Cytolytic T-cell Response Research Articles

HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not processed and presented

Human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies and a high percentage of cervical carcinoma cells express HPV-16 E6 and E7 oncoproteins. These proteins are attractive targets for cytolytic T lymphocyte (CTL) mediated immunotherapy. We screened peptides derived from the HPV-16 E7 protein for binding to HLA-A2 and tested their potential to induce specific CTL responses in chimeric HLA-A2/H2-Kb transgenic mice. From eight potential binding peptides four displayed binding and were tested for immunogenicity. CTL activity was tested using target cells pulsed with peptide or expressing E7 protein. While there was no CTL induction observed with the peptides 7–15, 66–74 and 82–90, CTL from mice immunized with 86–93 lysed targets presenting the peptide in the context of the HLA-A2/H2-Kb molecule or wild-type HLA-A2. In contrast, 86–93 induced CTL showed no cytolytic activity against cells expressing the protein E7 and vaccination with the E7 protein did not lead to cytotoxicity against targets pulsed with the 86–93 peptide. Therefore the peptide 86–93, which binds to HLA-A2, is able to induce CTL responses in context of HLA-A2, but the peptide appears not to be processed or presented by HPV type 16 infected cells.

Immune monitoring in cancer immunotherapy.

The one-century-old idea of turning the immense resources of the immune system against cancer cells as a tool in the treatment of cancer patients has been reinvigorated with the identification of the molecular targets recognized by tumor-specific cytolytic T lymphocytes (CTLs). Indeed, over 40 different antigenic peptides, derived from mostly non-mutated and normally expressed proteins, have been defined that mimic an equal number of CTL-defined tumor antigens. The possibility to chemically synthesize large quantities of well-defined antigenic peptides together with significant progress made in the understanding of in vivo induction of specific CTL responses, have paved the way to new approaches for the design of therapeutic cancer vaccines.

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4 + and CD8 + T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.

Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells.

C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.

Mimotopes of cytolytic T lymphocytes in cancer immunotherapy.

Rapid identification of the cytolytic T lymphocyte (CTL) epitopes that are presented by cancer cells will provide the basis for manipulation of tumor immunity but remains a major challenge in the field. By screening randomly synthesized peptide libraries, mimotopes for tumor-reactive CTLs can be readily constructed and they may be functionally equal to or potentially more potent than natural peptides in stimulating CTL responses; therefore, the peptide library approach provides great promise for design of cancer vaccines and adoptive tumor immunotherapy.

Use of parvovirus-like particles for vaccination and induction of multiple immune responses.

Expression of the VP2 gene of autonomous parvoviruses in insect cells with the use of the baculovirus system has led to the production of virus-like particles (VLPs) formed by the self-assembly of VP2. These VLPs are expressed at high levels and can easily be purified by salt fractionation. They are highly immunogenic in the corresponding host, being fully protective at doses as low as 1-2 microg of purified material per animal. No special adjuvants are required. An interesting property of these particles is their usefulness as a diagnostic reagent for ELISA kits, which have successfully replaced conventional methods for parvovirus diagnostics based on haemagglutination. Another application of the hybrid recombinant parvovirus-like particles of pig parvovirus (PPV) and canine parvovirus (CPV) is its use as an antigen delivery system. PPV:VLPs containing a CD8(+) epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein are able to evoke a potent cytolytic T-lymphocyte response and to protect mice against a lethal infection with LCMV. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a T helper response in mice. These T-cell epitopes were inserted into the N-terminus of the VP2 protein. Unfortunately, the N-terminus is not adequate for antibody responses because it is inside the particle. Recent findings have shown that fine tailoring of the point of insertion around the tip of loop 2 of the surface of CPV allowed the elicitation of a potent antibody response. Thus mice immunized with chimaeric C3:B CPV:VLPs were able to elicit a strong neutralizing antibody response (>3 log10 units) against poliovirus. We now have the possibility of using these particles to elicit different immune responses against single or multiple pathogens in a simple and economic way.

Limitations of adenovirus-mediated interleukin-2 gene therapy for oral cancer.

