Altered CD8(+) T-cell responses when immunizing with multiepitope peptide vaccines

Efforts to develop effective cancer vaccines often use combinations of immunogenic peptides to increase the applicability and effectiveness of the immunizations. The immunologic consequences of combining more than 1 self/tumor antigen in a single vaccine emulsion remain unclear, however. We performed 2 sequential clinical trials in patients at high risk for melanoma recurrence. Patients were given the highly immunogenic gp100:209-217(210M) peptide and the less immunogenic tyrosinase:368-376(370D) peptide once every 3 weeks for 4 weeks. This vaccination course was 12 weeks long, and patients were vaccinated for up to 4 courses (16 total vaccinations). In the first trial in 31 patients, the peptides were emulsified separately in incomplete Freund adjuvant and injected at 2 different sites. In the second trial in 33 patients, the peptides were emulsified together and injected at the same site. Cryopreserved lymphocytes were obtained by apheresis after each course and were evaluated for antipeptide activity using tetramer, enzyme-linked immunospot, and in vitro sensitization boost assays. When the peptides were injected at separate sites, robust specific reactivity to the native gp100:209-217 peptide was measured by each of the assays, whereas immunization with the tyrosinase:368-376(370D) peptide was far less effective. When the peptides were emulsified and injected together at the same site, immunization to the gp100:209-217(210M) epitope dropped precipitously, whereas reactivity to the tyrosinase:368-376(370D) peptide was enhanced. These cautionary data indicate that mixing peptides in the same emulsion can alter reactivity compared with peptides injected separately by mechanisms that may include the induction of localized nonspecific inflammation or competitive binding of peptides to major histocompatibility complex molecules.     

Weak anti-HIV CD8(+) T-cell effector activity in HIV primary infection

HIV-specific CD8(+) T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-gamma enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8(+) T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0-6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1-13). The frequency of HIV-specific CD8(+) T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8(+)CD28(-) terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination.     

Distinct structural TCR repertoires in naturally occurring versus vaccine-induced CD8+ T-cell responses to the tumor-specific antigen NY-ESO-1

Spontaneous immune responses to the cancer testis antigen NY-ESO-1 are frequently found in cancer patients bearing antigen-expressing tumors. In HLA-A2-expressing patients, naturally elicited NY-ESO-1-specific, tumor-reactive cytotoxic T lymphocytes (CTLs) are mostly directed against an immunodominant epitope corresponding to peptide NY-ESO-1 157-165. NY-ESO-1-specific CTLs can also be induced by synthetic peptide vaccines, but they are heterogeneous in terms of functional avidity and tumor reactivity. The authors investigated the structural bases of this phenomenon by analyzing the TCR features of natural and vaccine-induced NY-ESO-1-specific CTLs. The results indicate that CTLs from the two groups exhibit highly structurally conserved but distinct TCR features, suggesting that the synthetic peptides used for vaccination may fail to faithfully mimic the naturally processed antigen. Together, the results of this study underline the strength of TCR molecular monitoring and will be instrumental for the development and monitoring of vaccines aimed at eliciting CTLs with high tumor reactivity.     

CD8(+) T-cell selection, function, and death in the primary immune response in vivo

The primary immune response to Epstein Barr virus (EBV) is characterized by striking proliferation of EBV-specific CD8(+) T cells. In this study we have investigated the clonal composition and functional properties of the cells mediating this primary response and have analyzed the mechanisms that control the downregulation of the primary response and the selection of memory cells. We show that massively expanded T-cell clones often dominate the primary antigen-specific T-cell response. Despite the enormous extent of expansion, the virus-specific T cells express high levels of intracellular perforin and are potently cytotoxic. They are, however, functionally heterogeneous in their ability to secrete proinflammatory cytokines, with subpopulations of the antigen-specific T cells being hyporesponsive. The primary response is closely regulated, and the majority of cells are programmed to die via a cytokine-rescuable pathway, leaving only small populations of memory T cells surviving. Comparison of the clonal composition of primary and memory responses in vivo shows that the clones that dominate the primary response are relatively heavily culled during the downregulation of the primary response and the establishment of T-cell memory.     

CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.     

Selective expansion of cross-reactive CD8(+) memory T cells by viral variants.

