C by a CTL clone was demonstrated by lysis of the HLA class I-negative 721.221 cell line transfected with Cw*1601 cDNA. Because of the functional importance of Cw polymorphism, a PCR-SSO oligotyping procedure was set up allowing the resolution of 29 Cw alleles. Oligotyping of a panel of 382 individuals (including 101 patients and their 272 potential unrelated donors, 5 related donors and 4 platelet donors) allowed to determine HLA-C and HLA A-B-Cw-DRB1 allelic frequencies, as well as a number of A-Cw, B-Cw, and DRB1-Cw associations. Two new HLA-Cw alleles (Cw*02023 and Cw*0707) were identified by DNA sequencing of PCR-amplified exon 2-intron 2-exon 3 amplicons. Furthermore, we determined the degree of HLA-C compatibility in 287 matched pairs that could be formed from 73 patients and their 184 potential unrelated donors compatible for HLA-AB by serology and for HLA-DRB1/ B3/B5 by oligotyping. Cw mismatches were identified in 42.1% of these pairs, and AB-subtype oligotyping showed that 30% of these Cw-incompatible pairs were also mismatched for A or B-locus subtype. The degree of HLA-C incompatibility was strongly influenced by the linkage with B alleles and by the ABDR haplotypes. Cw alleles linked with B*4403, B*5101, B18, and B62 haplotypes were frequently mismatched. Apparently high resolution DNA typing for HLA-AB does not result in full matching at locus C. Since HLA-C polymorphism is recognized by alloreactive CTLs, such incompatibilities might be as relevant as AB-subtype mismatches in clinical transplantation.

Frequency of CTL precursors and effectors directed to a single peptide of melan-A/Mart-1

Frequency of CTL precursors and effectors directed to a single peptide of melan-A/Mart-1

Split tolerance induced by immunotoxin in a rhesus kidney allograft model.

Renal allografts were performed in rhesus monkeys using FN18-CRM9, a potent immunotoxin capable of depleting T cells to less than 1% of baseline levels in blood and lymph nodes, as a preparative agent. We have recently reported that animals pretreated with FN18-CRM9 1 week before transplantation without further immunosuppression had prolonged graft survival time compared with control animals, and frequently became tolerant. This report examines the alloimmune responses of recipient monkeys to the donor, including cytotoxic T lymphocyte precursor (CTLp) frequency, mixed lymphocyte response, and antidonor IgG response. CTLp frequencies declined significantly (P<0.01) after FN18-CRM9 treatment and renal transplantation. This decline in CTLp was initially nonspecific, as CTLp frequencies against third-party animals also declined (P<0.01). The decrease in CTLp was maintained in five of five animals tested 6 months after transplant. However, unresponsiveness was limited to the CTL arm of the immune response as antidonor IgG was detected in four of four animals tested, and the 5-day mixed lymphocyte response stimulation index and relative response were not significantly different before and after transplant. In long-term survivors (>150 days), an increase in anti-third-party CTLp was detected 1 month after grafting with third-party skin. No change was seen in the antidonor CTLp frequency after donor skin grafting, indicating that a specific defect in the antidonor CTL response had developed. These data suggest that FN18-CRM9 treatment of rhesus monkeys allows the development of specific down-regulation of antidonor CTL activity in renal allograft recipients.

Differential effect of HLA-C locus mismatches on the CTL precursor frequency (CTLp)

Differential effect of HLA-C locus mismatches on the CTL precursor frequency (CTLp)

Differential effect of HLA-C locus mismatches on the CTL precursor frequency (CTLp)

Differential effect of HLA-C locus mismatches on the CTL precursor frequency (CTLp)

Characteristics of direct and indirect activation of human T cells against allogeneic and porcine xenogeneic cells/peptides

