Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C > LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 > LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities

The hepatitis C virus (HCV)-specific CD8(+)-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8(+)-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8(+)-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8(+)-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.     

Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses

Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.     

Cytotoxic CD4+ T cell responses to EBV contrast with CD8 responses in breadth of lytic cycle antigen choice and in lytic cycle recognition

EBV, a B lymphotropic herpesvirus, encodes two immediate early (IE)-, >30 early (E)-, and >30 late (L)-phase proteins during its replication (lytic) cycle. Despite this, lytic Ag-induced CD8 responses are strongly skewed toward IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically infected cells. For comparison, we examined CD4(+) T cell responses to eight IE, E, or L proteins, screening 14 virus-immune donors to overlapping peptide pools in IFN-γ ELISPOT assays, and established CD4(+) T cell clones against 12 defined epitopes for target-recognition assays. We found that the lytic Ag-specific CD4(+) T cell response differs radically from its CD8 counterpart in that it is widely distributed across IE, E, and L Ag targets, often with multiple reactivities detectable per donor and with IE, E, or L epitope responses being numerically dominant, and that all CD4(+) T cell clones, whether IE, E, or L epitope-specific, show strong recognition of EBV-transformed B cell lines, despite the lines containing only a small fraction of lytically infected cells. Efficient recognition occurs because lytic Ags are released into the culture and are acquired and processed by neighboring latently infected cells. These findings suggested that lytic Ag-specific CD4 responses are driven by a different route of Ag display than drives CD8 responses and that such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering the lytic cycle.     

Analysis of CD4(+) T-cell responses to human papillomavirus (HPV) type 11 L1 in healthy adults reveals a high degree of responsiveness and cross-reactivity with other HPV types

Human papillomavirus type 11 (HPV-11) infection causes genital warts and recurrent respiratory papillomatosis. While there is compelling evidence that CD4(+) T cells play an important role in immune surveillance of HPV-associated diseases, little is known about human CD4(+) T-cell recognition of HPV-11. We have investigated the CD4(+) T-cell responses of 25 unrelated healthy donors to HPV-11 L1 virus-like particles (VLP). CD4(+) T-cell lines from 21 of 25 donors were established. Cell sorting experiments carried out on cells from six donors demonstrated that the response was located in the CD45RA(low) CD45RO(high) memory T-cell population. To determine the peptide specificity of these responses, epitope selection was analyzed by using 95 15-mer peptides spanning the entire HPV-11 L1 protein. No single region of L1 was immunodominant; responders recognized between 1 and 10 peptides, located throughout the protein, and peptide responses fell into clear HLA class II restricted patterns. Panels of L1 peptides specific for skin and genital HPV were used to show that the L1 CD4(+) T-cell responses were cross-reactive. The degree of cross-reactivity was inversely related to the degree of L1 sequence diversity between these viruses. Finally, responses to HPV-11 L1 peptides were elicited from ex vivo CD45RO(+) peripheral blood mononuclear cells, demonstrating that recognition of HPV-11 was a specific memory response and not due to in vitro selection during tissue culture. This is the first study of CD4(+) T-cell responses to HPV-11 in healthy subjects and demonstrates marked cross-reactivity with other skin and genital HPV types. This cross-reactivity may be of significance for vaccine strategies against HPV-associated clinical diseases.     

Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+-T-cell epitopes recognized

We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4(+)-T-cell responses. To better characterize the CD8(+)-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8(+)-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8(+)-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8(+)-T-cell epitopes were induced at a higher number after a fourth injection (P < 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8(+)-T-cell epitope recognition after the boost.     

Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.     

Requirement for CD40 Ligand, CD4+ T Cells, and B Cells in an Infectious Mononucleosis-Like Syndrome

Respiratory challenge with the murine gammaherpesvirus 68 (gammaHV-68) results in productive infection of the lung, the establishment of latency in B lymphocytes and other cell types, transient splenomegaly, and prolonged clonal expansion of activated CD8(+) CD62L(lo) T cells, particularly a Vbeta4(+) CD8(+) population that is found in mice with different major histocompatibility complex (MHC) haplotypes. Aspects of the CD8(+)-T-cell response are substantially modified in mice that lack B cells, CD4(+) T cells, or the CD40 ligand (CD40L). The B-cell-deficient mice show no increase in Vbeta4(+) CD8(+) T cells. Similar abrogation of the Vbeta4(+) CD8(+) response is seen following antibody-mediated depletion of the CD4(+) subset, through the numbers of CD8(+) CD62L(lo) cells are still significantly elevated. Virus-specific CD4(+)-T-cell frequencies are minimal in the CD40L(-/-) mice, and the Vbeta4(+) CD8(+) population remains unexpanded. Apparently B-cell-CD4(+)-T-cell interactions play a part in the gammaHV-68 induction of both splenomegaly and non-MHC-restricted Vbeta4(+) CD8(+)-T-cell expansion.     

