Cytotoxic T-lymphocyte Precursor Frequencies Research Articles

Does the emergence and persistence of donor-derived leukaemia-reactive cytotoxic T lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study.

The frequency of CTL precursors (CTLp) directed towards recipient-derived pre-transplant leukaemic blasts (LB) was measured in the peripheral blood of nine children with acute leukaemia and given BMT from either an HLA-identical sibling or a matched unrelated donor (MUD). Patients were evaluated at various time points between 1 month and more than 2 years after transplantation. A high frequency of donor-derived LB-reactive CTLp was detectable 2-6 months after BMT in all children and persisted for at least up to 18 months in the eight patients in haematological remission, while it rapidly declined in the only patient who relapsed. Generation of LB-reactive T cell clones obtained from some of these patients demonstrates that various T lymphocyte subsets, either HLA class I-restricted/TCR-dependent or HLA-unrestricted, contribute to this phenomenon. The in vitro GVL effect here described seems to be at least partially separated from GVHR, since no correlation was observed between the emergence of LB-reactive CTLp and the development and/or severity of GVHD. Development of LB-reactive CTL in the patients was independent of the frequency of these cells in the donor. These data suggest that donor-derived CTL activity specifically directed towards leukaemic blasts may develop in patients given allogeneic BMT and contribute to the maintenance of a state of haematological remission.

Predicting graft-versus-host disease in HLA-identical bone marrow transplant: a comparison of T-cell frequency analysis and a human skin explant model.

Graft-versus-host disease (GVHD) occurring after HLA-identical sibling bone marrow transplantation (BMT) is considered to be mainly caused by minor histocompatibility antigen (mHag) disparities between the recipient and donor. In our laboratory, a human skin explant model has been successfully used to predict acute GVHD in HLA-identical sibling BMT. More recently, the frequency analysis of host-reactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp, respectively) has been shown to have potential application for predicting GVHD. In the present study, HTLp and CTLp frequency analysis and the skin explant model were directly compared for their ability to predict acute GVHD in HLA-identical sibling BMT. Host-reactive HTLp and CTLp frequencies were determined using a combined limiting dilution assay. A human skin explant model was used to detect graft-versus-host reactions in vitro. The results from the skin explant model (graft-versus-host reaction grades I-IV) and T cell frequency analysis (>/< 1:100,000) were correlated with posttransplant GVHD outcome, respectively. The skin explant model correctly predicted GVHD outcome in 77% of cases (P=0.03). HTLp frequencies were very low in all patient/donor pairs tested. None of them exceeded 1:100,000, although 9/18 recipients developed GVHD (> or =clinical grade II) after transplant. In all patients tested, the relationship between either high (>1:100,000) or low (<1:100,000) CTLp frequency and occurrence of GVHD appeared to be random (P=1.0). HTLp and CTLp frequency analysis did not predict the occurrence of acute GVHD after HLA-identical sibling BMT. The human skin explant model, however, remained an accurate indicator of acute GVHD and probably detects mHag disparities.

Cellular alloreactivity of human cord blood cells detected by T-cell frequency analysis and a human skin explant model.

Recent clinical data have demonstrated the success of allogeneic stem cell transplantation using HLA-mismatched unrelated human umbilical cord blood (CB). The incidence and severity of acute graft-versus-host disease (GVHD) in these mainly pediatric transplants is low. The immunological mechanisms by which CB transplants may result in reduced GVHD is not completely clear. In this study, the functional cellular alloreactivity of CB cells was investigated, by measuring the frequency of alloreactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp, respectively) in CB and detecting the ability of CB cells to induce graft-versus-host (GVH) type alloreactivity in vitro. A human skin explant model was used to measure GVH type alloreactivity in vitro. A combined limiting dilution assay was carried out in parallel to determine alloreactive HTLp and CTLp frequencies. The cellular alloreactivity was compared between cord and HLA-haploidentical parental blood cells against the same HLA-mismatched unrelated stimulator. The results demonstrated that alloreactive CTLp frequency in CB mononuclear cells (CBMCs) was significantly lower (mean, 1:35,694, range, 1:1,667-<1:500,000) than that in adult peripheral blood mononuclear cells (PBMCs) (mean, 1:5,333, range, 1:544-1:47,619). Alloreactive HTLp frequencies, however, were comparable for CBMCs and PBMCs (mean, 1:7,586, range, 1:1,359-1:200,000; and mean, 1:5,976, range, 1:385-1:50,000, respectively). A significantly decreased ability to induce in vitro GVH type alloreactivity was observed for CBMCs and that was strongly associated with low alloreactive CTLp frequencies (P=0.001). The present study provides the first clear in vitro evidence to suggest that CBMCs are less able than PBMCs to induce skin GVH type alloreactivity in HLA-mismatched pairs. The severity of in vitro GVH type alloreactivity (graded as I-IV) was strongly associated with the levels of alloreactive CTLp frequencies. The low cellular alloreactivity of CBMCs detected in vitro suggests that in a proportion of cases HLA-mismatched unrelated CB may not give rise to severe GVHD in vivo after transplantation.