Abstract: The immunological consequences of direct versus indirect activation of T lymphocytes is of considerable interest both in allogeneic and in xenogeneic transplantation. In this paper, the activation of human T cells by allogeneic and xenogeneic porcine antigen‐presenting cells (APC) (direct activation), or by donor‐derived peptides presented on autologous APC (indirect activation) was studied in vitro. The direct activation of purified human T lymphocytes by allogeneic and xenogeneic porcine stimulator cells led to marked proliferative responses, IL‐2 and IFN‐γ production. The ratio of CD4/CD8 of responsive cells upon direct activation was the same for allogeneic and xenogeneic stimulation. Also, the CD25 expression on total and on CD8+ lymphoblasts was similar in allogeneic and xenogeneic responses. The frequency of alloreactive and xenoreactive CTLp following direct activation was 7.7 ± 1.4 / 104 and 3.7 ± 1.3 / 104, respectively. Direct activation of proliferation and IL‐2 and IFN‐γ production in both experimental situations was completely blocked by Cyclosporin A (CsA). 15‐deoxyspergualin (DSG), which is thought to prevent antigen processing, had little effect. Thus, when it comes to direct activation of T lymphocytes xeno‐ and allospecific responses were qualitatively and quantitatively comparable.However, some differences between allogeneic and xenogeneic responses were observed when indirect activation was studied using stimulator cells from peripheral blood depleted of both adherent cells and of SLA class II positive cells or when human allogeneic or xenogeneic fibroblasts were used as stimulators. Indirect activation following recognition of alio‐ or xenogeneic peptides in a responder HLA‐restricted fashion resulted in proliferation, IL‐2 and IFN‐y production, albeit lower than in direct combinations. Indirect activation also induced a lower proportion of CD25+ lymphoblasts than direct activation. Furthermore, indirect xenoactivation resulted preferentially in proliferation and CD25 expression in the CD4+ cell population, and in contrast to direct activation, not only CsA but also DSG inhibited activation. Thus, our findings indicate that direct and indirect activation of human responder T cells stimulated by allogeneic and/or xenogeneic porcine cells/peptides results in immune responses with similar characteristics. Only the proliferation of CD8+ cells and their expression of CD25 and of functional IL‐2 receptors was weaker following xeno‐ than alloactivation, reasons for which are discussed. We conclude that indirect activation of T cell reactivity against xenogeneic peptides leads to an efficient immune response, likely to be of clinical importance for rejection of xenogeneic transplants devoid of professional antigen‐presenting cells.

Epstein-Barr virus-specific cytotoxic T cell responses in HIV-1 infection: different kinetics in patients progressing to opportunistic infection or non-Hodgkin's lymphoma.

Although the high incidence of EBV-associated diffuse large cell lymphomas (DLCL) in HIV-1 infection is believed to be related to loss of immune control due to HIV-induced immune deficiency, it has been claimed that cytotoxic T lymphocyte (CTL) responses to EBV are longer lasting in HIV-1-infected persons than CTL directed against HIV-1 itself. We approached this apparent paradox by performing the first longitudinal study into the kinetics of EBV and HIV-specific CTL responses in HIV-infected patients progressing either to AIDS with non-Hodgkin's lymphoma (NHL) or AIDS with opportunistic infection (OI). Multiple samples were tested from HIV-1 seroconversion to AIDS-diagnosis. Four out of six patients that were either long-term asymptomatic or progressing to OI showed declining HIV-1 CTL precursor (CTLp) frequencies whereas EBV-CTLp remained stable, suggestive for HIV-1-specific immune exhaustion. In two patients rapidly progressing to AIDS-OI, a parallel decline of HIV-1- and EBV-CTL responses was seen, indicative for total collapse of cellular immunity. In all these six patients EBV-load remained low. However, in four out of five patients that progressed to DLCL, EBV-load was high and increasing several months preceding the NHL. In all five patients, EBV-CTLp decreased before the emergence of the NHL. Thus, our data show that in HIV-1 infection loss of HIV-1-specific T cell immunity is not necessarily paralleled by loss of EBV-specific T cell responses. The occurrence of AIDS-related DLCL is preceded by decreasing EBV-CTLp and increasing EBV load. Failing EBV-control might therefore be an important step in the pathogenesis of AIDS-related DLCL.

Gag-Specific Immune Responses after Immunization with pl7/p24:Ty Virus-Like Particles in HIV Type 1-Seropositive Individuals

Gag-specific immune responses and changes in HIV-1 RNA levels were evaluated in eight HIV-1-infected persons, in order to assess the immunotherapeutic potential HIV-1 p17/p24: Ty virus-like particles (p24-VLP). All treated subjects showed transient and dose-dependent proliferative responses to the Ty-VLP carrier (stimulation index [SI], 2.0-119.5). Three of four individuals who received either 500 or 1,000 micrograms of p24-VLP also showed proliferative responses to p17 or p24 (SI, 2.0-15.7). In 2 subjects who were treated with either 500 or 1,000 micrograms of p24-VLP, enhanced Gag-specific CTL precursor (CTLp) frequencies were observed after immunization (10- to 14-fold). Both subjects had low baseline Gag-specific CTL activity (< 25 cTLp/10(6) PBMCs). In the other participants studied no significant boosting of preexisting Gag-specific CTL responses was observed. Short-term elevation of HIV-1 RNA levels at weeks 2 and 4 was observed in two subjects treated with the highest dose of p24-VLP. However, HIV-1 RNA levels at week 24 did not significantly differ from those found in the placebo group. In conclusion, p24-VLP induced marginal Gag-specific immune responses in limited numbers of HIV-1-seropositive individuals, with some showing transient elevation of HIV-1 viral load. Further studies are needed to establish potential clinical effects of these observations.

Precursor frequency analysis of cytotoxic T lymphocytes to pre-erythrocytic antigens of Plasmodium falciparum in West Africa.