Adenoviral interleukin-2 (AdV-IL-2) gene therapy has previously not proven effective in treating established murine oral cancer. We hypothesize that the intratumoral level of IL-2 expression is a major limiting factor in treatment outcome. A microscopic disease and established oral cancer murine model was used to test this hypothesis. IL-2 gene transfer was performed with a recombinant adenovirus vector. Tumor cells were transduced in vitro with AdV-IL-2 and subsequently implanted into the floor of the mouth in C3H/HeJ mice. IL-2 expression in vitro ranged from 990 to 1,050 pg/10(6) tumor cells. This microscopic disease treatment resulted in either complete tumor regression or a dramatic decrease in tumor progression. Cytolytic T-cell (CTL) assays demonstrated a predominance of CD8-specific, T-cell-mediated tumor killing. Reducing IL-2 expression by half with a mixture of 1:1 transduced to nontransduced tumor cells eliminated the antitumor effect and decreased the CTL response. These findings support the presence of a critical "threshold" of IL-2 expression. Adenovirus repurification and amplification allowed isolation of a twofold-higher-titer AdV-IL-2 vector. Treatment of established tumors with the higher-titer AdV-IL-2 at a new maximal dose of 1.4 x 10(9) plaque-forming units (pfu) increased in vivo IL-2 expression to 1,127 pg/10(6) cells and generated a significant antitumor response. Complete regression of established tumors, however, could not be achieved, and we noted a decrease in IL-2 expression well below the threshold at 1 week after treatment. Upon repeat maximal AdV-IL-2 injection in vivo, a greater antitumor effect and increased CTL response was seen, but also, 28% of the animals died of IL-2 toxicity. Although limited by expression and toxicity as a single-treatment strategy for established tumors, AdV-IL-2 gene therapy should be considered a potential component of combination therapy strategies.

Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway.

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.

Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by geneMAGE-3 and presented by HLA-A1

Thirty-nine tumor-bearing patients with metastatic melanoma were treated with 3 subcutaneous injections of the MAGE-3.A1 peptide at monthly intervals. No significant toxicity was observed. Of the 25 patients who received the complete treatment, 7 displayed significant tumor regressions. All but one of these regressions involved cutaneous metastases. Three regressions were complete and 2 of these led to a disease-free state, which persisted for more than 2 years after the beginning of treatment. No evidence for a cytolytic T lymphocyte (CTL) response was found in the blood of the 4 patients who were analyzed, including 2 who displayed complete tumor regression. Our results suggest that injection of the MAGE-3.A1 peptide induced tumor regression in a significant number of the patients, even though no massive CTL response was produced.

Antitumor Immunity at Work in a Melanoma Patient

This review covers the results obtained so far with a chronological analysis of the antitumor cytolytic T lymphocyte (CTL) cell response of a melanoma patient who enjoys an unusually favorable evolution. Two melanoma cell lines, MEL.A and MEL.B, were derived from metastases removed from the patient in 1988 and 1993, respectively. The patient developed a very strong CTL response against the MEL.A cells. Several antigens on these cells, presented by various HLA class I molecules, result from point mutations present in the genome of the tumor. The MEL.B cells, on the other hand, resist lysis by these CTLs because they have lost expression of most HLA class I molecules, suggesting that they were selected in vivo by the anti-MEL.A CTLs. New CTLs recognize MEL.B cells specifically, however. Analysis of their specificity indicates that they carry inhibitory receptors similar to those present on natural killer (NK) cells. These results illustrate the relationship between a tumor and the immune system in vivo over a period of several years. They are discussed in the context of the recent identification of many human tumor antigens recognized by CTLs, and the perspectives of specific immunotherapy opened up by these discoveries.

Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes.

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Oral Immunization with Enterocoated Microbeads Induces Antigen-Specific Cytolytic T-Cell Responses

A cytolytic T-lymphocyte (CTL) response has been generated to a protein antigen via oral administration in an experimental murine system. This was achieved by the formulation of the test antigen ovalbumin (OVA) in a pH-sensitive microsphere particle (microbead) with an acrylic-based coating to avoid its degradation by gastric enzymes. Comparative studies of orally administered enterocoated microbead-OVA particles, versus the more traditional formulation of OVA in adjuvant (DETOX-PC) given subcutaneously as immunogen, were conducted; both elicited comparable responses in the generation of antigen-specific lymphoproliferative and CTL responses. CTL lines generated via oral administration of antigen were shown to be CD3+, CD4−, and CD8+. CTL lysis of OVA peptide-pulsed targets was shown to be inhibited by anti-CD8 antibody. Whereas oral administration of pH-sensitive enterocoated microbeads containing proteins has previously been shown to elicit antibody and lymphoproliferative T-cell responses to protein antigens, this is the first demonstration of the generation of an antigen-specific cytolytic T-cell response via oral administration of a protein immunogen formulated in such microbeads.

Control of murine cytomegalovirus in the lungs: relative but not absolute immunodominance of the immediate-early 1 nonapeptide during the antiviral cytolytic T-lymphocyte response in pulmonary infiltrates.