Beyond help: direct effector functions of human immunodeficiency virus type 1-specific CD4(+) T cells

The immune correlates of protection in human immunodeficiency virus type 1 (HIV-1) infection remain poorly defined, particularly the contribution of CD4(+) T cells. Here we explore the effector functions of HIV-1-specific CD4(+) T cells. We demonstrate HIV-1 p24-specific CD4(+)-T-cell cytolytic activity in peripheral blood mononuclear cells directly ex vivo and after enrichment by antigen-specific stimulation. We further show that in a rare long-term nonprogressor, both an HIV-1-specific CD4(+)-T-cell clone and CD4(+) T cells directly ex vivo exert potent suppression of HIV-1 replication. Suppression of viral replication was dependent on cell-cell contact between the effector CD4(+) T cells and the target cells. While the antiviral effector activity of CD8(+) T cells has been well documented, these results strongly suggest that HIV-1-specific CD4(+) T cells are capable of directly contributing to antiviral immunity.     

CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection

Although the CD4(+)- and CD8(+)-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8(+) cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8(+)-T-cell response.     

Distinct cycling CD4(+)- and CD8(+)-T-cell profiles during the asymptomatic phase of simian immunodeficiency virus SIVmac251 infection in rhesus macaques

Elevated CD4 T-cell turnover may lead to the exhaustion of the immune system during human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, this hypothesis remains controversial. Most studies of this subject have concerned the blood, and information about the lymph nodes is rare and controversial. We used Ki67 expression to measure cycling T cells in the blood and lymph nodes of uninfected macaques and of macaques infected with a pathogenic SIVmac251 strain or with a nonpathogenic SIVmac251Deltanef clone. During the asymptomatic phase of infection, the number of cycling CD8(+) T cells progressively increased (two- to eightfold) both in the blood and in the lymph nodes of macaques infected with SIVmac251. This increase was correlated with viral replication and the progression to AIDS. In contrast, no increases in the numbers of cycling CD4(+) T cells were found in the blood or lymph nodes of macaques infected with the pathogenic SIVmac251 strain in comparison with SIVmac251Deltanef-infected or healthy macaques during this chronic phase. However, the lymph nodes of pre-AIDS stage SIVmac251-infected macaques contained more cycling CD4(+) T cells (low baseline CD4(+)-T-cell counts in the blood). Taken together, these results show that the profiles of CD4(+)- and CD8(+)-T-cell dynamics are distinct both in the lymph nodes and blood and suggest that higher CD4(+)-T-cell proliferation at the onset of AIDS may lead to the exhaustion of the immune system.     

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.     

Cytolytic CD4(+)-T-cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell proliferation

In the absence of immune surveillance, Epstein-Barr virus (EBV)-infected B cells generate neoplasms in vivo and transformed cell lines in vitro. In an in vitro system which modeled the first steps of in vivo immune control over posttransplant lymphoproliferative disease and lymphomas, our investigators previously demonstrated that memory CD4(+) T cells reactive to EBV were necessary and sufficient to prevent proliferation of B cells newly infected by EBV (S. Nikiforow et al., J. Virol. 75:3740-3752, 2001). Here, we show that three CD4(+)-T-cell clones reactive to the latent EBV antigen EBNA1 also prevent the proliferation of newly infected B cells from major histocompatibility complex (MHC) class II-matched donors, a crucial first step in the transformation process. EBNA1-reactive T-cell clones recognized B cells as early as 4 days after EBV infection through an HLA-DR-restricted interaction. They secreted Th1-type and Th2-type cytokines and lysed EBV-transformed established lymphoblastoid cell lines via a Fas/Fas ligand-dependent mechanism. Once specifically activated, they also caused bystander regression and bystander killing of non-MHC-matched EBV-infected B cells. Since EBNA1 is recognized by CD4(+) T cells from nearly all EBV-seropositive individuals and evades detection by CD8(+) T cells, EBNA1-reactive CD4(+) T cells may control de novo expansion of B cells following EBV infection in vivo. Thus, EBNA1-reactive CD4(+)-T-cell clones may find use as adoptive immunotherapy against EBV-related lymphoproliferative disease and many other EBV-associated tumors.     

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.     

Hormonal regulation of CD4(+) T-cell responses in coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Ealpha mice. This gender difference in disease susceptibility correlates with selective induction of CD4(+) Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4(+) Th2 (interleukin-4 positive [IL-4(+)]) cell responses. Differences in immune deviation of CD4(+) T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69(+)) T cells expressing the gammadelta T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of gammadelta+ cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4(+) Th1 cell response and myocarditis by promoting increased gammadelta+ cell activation.     

Multiple epitopes in the murine cytomegalovirus early gene product M84 are efficiently presented in infected primary macrophages and contribute to strong CD8+-T-lymphocyte responses and protection following DNA immunization

We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8(+) T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8(+) T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8(+)-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8(+)-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8(+)-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8(+)-T-cell responses during MCMV infection.     

Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis

BACKGROUND: CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS: CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS: In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION: The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.