The murine buccal mucosa is an inductive site for priming class I-restricted CD8+ effector T cells in vivo

An isometer, a highly compliant spring-scale device for measuring suture displacement, has been used intraoperatively by surgeons to select the optimal placement of the femoral tunnel for an anterior cruciate ligament graft. The isometer measures the displacement of a suture centered in a tibial tunnel and attached to an intraarticular location on the femur before the femoral tunnel is drilled. Because the placement of the femoral tunnel strongly impacts the tensile behavior of an anterior cruciate ligament graft and because surgeons have used the amount of suture displacement to guide the placement of the femoral tunnel, the objective of this study was to determine the ability of an isometer to predict graft tension. In 14 patients undergoing reconstructive surgery of the anterior cruciate ligament, an isometer was used to measure suture displacement during passive knee motion for a provisional femoral tunnel location. An electrogoniometer recorded the flexion angle of the knee. The femoral tunnel was drilled. A double-looped semitendinosus and gracilis autograft was inserted around a post in the femoral tunnel, and the tension in the four limbs of the graft exiting the tibial tunnel was measured during passive knee motion. Graft-tension versus knee-flexion-angle curves revealed that each knee exhibited one of two distinct curve shapes: L-shaped, characterized by the maximum tension occurring at full extension and a nearly flat profile from 35 to 90 degrees of flexion, or U-shaped, with elevated tensions at 80-90 degrees of flexion (p < 0.001) reaching at least half of the tension in full extension. Because the shapes of the suture-displacement versus flexion-angle curves were more consistently L-shaped, the intraoperative measurement of suture displacement was not a useful predictor of either the increase in tension in the graft with flexion or the maximum tension in the graft.     

Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes

It has previously been shown that bone marrow-generated dendritic cells (DC) are potent stimulators in allogeneic mixed leukocyte reactions and are capable of activating naive CD4+ T cells in situ in an antigen-specific manner. In this study we have investigated whether bone marrow-generated DC are capable of inducing antigen-specific CD8+ T cell responses in vivo. Initial attempts to induce specific cytotoxic T lymphocyte (CTL) responses in mice injected with bone marrow-generated DC pulsed with ovalbumin (OVA) peptide were frustrated by the presence of high levels of nonspecific lytic activity, which obscured, though not completely, the presence of Ag-specific CTL. Using conditions that effectively differentiate between antigen-specific and nonspecific lytic activity, we have shown that bone marrow-generated DC pulsed with OVA peptide are potent inducers of OVA-specific CTL responses in vivo, compared with splenocytes or RMA-S cells pulsed with OVA peptide, or compared with immunization with free OVA peptide mixed with adjuvant. Antibody-mediated depletion experiments have shown that the cytotoxic effector cells consist primarily of CD8+ cells, and that induction of CTL in vivo is dependent on CD4+ as well as on CD8+ T cells. These results provide the basis for exploring the role of bone marrow-generated DC in major histocompatibility class I-restricted immune responses, and they provide the rationale for using bone marrow-generated DC in CTL-mediated immunotherapy of cancer and infectious diseases.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28- T cells

Specific suppression of the host's immune response to donor HLA antigens remains the ultimate goal for clinical transplantation. In spite of considerable effort, however, allospecific human suppressor T cells (Ts) have been difficult to generate. Here we show that allospecific and xenospecific Ts can be raised by multiple priming of human T cells in mixed lymphocyte cultures. Ts derive from the CD8+CD28- subset and recognize specifically the MHC class I antigens expressed by antigen-presenting cells (APC) used for in vitro immunization. Allospecific Ts prevent the up-regulation of B7 molecules on target APC, interfering with the CD28-B7 interaction required for T helper (Th) activation. These findings provide a basis for the development of specific immunosuppressive therapy.     

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.     

Anthrax toxin-mediated delivery in vivo and in vitro of a cytotoxic T-lymphocyte epitope from ovalbumin

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264). These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.     

Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas

A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen-specific stimulation of class I-restricted T cell hybridomas, as assessed by IL-2 release, does not depend on CD8.     

In vivo treatment with a MHC class I-restricted blocking peptide can prevent virus-induced autoimmune diabetes

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-gamma secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-gamma and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.     

Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4+ T cells

Epithelial cell mucin MUC1 is expressed on adenocarcinomas in an underglycosylated form that serves as a tumor antigen in breast, pancreatic, ovarian, and other tumors. Two predominant MUC1-specific immune responses are found in patients: CD8+ CTLs, which recognize tandemly repeated epitopes on the MUC1 protein core, and IgM antibodies. There have been no reports to date of MUC1-specific CD4+ T-helper cells in cancer patients. We show here that MUC1-specific CD4+ T cells are neither deleted nor tolerized and that CD4+ T cell responses can be generated when an appropriate soluble form of MUC1 is used. Naive CD4+ T cells from healthy donors were primed in vitro to a synthetic MUC1 peptide of 100 amino acids, representing five unglycosylated tandem repeats, presented by dendritic cells. They produced IFN-gamma and had moderate cytolytic activity. We identified one core peptide sequence, PGSTAPPAHGVT, that elicits this response when it is presented by HLA-DR3.     

IL-10 inhibits alloreactive cytotoxic T lymphocyte generation in vivo

In this report, we present evidence that the CTL response directed against MHC Class I allo-determinants can be inhibited as a result of IL-10 expression in vivo. The presence of localized IL-10 secretion at the site of allogeneic tumor cell challenge resulted in marked inhibition of the CTL response and allowed growth of the tumor in the allogeneic host. Using purified CD4+ T cells from mice immunized in the presence or absence of IL-10, we have shown that the loss of alloreactivity as a consequence of IL-10 expression results from the inhibition of CD4+ T cell function. The expression of either IL-2 or IFN-gamma with IL-10 locally at the time of allogeneic cell challenge completely restored CTL alloreactivity, suggesting that the action of IL-10 could be bypassed by providing helper T lymphocyte-derived cytokines of the Th1 type at the site of immunization. Inhibition of alloreactivity by IL-10 was observed using either purified macrophages or dendritic cells as APC in an in vitro assay. Thus, the expression of IL-10 following antigenic challenge (such as that observed in Th2-like immune responses) may profoundly limit the ability for generating functional CTL in vivo.     

Extensive cross-reactivity of adenovirus-specific cytotoxic T cells

Although adenovirus is a major source of morbidity for immunocompromised individuals and a popular vector for gene therapy, little is known about the cellular immune responses it evokes in humans. Initial trials using adenovirus vectors have been disappointing, probably owing both to a preexisting immune response to Ad2 and Ad5, the most commonly used vector backbones, and to a response to the transgene. The former problem might be overcome by switching from the common type C adenoviruses, of which Ad2 and Ad5 are members, to other less common serotypes. Evidence for the feasibility of this approach has been provided by a rat model system. However, its success in humans depends on there being no immunological cross-reactivity between groups at the humoral or cellular level. Here, we examine the cross-reactivity of the cellular immune response to adenovirus in a human system, and find that human cytotoxic T lymphocytes (CTLs) prepared in vitro against an adenovirus from two of the six subgroups can lyse cells infected with adenoviruses from the other subgroups. Hence, the proposed use of adenovirus vectors from uncommon subgroups to evade memory immune response to subgroup C adenoviruses may not be successful. However, this same cross-reactivity indicates that adoptive transfer of CTLs generated in vitro against one adenovirus serotype may protect immunocompromised patients from infections by adenoviruses of all serotypes.     

MHC class I-restricted lysis of human oligodendrocytes by myelin basic protein peptide-specific CD8 T lymphocytes

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110-118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.     

Retroviral-mediated expression of an MHC class I-restricted T cell receptor in the CD8 T cell compartment of bone marrow-reconstituted mice