The lungs are a major organ site of cytomegalovirus (CMV) infection, pathogenesis, and latency. Interstitial CMV pneumonia represents a critical manifestation of CMV disease, in particular in recipients of bone marrow transplantation (BMT). We have employed a murine model for studying the immune response to CMV in the lungs in the specific scenario of immune reconstitution after syngeneic BMT. Control of pulmonary infection was associated with a vigorous infiltration of the lungs, which was characterized by a preferential recruitment and massive expansion of the CD8 subset of alpha/beta T cells. The infiltrate provided a microenvironment in which the CD8 T cells differentiated into mature effector cells, that is, into functionally active cytolytic T lymphocytes (CTL). This gave us the opportunity for an ex vivo testing of the antigen specificities of CTL present at a relevant organ site of viral pathogenesis. The contribution of the previously identified immediate-early 1 (IE1) nonapeptide of murine CMV was evaluated by comparison with the CD3epsilon-redirected cytolytic activity used as a measure of the overall CTL response in the lungs. The IE1 peptide was detected by pulmonary CTL, but it accounted for a minor part of the response. Interestingly, no additional viral or virus-induced antigenic peptides were detectable among naturally processed peptides derived from infected lungs, even though infected fibroblasts were recognized in a major histocompatibility complex-restricted manner. We conclude that the antiviral pulmonary immune response is a collaborative function that involves many antigenic peptides, among which the IE1 peptide is immunodominant in a relative sense.

Estimation of the frequencies of anti-MAGE-3 cytolytic T-lymphocyte precursors in blood from individuals without cancer.

Attempts to detect a cytolytic T-lymphocyte (CTL) response in melanoma patients vaccinated with MAGE-3 peptides have been negative so far, even though some tumor regressions have been observed. The detection of such responses may require very sensitive detection assays for CTL precursors. To this end, we set up a method whereby a large number of CD8+ T-cell microcultures are stimulated with autologous antigen-presenting cells incubated with a peptide, in the presence of interleukin (IL)-6 and IL-12 during the first week, and IL-2 and IL-7 from the second week. We report here that not only monocyte-derived dendritic cells but also activated T cells incubated with the MAGE-3 antigenic peptide presented by HLA-A2 were effective in activating specific CTL precursors present in the blood of individuals without cancer. These precursors were detected in the CD8+ CD45RA+ subpopulation of T cells. Among the CD8+ T-lymphocyte population of blood donors, the frequency of CTL precursors specific for the MAGE-3.A2 antigen ranged from 4 to 17 x 10(-7). For the MAGE-3 antigenic peptide presented by HLA-A1, this frequency ranged from 0.4 to 3 x 10(-7). Knowing that several parameters of this procedure still have to be optimized, we will begin to use it to evaluate the CTL precursor frequencies of cancer patients before and after injection of MAGE peptides.

Characterization of the cytolytic T-lymphocyte response to a candidate vaccine strain of equine herpesvirus 1 in CBA mice.

The cytolytic T-lymphocyte (CTL) response to respiratory infection with equine herpesvirus 1 (EHV-1) in CBA (H-2(k)) mice was investigated. Intranasal (i.n.) inoculation of mice with the attenuated EHV-1 strain KyA resulted in the generation of a primary virus-specific CTL response in the draining mediastinal lymph nodes 5 days following infection. EHV-1-specific CTL could be restimulated from the spleen up to 26 weeks after the resolution of infection, indicating that a long-lived memory CTL population was generated. Depletion of CD8+ T cells by treatment with antibody and complement prior to assay eliminated CTL activity from both primary and memory populations, indicating that cytolytic activity in this model was mediated by class I major histocompatibility complex-restricted, CD8+ T cells. A single i.n. inoculation with KyA induced protective immunity against infection with the pathogenic EHV-1 strain, RacL11. The adoptive transfer of splenocytes from KyA-immune donors into sublethally irradiated recipients resulted in a greater than 250-fold reduction in RacL11 in the lung. The elimination of both CD4+ and CD8+ T cells from the transferred cells abrogated clearance of RacL11, while the selective depletion of either subpopulation alone had little effect. These results suggested that both lymphocyte subpopulations contribute to viral clearance, with either subpopulation alone being sufficient.

Stress-induced neuroendocrine modulation of viral pathogenesis and immunity.