The Impact of Variation in the Number of CD8+T-Cell Precursors on the Outcome of Virus Infection

We investigated the role of varying the initial number of naive antiviral CTL precursors on the dynamics of LCMV–DOCILE infection. C57BL/6 mice, exhibiting LCMV-specific CTLp frequencies of about 50, are protected against virus persistence over a range of infectious doses up to 104pfu. With 10-fold higher doses, a 100-fold increase in CTLp is required to restore virus control. With doses above 106pfu, elevation of the initial CTLp number leads only to lethal immunopathology. Similarly, a 1000-fold increase in the number of initial naı̈ve CTLp enhances the overall kinetics of virus elimination, but cannot limit early virus spread within the first 48 h after low-dose infection (500 pfu). Increases in initial naı̈ve virus-specific CTLp numbers are of limited benefit in antiviral control. In addition to the number of virus-specific T cells, the time period needed to reach cytolytic effector function is a limiting parameter.

Donor-specific hyporeactivity after liver transplantation: prominent decreases in donor-specific cytotoxic T lymphocyte precursor frequencies independent of changes in helper T lymphocyte precursor frequencies or suppressor cell activity.

The development of immunological donor-specific hyporeactivity may account for the low incidence of chronic rejection after clinical liver transplantation. We investigated whether hyporeactivity commonly develops after liver transplantation by analyzing precursor frequencies of donor-reactive cytotoxic (CTLp) and helper (HTLp) T lymphocytes and mixed lymphocyte culture (MLC) reactivity in liver allograft recipients. We further studied whether CTLp hyporeactivity correlated with changes in donor-specific HTLp frequencies or suppressor cell activity. CTLp and HTLp frequencies and MLC reactivity against donor and third-party spleen cells were determined in pre- and posttransplantation peripheral blood samples from 18 recipients with good graft function 2 years after transplantation. By mixing posttransplantation samples (with "putative" suppressor cell activity) with pretransplantation samples (in which normal CTL activity with no suppressor cell activity is expected), the presence of suppressor cell activity in peripheral blood was analyzed. Two years after transplantation, all but one (94%) of the recipients had developed CTLp hyporeactivity as evidenced by reduced donor-specific CTLp frequencies. The development of hyporeactivity was not specific for any particular underlying disease. The occurrence of HTL hyporeactivity, however, was less frequent: 38% and 20% of recipients were HTLp and MLC hyporeactive, respectively. Decreases in CTLp frequencies did not correlate with decreased donor-specific HTL function or suppressor cell activity in peripheral blood samples. Donor-specific CTLp hyporeactivity can develop in the majority of liver allograft recipients, irrespective of underlying disease. Donor-specific HTL hyporeactivity, however, occurs infrequently. A reduction in donor-specific CTLp frequencies was found to be independent of changes in donor-specific HTLp or suppressor cell activity, suggesting that other mechanisms (e.g., clonal deletion) are operative in the reduction of donor-specific CTLp after liver transplantation.

Contribution of virus-specific CD8+ cytotoxic T cells to virus clearance or pathologic manifestations of influenza virus infection in a T cell receptor transgenic mouse model.

The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8+ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8+ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8+ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8+ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8+ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.

Donor-specific CTL frequencies in peripheral blood in relation to graft vascular disease after clinical heart transplantation.

Cellular mechanisms may play a role in the development of graft vascular disease (GVD). We previously demonstrated that GVD correlated with an increase of donor-specific T-helper 1 cytokine production by graft-infiltrating lymphocytes but not by peripheral blood mononuclear cells (PBMC). These T-helper 1 cytokines aid the generation of cytotoxic T-lymphocytes (CTL). In the present report, we investigated whether there is a relationship between the frequency of donor-specific CTL precursors (pCTL) in PBMC and the development of GVD. We tested PBMC samples of five patients with GVD and five patients without GVD in the periods 3-6 months, 1 year, and 3 years after heart transplantation. At all time points, GVD was not related to the number of pCTL. In conclusion, donor-specific cellular tests in peripheral blood could not be related to GVD. Apparently, donor-specific reactions associated with the induction of GVD can only be monitored in the graft.

DNA immunization with minigenes: low frequency of memory cytotoxic T lymphocytes and inefficient antiviral protection are rectified by ubiquitination.

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.

Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia

AbstractPatients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus (ALVAC)

The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus (ALVAC) expressing HCV-capsid/E1/E2/NS2/NS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1/2663 and 1/27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV–CTL response and virological or histological status.

