High avidity antigen-specific CTL identified by CD8-independent tetramer staining

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.     

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.     

Reduced functional avidity promotes central and effector memory CD4 T cell responses to tumor-associated antigens

The effect of TCR signals on the differentiation of memory T cells is poorly defined. Conventional wisdom suggests that high-avidity interactions are best for the selection of vaccine Ag candidates or T cell specificities for adoptive T cell therapy to stimulate robust responses. However, in conditions of Ag persistence, high-avidity clones might exhaust and fail to form long-lived protective memory. We have manipulated the functional avidity of CD4 T cells by reducing expression of Lck, a key kinase involved in TCR triggering. Using a mouse model, we followed tetramer-positive T cells responding to a tumor Ag expressed by an adenocarcinoma. We show that reducing the functional avidity increased effector-effector memory responses and improved the generation of self-renewing, recirculating, tumor Ag-specific memory phenotype CD4 T cells. Moreover, such cells together with wild type CD8 T cells were better able to control tumor growth. Mechanistically, reducing Lck prolonged IL-2 production and cell turnover in the central memory population while reducing expression of exhaustion markers in the face of chronic Ag. Our data indicate that, in situations of persistent Ag challenge, generating T cells with reduced functional avidity may elicit more effective immune responses.     

Selective induction of high avidity CTL by altering the balance of signals from APC

High avidity CTL are most effective at clearing viruses and cancer cells. Therefore, understanding the mechanisms involved in induction of high avidity CTL is critical for effective vaccines. However, no vaccine approach to selectively induce high avidity CTL in vivo has been discovered. In a new approach, signals from MHC class I (signal 1) and costimulatory molecules (signal 2) were adjusted by varying Ag dose and by use of recombinant poxvirus expressing a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3), respectively. Independent of CTL avidity, a strong signal 1 resulted in an increased frequency of CD8(+) CTL. However, a strong signal 2 was necessary for the induction of high avidity CD8(+) CTL that killed target cells more efficiently, and signal 2 played a more crucial role in the absence of a strong signal 1. Only CTL induced with strong signal 2 killed tumor cells endogenously expressing low levels of Ag. Signal 2 contributed to the induction of high avidity CD8(+) CTL in both primary and secondary responses. Thus, although signal 2 has been known to increase the quantity of CTL response, in this study we show that it also improves the quality of CTL response. Our data also suggested that dendritic cells play an important role in induction of high avidity CD8(+) CTL in vivo. This strategy to selectively induce higher avidity CTL may lead to more effective vaccines for viruses and cancer.     

High avidity CD8+ T cells are the initial population elicited following viral infection of the respiratory tract

Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were approximately 50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.     

Accumulation of CD8+ T cells in advanced-stage tumors and delay of disease progression following secondary immunization against an immunorecessive epitope

Self-reactive T cells that survive the process of positive and negative selection during thymocyte development represent potential effector cells against tumors that express these same self-Ags. We have previously shown that CD8+ T lymphocytes (T(CD8)) specific for an immunorecessive epitope, designated epitope V, from the SV40 large T Ag (Tag) escape thymic deletion in line SV11 Tag-transgenic mice. In contrast, these mice are tolerant to the three most dominant Tag epitopes. The majority of the residual epitope V-specific T(CD8) have a low avidity for the target epitope, but a prime/boost regimen can expand higher avidity clones in vivo. Whether higher avidity T(CD8) targeting this epitope are affected by Tag-expressing tumors in the periphery or can be recruited for control of tumor progression remains unknown. In the current study, we determined the fate of naive TCR-transgenic T(CD8) specific for Tag epitope V (TCR-V cells) following transfer into SV11 mice bearing advanced-stage choroid plexus tumors. The results indicate that TCR-V cells are rapidly triggered by the endogenous Tag and acquire effector function, but fail to accumulate within the tumors. Primary immunization enhanced TCR-V cell frequency in the periphery and promoted entry into the brain, but a subsequent booster immunization caused a dramatic accumulation of TCR-V T cells within the tumors and inhibited tumor progression. These results indicate that epitope V provides a target for CD8+ T cells against spontaneous tumors in vivo, and suggests that epitopes with similar properties can be harnessed for tumor immunotherapy.     