Physical restraint (RST) was used to examine the interactions among the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic nervous system, and the immune response to infection. In these studies, mice were infected with either herpes simplex virus (HSV) or influenza A/PR8 virus so that the impact of neuroendocrine activation could be assessed on disease pathophysiology and anti-viral immunity. RST suppressed lymphadenopathy in draining lymph nodes, reduced mononuclear cellular infiltration in the lungs, and suppressed virus-specific cytokine and cytolytic T-cell responses. Blockade of type II glucocorticoid receptors (by RU486) restored cellularity and cytokine responses to both organs in restraint-stressed, infected mice. Thus, the HPA axis modulated cell trafficking and T-cell cytokine responses. However, RU486 treatment failed to restore cytolytic T-cell responses. Blockade of beta-adrenergic receptors (by nadolol), in combination with RU486 treatment, fully restored cytolytic T-cell responses, suggesting that catecholamines were involved in suppressing the virus-specific CD8+ cytolytic T-cell response. RST also modulated the local development or expression of antibody-secreting cells (ASC) in the lungs draining lymph nodes, and spleen following infection of restrained mice. RST significantly suppressed the number of virus-specific ASC (IgM, IgG and subclasses IgG1 and IgG2a) in the lungs, mediastinal (MLN) lymph nodes and spleen, while it enhanced the responses in the superficial cervical (SCV) lymph nodes. This observation of differential modulation of ASC responses in the MLN and SCV lymph nodes supports the concept of tissue-specific immunoregulation in response to stress.

Assessment and characterization of the cytolytic T lymphocyte response against Epstein-Barr virus in patients with non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation.

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.

B lymphocytes secreting IgG linked to latent transforming growth factor-beta prevent primary cytolytic T lymphocyte responses.

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency-associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG-TGF-beta is taken up through Fc receptors for IgG and that active TGF-beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.

The Distinctive Features of Influenza Virus Infection of Dendritic Cells

CD8 + cytolytic T lymphocytes (CTLs) are considered to be critical mediators for resistance to influenza virus infection. We have previously demonstrated that dendritic cells are potent antigen presenting cells in the development of anti-influenza CTLs. Here we identify distinctive features of the interaction of influenza virus with dendritic cells. Exposure of dendritic cells to influenza virus at MOIs of 2–4:1 leads to >90% infection, as manifested by the expression of the viral proteins HA and NS1. The infection is non-toxic as viral protein expression is sustained for >2 days with retention of viablility, but little infectious virus is produced. Substantial induction of the anti-viral cytokine IFN-α also occurs. Influenza infection of macrophages also results in viral protein expression in a majority of cells, and synthesis of IFN-α. In contrast to dendritic cells, macrophages display evidence of apoptosis within 10–12 hours, and the majority of cells die within 24–36 hours. During this interval macrophages synthesize >10-fold higher levels of virus than dendritic cells. Infected dendritic cells but not macrophages, can induce substantial CTL responses from purified blood CD8 + T cells in the absence of exogenous cytokines such as IL-2. Low levels of infection (MOIs of 0.02) are sufficient to generate potent CTL responses. Influenza virus expressing non-cleaved HA does not elicit CTLs indicating that virus must access the cytoplasm of dendritic cells to utilize traditional class I processing pathways. These observations indicate that DCs are distinct in their handling of influenza virus and for the induction of anti-viral immunity.

Immunodominant minor histocompatibility antigen peptides recognized by cytolytic T lymphocytes primed by indirect presentation.

Indirect presentation of minor histocompatibility antigens (HA) as revealed by cross-priming of H2 heterozygous recipients effectively primes minor HA-specific cytolytic T lymphocytes (CTLs). However, it is not known if indirect priming generates CTLs specific for the same set of immunodominant minor HA recognized by CTLs primed by direct spleen cell injections. In order to indirectly prime minor HA-specific CTLs, we implanted (C57BL/6 x B6.C-H2d)F1 recipients with BALB.B and BALB/c splenocytes loaded into immunoisolation devices that effectively preclude direct donor:host contact. Responder spleen cells from these recipients were stimulated in vitro to expand BALB.B- and BALB/c-specific CTLs to reveal classical cross-priming. Tests of CTL specificity using (1) CXB recombinant inbred strain targets that express different arrays of BALB/c minor HA and (2) high-performance liquid chromatography fractions of peptides from BALB.B Kb and Db molecules revealed that anti-BALB.B CTLs were specific for two previously identified dominant peptides, CTT-2 and CTT-5, presented by Kb molecules. Variation of responders and priming cells resulted in CTL responses to additional dominant peptides that had been identified previously with CTLs generated by direct priming with spleen cell injections. Indirect priming was not limited to this set of peptides recognized by CTLs in vitro because devices loaded with cells devoid of the CTL-detected peptides primed for accelerated skin allograft rejection. Indirect presentation of minor HA in vivo stimulates the generation of CTLs specific for a subset of dominant minor HA peptides recognized by CTLs primed by direct presentation.