The introduction of cloned T cell receptor (TCR) genes into bone marrow cells could provide a way to increase the frequency of tumor- or pathogen-specific cytotoxic T lymphocyte (CTL) precursors. We demonstrate here the ability of a retroviral vector to direct expression of a Valpha15/Vbeta13 MHC class I-restricted TCR in lethally irradiated mice reconstituted with transduced bone marrow cells. We have detected retroviral-mediated TCR expression by flow cytometry 6-19 weeks after transplantation in C57L (Vbeta13(-/-)) and Rag1(-/-) bone marrow-reconstituted mice, and in C57BL/6 hosts reconstituted with transduced C57BL/6-Rag1(-/-) bone marrow. Southern analysis confirmed the presence of integrated provirus and revealed that the frequency of transduction is greater than the frequency of cell surface TCR expression. Although TCR expression on Vbeta13+ transduced cells is lower than endogenous TCR levels, it is largely confined to CD4+CD8+ (thymus) and CD8+ (thymus and spleen) T cells. In Rag1(-/-) mice, which display a developmental arrest of thymocytes at the immature CD4-CD8- stage, retrovirus-mediated TCR expression selectively rescues CD4+CD8+ and CD8+ populations. These results indicate that the ectopically expressed TCR is functional during T cell development. Furthermore, we have observed Vbeta13+ TCR expression by up to 13% of peripheral CD8+ T cells in C57L and C57BL/6 hosts. This represents a substantial increase relative to total Vbeta13 frequency in normal C57BL/6 mice (3-5%), and an even greater increase over the estimated frequency of CTL precursors of a defined specificity (10(-5)-10(-4)). Our findings indicate that TCR gene transfer can be used to develop new approaches to immunotherapy, and provide the basis for further studies examining the contribution of retrovirus-mediated TCR expression to an antigen-specific CTL response.     

In vivo detection of fluorescent tumor-specific cytotoxic T cell clones

A procedure for detecting mumps virus in under 48 h was developed using the PCR. The sensitivity of the PCR amplification reaction and of the detection of the PCR product was significantly improved by: (i) enriching for viral template RNAs by overnight culture of the virus in Vero cells and (ii) substitution of polyacrylamide gel analysis for agarose gel electrophoresis. The technique was capable of detecting 1-20 infectious units of virus or an equivalent of 1-10 pg of mumps virus-specific plasmid DNA.     

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

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

Immune CD8+ T lymphocytes lyse Listeria monocytogenes-infected hepatocytes by a classical MHC class I-restricted mechanism

Hepatocytes constitute the principal site of listerial replication in the livers of mice infected i.v. CD8+ T lymphocytes play a predominant role in the host defenses to Listeria monocytogenes. In vitro experiments by others undertaken to delineate the functions of CD8+ T lymphocytes have focused primarily on their interaction with Listeria-infected macrophages. Such experiments do not address directly the role of CD8+ T lymphocytes in eliminating the bulk of Listeria replicating within the liver. Here, we report that immune CD8+ T cells at an E:T cell ratio > or = 10:1 lysed Listeria-infected hepatocytes as judged by the following two criteria. Aspartate aminotransferase activity in the culture supernatants, indicative of hepatocyte damage, increased significantly. Conversely, infected hepatocytes cocultured with immune CD8+ T cells exhibited a marked reduction in viable intracellular Listeria assessed by CFUs. Neither immune CD4+ T cells nor nonimmune CD8+ T cells caused a similar increase in aspartate aminotransferase activity released or a decrease in intracellular bacteria. Immune CD8+ T cell-mediated lysis of infected hepatocytes was restricted by classical MHC class I (H-2Kb) molecules and was inhibited by the presence of either brefeldin A or mAb specific for CD8. These results suggest that the predominant role of CD8+ T lymphocytes in host resistance to listerial infections of the liver may be due to their capacity to lyse infected hepatocytes.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE2, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO- CD31+ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE2 primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and TNF-beta. PGE2 does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-gamma. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE2, prevents the development of IL-4-producing cells but does not abolish the effects of PGE2 on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE2 on naive T cell maturation. Thus PGE2 does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE2 suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells

Intracoronary thrombosis plays a key role in the pathogenesis of acute myocardial infarction (AMI), and the formation of an occlusive thrombus usually precedes the development of myocardial damage. Therefore we evaluated and compared the early sensitivities of thrombin-antithrombin III complex (TAT), D-dimer, myoglobin, creatine kinase (CK) MB mass concentration, and cardiac troponin T (cTnT) on admission to a coronary care unit (CCU) before heparin or thrombolytic therapy was started. We investigated 31 consecutive patients admitted to CCU for evolving AMI within 6 hours from the onset of infarct-related symptoms; the median delay from chest pain onset to CCU admission was 135 minutes. Of all biochemical markers tested TAT had the highest early sensitivity on admission to the CCU, and TAT was significantly more sensitive than cTnT, CKMB mass, myoglobin, and D-dimer. However, TAT increases give no information about the location of clot formation in the body, and the diagnosis of AMI must be subsequently verified by an increase in more cardiac specific proteins, such as troponins or CKMB.