T cells undergo rapid ON/OFF but not ON/OFF/ON cycling of cytokine production in response to antigen

Inflammatory cytokines such as IFN-gamma and TNF produced by Ag-stimulated CD4(+) and CD8(+) T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8(+) T cells maintain IFN-gamma production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8(+) T cells could rapidly cycle IFN-gamma production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8(+) T cells, our results show that Listeria monocytogenes-specific CD4(+) T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-gamma and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4(+) T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8(+) T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8(+) T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-gamma after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-gamma secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.     

Functional Exhaustion of CD4+ T Lymphocytes during Primary Cytomegalovirus Infection

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4(+) T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4(+) T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-specific CD4(+) T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-specific CD4(+) T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28(-) and CD28(+)CD4(+) T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4(+) T cells produced lower amounts of IFN-γ and TNF-α and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28(+) CMV-specific CD4(+) T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-specific CD4(+) T cells displaying low functional avidity for viral Ags.     

B cells and TCR avidity determine distinct functions of CD4+ T cells in retroviral infection

The T cell-dependent B cell response relies on cognate interaction between B cells and CD4(+) Th cells. However, the consequences of this interaction for CD4(+) T cells are not entirely known. B cells generally promote CD4(+) T cell responses to pathogens, albeit to a variable degree. In contrast, CD4(+) T cell responses to self- or tumor Ags are often suppressed by B cells. In this study, we demonstrated that interaction with B cells dramatically inhibited the function of virus-specific CD4(+) T cells in retroviral infection. We have used Friend virus infection of mice as a model for retroviral infection, in which the behavior of virus-specific CD4(+) T cells was monitored according to their TCR avidity. We report that avidity for Ag and interaction with B cells determine distinct aspects of the primary CD4(+) T cell response to Friend virus infection. Virus-specific CD4(+) T cells followed exclusive Th1 and T follicular helper (Tfh) differentiation. High avidity for Ag facilitated expansion during priming and enhanced the capacity for IFN-γ and IL-21 production. In contrast, Tfh differentiation was not affected by avidity for Ag. By reducing or preventing B cell interaction, we found that B cells promoted Tfh differentiation, induced programmed death 1 expression, and inhibited IFN-γ production by virus-specific CD4(+) T cells. Ultimately, B cells protected hosts from CD4(+) T cell-mediated immune pathology, at the detriment of CD4(+) T cell-mediated protective immunity. Our results suggest that B cell presentation of vaccine Ags could be manipulated to direct the appropriate CD4(+) T cell response.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5?mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.     

Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neu-expressing tumors after depletion of effector/memory-like regulatory T cells

Background

Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.

Methodology/Principal Findings

High and low avidity CD8⁺ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4⁺Foxp3⁺CD25^low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.

Conclusion/Significance

Depletion of CD25^low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.     

A T cell clone's avidity is a function of its activation state

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-gamma release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag ("functional avidity") is not a constant parameter of a T cell clone but a function of the T cell's history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.     

Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells

Memory T cells (T(M)) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as T(M) undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 T(M) by comparing them to defective CD8 T(M) generated in the absence of CD4 T cells. Unhelped CD8 T(M) are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-gamma. Our data show that this defect in IL-2 and IFN-gamma production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-gamma loci and substantial histone acetylation at the IFN-gamma promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-gamma locus. Thus, CD4 T cell help influences epigenetic modification during CD8 T(M) differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 T(M).     

Peripheral "CD8 tuning" dynamically modulates the size and responsiveness of an antigen-specific T cell pool in vivo

In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8(+) T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8(low) T cells produced after peripheral HY-D(b) MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8(int) T cells from heterozygote CD8(+/0) mice. Finally, we used pre-existing nonresponsive CD8(low) T cells from male HY TCR transgenic mice. In CD8(low) and CD8(high) mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (K(D)) and dissociation constant (T(1/2)) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8(high) T cells of the same specificity. Additionally, direct injections of wild-type HY-D(b) and C2A-D(b) tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8(low) T cells, yet C2A-D(b) was superior in inducing a primed CD8(+)CD44(+) memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by "CD8 tuning" of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.     