D6STNFa Microsatellite Locus Correlates with CTLp Frequency in Unrelated Bone Marrow Donor-Recipient Pairs

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings, primarily due to an increased rate of graft-versus-host-disease. HLA matching for donors and recipients is the most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA antigens without considering potential incompatibility for other MHC loci. Cellular assays have been developed to predict incompatibility that cannot be detected by current typing methods. The CTLp frequencies correlate with the degree of incompatibility of patient/donor and the clinical grade of GVHD. Since the CTLp assay is expensive and time consuming, an alternative is wanted. We studied the means of matching for microsatellites in determining MHC identity and possible correlation with CTLp frequencies. Therefore, 26 recipient/donor pairs were analysed for eleven microsatellite loci within and around the MHC region. Our study provides evidence that the D6STNFa locus correlates with CTLp frequency. The D6STNFa locus provides an additional marker that may help to improve the matching of unrelated donors and bone marrow recipients.

Tolerance induction by "megadose" hematopoietic transplants: donor-type human CD34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture.

Recently, the use of megadoses of CD34+ hematopoietic progenitors has been reported to abrogate resistance to engraftment, thus overcoming major histocompatibility barriers in bone marrow transplantation in leukemia patients. The ability of human CD34+ cells to possess potent tolerizing activity was studied by limiting dilution analysis of cytotoxic T lymphocyte (CTL) precursors (CTL-p) in human peripheral blood lymphocytes after addition of purified CD34+ cells. The addition of purified human CD34+ cells to primary mixed lymphocyte culture led to a marked reduction of antiallogeneic CTL-p frequency against stimulator cells of the same origin, compared with the response against cells of third-party origin. The CD34+ cells caused a marked inhibition of the CTL activity, when added at an equal number with the responder T cells, and they were still present after the mixed lymphocyte culture, which suggests that no significant killing of CD34+ cells had occurred. The tolerizing activity is abrogated by irradiation and requires cell contact. This pattern of tolerization most closely resembles what has been ascribed to veto cells in other systems. Phenotypic analysis of the purified CD34+ cells showed that they express MHC class I and class II antigens, but do not express costimulatory molecules of the B7 family. It is possible, that CD34+ cells in the megadose transplants-perhaps by their inability to provide costimulatory molecules-are actively reducing the frequency of CTL-p directed against their antigens, and thereby help to overcome allogeneic rejection, and enhance their own engraftment.

Mice Expressing the E7 Oncogene of HPV16 in Epithelium Show Central Tolerance, and Evidence of Peripheral Anergising Tolerance, to E7-Encoded Cytotoxic T-Lymphocyte Epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 Kbtransgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2b. F1 mice (denoted K14E7×A2.1) expressed E7 in the thymus at least as early as 2–5 days before birth. Immunisation of FVB×A2.1 control mice (transgenic for HLA A*0201 and H-2bbut not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2b-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7×A2.1 mice, the CTL responses to the H-2bepitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cytotoxicity assays, and by CTL precursor frequency analysis. In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7×A2.1 mice was replaced by a FVB×A2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with thesein vitrofindings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7×A2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin.

Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge.

The aim of this study was to evaluate the role of CTLs in the protection from challenge with pathogenic SHIV in macaques vaccinated with attenuated virus. More specifically, we have analyzed the CTL response in cynomolgus macaques vaccinated/infected with the attenuated SIVmacC8 or the wild-type SIVmacJ5 and correlated these responses to the protection from SHIV89.6P challenge. SIVmacC8-vaccinated monkeys demonstrated a broader CTL response than the SIVmacJ5-infected animals. Nevertheless, CTL against some proteins in SIVmacC8-vaccinated monkeys became progressively more difficult to detect through the day of challenge. In regards to protection from superinfection with SHIV89.6P, neither the presence of circulating CTL nor the CTL precursor frequency against any of the tested proteins correlated with the outcome of the challenge when SIVmacJ5- and SIVmacC8-infected animals were analyzed together. By analyzing the SIVmacC8-vaccinated animals separately, only the protected animal had detectable CTL precursors with moderate frequencies against all three tested proteins at the day of challenge.

Cyclophosphamide enhances the CTL precursor frequency in mice immunized with MUC1-mannan fusion protein (M-FP).

We have previously described the induction of murine CD8+ major histocompatibility complex class I restricted cytotoxic T cells to the 20 amino acid repeat region of human Mucin 1 (MUC1) variable number of tandem repeats region--a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. Mannan-MUC1 immunization protocol induces a high cytotoxic T lymphocyte (CTL) frequency, and some protection of mice against a tumor challenge. The CTL frequency can be substantially increased using cyclophosphamide (Cy), from 1/84,900 without Cy to 1/8,100 with Cy. Furthermore, in the presence of Cy, established tumors are rapidly eradicated, which does not happen in its absence. Cy clearly gives a major increase in the frequency of CTL precursors (CTLp) to MUC1 and could be of therapeutic value in patients.