Some individuals living in malaria-endemic areas have CTL to Plasmodium falciparum liver stage Ags. We have quantified these CTL responses using limiting dilution analysis studies on the peripheral blood cells of naturally exposed Gambian donors. CTL precursor frequencies were determined to a wide range of epitopes derived from different liver stage Ags (liver stage protein 1, circumsporozoite protein, thrombospondin-related anonymous protein, and sporozoite threonine/asparagine-rich protein) restricted through common HLA alleles present in this population (HLA-A2.1, -A2.2, -B7, -B8, -B35, and B53). Precursor frequencies were between 17 and 98/million PBMC and correlated with the levels of specific lysis in parallel bulk cultures. The quantitative nature of limiting dilution assay analysis revealed varying degrees of immunodominance in the CTL responses to different epitopes within single proteins (thrombospondin related anonymous protein: tr42, tr43, tr26, tr29, and tr39; circumsporozoite protein: cp6, cp26, and cp29) and within individual donors. The temporal stability of some of these CTL responses was determined over a 4-yr period. This is the first quantitative study of CTL specific for any plasmodial species or nonviral pathogen in humans and provides a basis for a multiepitope approach to malaria vaccination.

The ability of peptides to induce cytotoxic T cells in vitro does not strongly correlate with their affinity for the H-2L d molecule: Implications for vaccine design and immunotherapy

The hypothesis that the ability of a peptide to bind to a class I molecule correlates with its immunogenicity is controversial. In this paper we have measured the affinity constants of nine synthetic peptides, which have been previously identified as binding to H-2L d molecules, and have determined their immunogenicity in an in vitro cytotoxic T lymphocyte (CTL) induction assay. We find that six peptides bind with high affinity ( K a > 10 7 M ); of these, four are of viral origin but only two elicit potent CTLs, one is a self peptide which is not immunogenic, while the sixth is of bacterial origin and also does not generate effective CTLs. Two peptides bind with intermediate affinity ( K a > 10 6 M ); one of these elicits a moderate CTL response, while the other, a tumor-derived epitope, is highly immunogenic. Intriguingly, the peptide with lowest affinity (p2Ca) is exceedingly effective at eliciting CTLs. The efficacy of peptides with modest affinity for their restriction elements appears to corrlate well with the CTL precursor frequency. We have also examined intrinsic parameters of some of the peptides such as solubility and stability. Taken together, our results underscore the relevance of factors other than affinity which affect immunogenicity and which may be critical in the design of peptide-based vaccines as well as tumor immunotherapy approaches.

Protection against immunopathological consequences of a viral infection by activated but not resting cytotoxic T cells: T cell memory without "memory T cells"?

Immunological memory is a key characteristic of specific immune responses. Persistence of increased levels of precursor T cells is antigen-independent and is often used as an indicator of T cell memory. This study documents that, depending on the chosen readout, cytotoxic T lymphocyte (CTL) memory against lymphocytic choriomeningitis virus (LCMV) appears long- or short-lived in the absence of persisting antigen. To study T cell memory in the absence of persisting antigen, either short-lived antigens were used for immunization or adoptive transfer methods were used to eliminate possibly persisting antigen. These experiments revealed that increased specific precursor frequencies and CTL-mediated protection against an i.v. infection with LCMV were long-lived. In contrast, CTL-mediated protection against a peripheral infection of the skin with LCMV, or of the ovary with recombinant vaccinia virus, was short-lived. These results show that maintenance of increased specific CTL precursor frequencies and central T cell memory in lymphoid tissue (where preexisting neutralizing antibodies usually provide protection anyway) is long-lived and antigen-independent. In contrast, in protection against peripheral viral infections, where the relative kinetics of virus growth and virus elimination by T cells are of key importance, T cell memory is short-lived in the absence of antigen. This indicates that peripheral T cell memory in antibody-inaccessible tissues is mediated by antigen-activated effector T cells and apparently not by specialized memory T cells.

Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines.

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are present in the peripheral blood and liver of chronically infected patients. The current study was performed to study the relationship between the strength of the CTL response, liver disease severity, and viral load. The results may be summarized as follows: first, using CTL precursor frequency (CTLpf) analysis to quantitate the peripheral blood CTL response, chronically infected patients were less strongly sensitized to a panel of well-defined HCV epitopes than they were to an epitope within the influenza matrix protein. Second, HCV-specific CTLpf did not correlate with disease activity or viral load in the majority of patients on a cross-sectional basis, although it did increase in three patients concomitant with sharp increases in liver disease. Finally, interferon therapy did not enhance the CTLpf against the HCV epitopes studied in these patients, indicating that its antiviral effect is independent of the CTL response. Since the HCV-specific CTLpf in the blood is actually quite low, the CTL may contribute to ongoing liver disease in these patients while being quantitatively inadequate to destroy all of the infected hepatocytes, thereby facilitating HCV persistence and contributing to chronic liver disease.