The role of memory T cells during the immune response against random antigenic variants has not been resolved. Here, we show by simultaneous staining with two tetrameric major histocompatibility complex (MHC)-peptide molecules, that the polyclonal CD8(+) T cell response against a series of natural variants of the influenza A nucleoprotein epitope is completely dominated by infrequent cross-reactive T cells that expand from an original memory population. Based on both biochemical and functional criteria, these cross-reactive cytotoxic T cells productively recognize both the parental and the mutant epitope in vitro and in vivo. These results provide direct evidence that the repertoire of antigen-specific T cells used during an infection critically depends on prior antigen encounters, and indicate that polyclonal memory T cell populations can provide protection against a range of antigenic variants.     

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.     

Aging leads to disturbed homeostasis of memory phenotype CD8(+) cells

Examining the rate of in vivo T cell turnover (proliferation) in aged mice revealed a marked reduction in turnover at the level of memory-phenotype CD44(hi) CD8(+) cells relative to young mice. Based on adoptive transfer experiments, the reduced turnover of aged CD44(hi) CD8(+) cells reflected an inhibitory influence of the aged host environment. Aged CD44(hi) CD8(+) cells also showed poor in vivo responses to IL-15 and IL-15-inducing agents, but responded well to IL-15 in vitro. Two mechanisms could account for the reduced turnover of aged CD44(hi) CD8(+) cells in vivo. First, aging was associated with a prominent and selective increase in Bcl-2 expression in CD44(hi) CD8(+) cells. Hence, the reduced turnover of aged CD44(hi) CD8(+) cells may in part reflect the antiproliferative effect of enhanced Bcl-2 expression. Second, the impaired in vivo response of aged CD44(hi) CD8(+) cells to IL-15 correlated with increased serum levels of type I interferons (IFN-I) and was largely reversed by injection of anti-IFN-I antibody. Hence the selective reduction in the turnover of aged CD44(hi) CD8(+) cells in vivo may reflect the combined inhibitory effects of enhanced Bcl-2 expression and high IFN-I levels.     

Potency of mature CD40L RNA electroporated dendritic cells correlates with IL-12 secretion by tracking multifunctional CD8(+)/CD28(+) cytotoxic T-cell responses in vitro

Electroporation of mature dendritic cells (DC) with RNA-encoding CD40L greatly enhances the production of interleukin (IL)-12, a proinflammatory cytokine necessary for the induction of T-cell immunity. Results presented herein reveal a correlation between the priming of CD28(+) antigen-reactive effector memory cytotoxic T lymphocytes (CTL) displaying 3 or 4 simultaneous effector functions and the quantity of IL-12 produced by postmaturation electroporation-CD40L DC. By using multiparameter flow cytometry, the quantities of IL-12 needed to prime naive antigen-reactive T cells to simultaneously produce interferon-γ and tumor necrosis factor-α in the presence or absence of IL-2 secretion in conjunction with lytic activity defined by CD107a expression can be used to determine the overall potency of a DC product. In the presence of IL-12, CTL differentiation toward lytic function is not accompanied by a reduction in the secretion of interferon-γ and tumor necrosis factor-α. Therefore, by measuring the availability of IL-12 one can predict the potency of a DC immunotherapeutics in relation to its ability to drive distinct effector memory CTL subsets with multifunctional activities.     