Generation of leukemia-reactive cytotoxic T lymphocytes from HLA-identical donors of patients with chronic myeloid leukemia using modifications of a limiting dilution assay.

Donor leukocyte transfusions (DLT) have an anti-leukemic effect in most patients with a relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. However, DLT are often complicated by graft-versus-host disease. Selection of donor lymphocytes with a relative specificity for leukemic cells is desirable. The generation of leukemia-reactive cytotoxic T lymphocyte (CTL) responses between HLA-identical donors and patients in bulk cultures showed major variations, and false negative results were observed. In a modification of a limiting dilution analysis (LDA) two-fold serial dilutions of HLA-identical donor mononuclear cells (MNC) were cultured in the presence of CML cells. The anti-leukemic CTL precursor frequencies in these donors varied between <1 and 9 per 106 MNC. HLA-restricted CD4+ or CD8+ lymphocytes as well as MHC non-restricted gammadelta T cells were responsible for the anti-leukemic responses. A positive correlation between cytotoxicity in the various wells after 3, 4 and 5 weeks of culture could be found. The LDA may be superior to bulk cultures in selecting stable immune responses and in separating multiple different anti-leukemic T cell responses in each donor-patient combination.

DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice

The potential of building multi-cytotoxic T lymphocyte (CTL) epitope antigens in combination with the nucleic acid immunization technology is explored for development of acquired immunodeficiency syndrome (AIDS) and malaria vaccines. A novel minimal vector pTH for direct gene transfer was constructed for efficient expression of vaccine antigens and used as a vehicle for human immunodeficiency virus (HIV)- and Plasmodium falciparum-derived polyepitope genes. Two murine epitopes were included into these constructs to allow for testing of vaccine immunogenicity in small animals. The results showed that a single DNA injection generated CTL responses in all 15 vaccinated mice. The elicited CTL precursor frequencies were estimated in an interferon-γ (IFN-γ)-based ELISPOT assay and found to be an average of 300 (range 4–1346) peptide-responding cells per 10 6 splenocytes.

Parameters for using mannan-MUC1 fusion protein to induce cellular immunity.

We have previously reported preclinical studies in mice of the human mucin 1 (MUC1) antigen covalently linked to the yeast cell-wall mannan polysaccharide (MFP), and shown strong cellular responses of the T1 type using mice. We now describe the optimum parameters for administration of MFP to obtain cellular immunity [as measured by the cytotoxic T cell precursor (CTLp) frequency]. In dose/response studies, in which 1 microg-150 microg was given by the i.p. route, it was clear that doses of 1-7 microg led to cellular and not humoral immunity; at doses above 7 microg humoral immunity prevailed with little cellular immunity increasing doses giving greater amounts of antibody. The most favoured routes of administration were intraperitoneal or intradermal immunisation, which were substantially better than i.m., i.v.; s.c. administration was the worst. Three immunisations were necessary for a maximum cellular response, further immunisation decreasing the CTLp frequency. Six different adjuvants were used with MFP [complete and incomplete Freund's adjuvant (CFA, IFA) Alum, Adjuprime, muramyl dipeptide (MDP) and glutaminyl-muramyl dipeptide (GMDP)]; Alum, GMDP, MDP and IFA moderately increased the CTLp frequency, IFA being the best. Even though preclinical studies of the immunogen in mice may not necessarily mirror the behaviour of the immunogen in humans, these studies demonstrate the factors to be taken into account for phase I/II clinical trials.

Recruitment, activation and proliferation of CD8+ memory T cells in an immunoprivileged site.

The capacity of a memory cytotoxic T lymphocyte (CTL) population to protect against viral infections is well established, but the processes underlying this protection are less well understood. We have used heterotypic intranasal immunization with influenza A/X31 (H3N2) to protect against a subsequent infection with the neurovirulent influenza A/WSN (H1N1) in either the cerebrospinal fluid or the immunoprivileged brain parenchyma. Viral clearance from both sites was associated with a local infiltration and proliferation of A/WSN-specific CD8+ T cells. Infection in the cerebrospinal fluid elicited a proliferative response in the draining lymph nodes, an anti-H1N1 serum antibody response and an increase in the extracerebral A/WSN-specific CTL precursor frequency. In contrast, infection in the brain parenchyma elicited no lymph node proliferative response or serum antibody response and caused a transient decrease in the extracerebral CTL precursor frequency. Thus the memory CTL population protected against an intracerebral viral infection independent of any immune response occurring in systemic lymphoid tissue.