High avidity CD8+ T cells generated from CD28-deficient or wildtype mice exhibit a differential dependence on lipid raft integrity for activation

CD28 has been shown to play an important role in T cell activation. Among the downstream events associated with CD28 engagement is the reorganization of the cytoskeleton resulting in lipid raft aggregation. In our previous studies we investigated the involvement of lipid rafts in the activation of high avidity CD8+ T lymphocytes, which recognize cells bearing very low levels of peptide antigen, versus low avidity cells, which require high levels of peptide antigen. In these studies we found that high avidity cells were much more sensitive to lipid raft disruption compared to low avidity cells. Given the important role for CD28 in lipid raft reorganization and our previous finding that high avidity cells are extremely dependent on lipid raft integrity, we hypothesized that high avidity cells could not be generated in the absence of CD28. Surprisingly, we have found that the absence of CD28 does not alter the ability to generate high or low avidity CD8+ T cells. In fact high and low avidity lines generated in parallel from CD28-deficient and WT mice exhibited very similar requirements for peptide antigen. We next compared the effect of lipid raft disruption on the activation of high versus low avidity cells from CD28-deficient and WT mice. While high avidity cells generated from WT mice exhibited the expected dependence on lipid raft integrity, high avidity cells from CD28-deficient mice were not affected. These data suggest that the lines generated from the CD28-deficient mice have developed alternative strategies to promote high sensitivity to peptide antigen.     

Modulation of CD8+ T cell avidity by increasing the turnover of viral antigen during infection

The increased potency of high avidity CD8+ T cells for the clearance of viral infections has been well documented. We have previously reported the novel finding that intranasal infection with the paramyxovirus SV5 induces a CD8+ T cell response to the SV5 P protein that is almost exclusively of high avidity. Based on our results that the level of peptide presentation is a critical factor in the selective expansion of high versus low avidity cells in vitro, we hypothesized that the avidity of the anti-viral response generated in vivo could be altered by increasing the turnover of the P protein during viral infection through linkage to ubiquitin (UbP). Infection with a virus expressing UbP (VV-UbP) elicited a significant increase in low avidity cells in both BALB/c and C3H mice compared to the almost exclusively high avidity response elicited by VV-P. Our results are the first demonstration of the control of avidity during the antiviral response through an engineered change to a viral antigen. The implications of our findings for vaccine development are discussed.     

A reduced antigen load in vivo, rather than weak inflammation, causes a substantial delay in CD8+ T cell priming against Mycobacterium bovis (bacillus Calmette-Guérin)

Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses.     

Plasmid DNA encoding CCR7 ligands compensate for dysfunctional CD8+ T cell responses by effects on dendritic cells

Lymphotoxin alpha-deficient (LTalpha-/-) mice, which lack lymph nodes and possess a disorganized spleen, develop dysfunctional CD8+ T cells upon HSV infection and readily succumb to herpes encephalitis. Such mice do develop apparently normal peptide-specific CD8+ T cell responses, as measured by MHC class I tetramer staining, but the majority of cells fail to become cytotoxic or express peptide-induced IFN-gamma production. In the present study, we demonstrate that functional defects of CD8+ T cells in LTalpha-/- mice can be largely rectified by the administration of plasmid DNA encoding CCR7 ligands before HSV infection. Treated mutant mice developed increased peptide-specific cytotoxic responses, enhanced numbers of CD8+ T cells capable of producing IFN-gamma, as well as improved resistance to HSV challenge. The corrective effect of chemokine treatment appeared to result from improved dendritic cell-mediated Ag presentation. Thus, a major consequence of the treatment was an increase in splenic dendritic cell number in CCR7 ligand-treated LTalpha-/- mice with such splenocyte populations showing improved APC activity in vitro. Our results document that functional defects of CD8+ T cells can be corrected, and indicate the value of plasmid vector encoding appropriate chemokines to achieve such immunotherapy.