Migratory properties of naive, effector, and memory CD8(+) T cells

It has been proposed that two different antigen-experienced T cell subsets may be distinguishable by their preferential ability to home to lymphoid organs (central memory cells) or nonlymphoid tissues (effector memory/effector cells). We have shown recently that murine antigen-primed CD8(+) T cells cultured in interleukin (IL)-15 (CD8(IL-15)) resemble central memory cells in phenotype and function. In contrast, primed CD8(+) T cells cultured in IL-2 (CD8(IL-2)) become cytotoxic effector cells. Here, the migratory behavior of these two subsets was investigated. Naive, CD8(IL-15) cells and, to a lesser degree, CD8(IL-2) cells localized to T cell areas in the spleen, but only naive and CD8(IL-15) cells homed to lymph nodes (LNs) and Peyer's patches. Intravital microscopy of peripheral LNs revealed that CD8(IL-15) cells, but not CD8(IL-2) cells, rolled and arrested in high endothelial venules (HEVs). Migration of CD8(IL-15) cells to LNs depended on L-selectin and required chemokines that bind CC chemokine receptor (CCR)7. Both antigen-experienced populations, but not naive T cells, responded to inflammatory chemokines and accumulated at sites of inflammation. However, CD8(IL-2) cells were 12 times more efficient in migrating to inflamed peritoneum than CD8(IL-15) cells. Furthermore, CD8(IL-15) cells proliferated rapidly upon reencounter with antigen at sites of inflammation. Thus, central memory-like CD8(IL-15) cells home avidly to lymphoid organs and moderately to sites of inflammation, where they mediate rapid recall responses, whereas CD8(IL-2) effector T cells accumulate in inflamed tissues, but are excluded from most lymphoid organs.     

Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection

Primary virus infection often elicits a large CD8(+) T cell response which subsequently contracts to a smaller memory T cell pool; the relationship between these two virus-specific populations is not well understood. Here we follow the human CD8(+) T cell response to Epstein-Barr virus (EBV) from its primary phase in infectious mononucleosis (IM) through to the persistent carrier state. Using HLA-A2.1 or B8 tetramers specific for four lytic cycle and three latent cycle epitopes, we find marked differences in the epitope-specific composition of the T cell populations between the two phases of infection. The primary response is dominated by lytic epitope specificities which are severely culled (and in one case extinguished) with resolution of the acute infection; in contrast latent epitope specificities are less abundant, if present at all, in acute IM but often then increase their percentage representation in the CD8 pool. Even comparing epitopes of the same type, the relative size of responses seen in primary infection does not necessarily correlate with that seen in the longer term. We also follow the evolution of phenotypic change in these populations and show that, from a uniform CD45RA(-)RO(+)CCR7(-) phenotype in IM, lytic epitope responses show greater reversion to a CD45RA(+)RO(-) phenotype whereas latent epitope responses remain CD45RA(-)RO(+) with a greater tendency to acquire CCR7. Interestingly these phenotypic distinctions reflect the source of the epitope as lytic or latent, and not the extent to which the response has been amplified in vivo.     

Characterization of HLA-A2-restricted HPV-16 E7-specific CD8(+) T-cell immune responses induced by DNA vaccines in HLA-A2 transgenic mice

We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.     

Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells.

Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4(+) or CD8(+) T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti-E- and -P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon gamma-inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand.     

A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T-cell and humoral responses in mice.

The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.     

Inflammatory chemokine receptors regulate CD8(+) T cell contraction and memory generation following infection

The development of T cell memory from naive precursors is influenced by molecular cues received during T cell activation and differentiation. In this study, we describe a novel role for the chemokine receptors CCR5 and CXCR3 in regulating effector CD8(+) T cell contraction and memory generation after influenza virus infection. We find that Ccr5(-/-) Cxcr3(-/-) cells show markedly decreased contraction after viral clearance, leading to the establishment of massive numbers of memory CD8(+) T cells. Ccr5(-/-) Cxcr3(-/-) cells show reduced expression of CD69 in the lung during the peak of infection, which coincides with differential localization and the rapid appearance of memory precursor cells. Analysis of single chemokine receptor-deficient cells revealed that CXCR3 is primarily responsible for this phenotype, although there is also a role for CCR5 in the enhancement of T cell memory. The phenotype could be reversed by adding exogenous antigen, resulting in the activation and contraction of Ccr5(-/-) Cxcr3(-/-) cells. Similar results were observed during chronic Mycobacterium tuberculosis infection. Together, the data support a model of memory CD8(+) T cell generation in which the chemokine-directed localization of T cells within infected tissues regulates antigen encounter and controls the extent of CD8(+) T cell activation and differentiation, which ultimately regulates effector versus memory cell fate decisions.     

CD8(+) T-cell autoreactivity to an HLA-B27-restricted self-epitope correlates with ankylosing spondylitis

HLA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705(+) and B*2709(+) subjects possess LMP2 236-244-specific, HLA-B27-restricted T cells, whereas only the B*2705(+) individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-gamma ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408-specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative "arthritogenic" peptide.     

Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here, we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.