The attachment (G) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both Th1 and Th2 CD4+ T cell responses

BALB/c mice immunized with a vaccinia virus expressing the attachment (G) glycoprotein of respiratory syncytial virus (RSV) develop a virus-specific CD4(+) T cell response that consists of a mixture of Th1 and Th2 CD4(+) T cells following intranasal infection with live RSV. Recent work has shown that both Th1 and Th2 CD4(+) T cells are elicited to a single region comprising aa 183-197 of the G protein. To more precisely define the CD4(+) T cell epitope(s) contained within this region, we created a panel of amino- and carboxyl-terminal truncated as well as single alanine-substituted peptides spanning aa 183-197. These peptides were used to examine the ex vivo cytokine response of memory effector CD4(+) T cells infiltrating the lungs of G-primed RSV-infected mice. Analysis of lung-derived memory effector CD4(+) T cells using intracellular cytokine staining and/or ELISA of effector T cell culture supernatants revealed a single I-E(d)-restricted CD4(+) T cell epitope with a core sequence mapping to aa 185-193. In addition, we examined the T cell repertoire of the RSV G peptide-specific CD4(+) T cells and show that the CD4(+) T cells directed to this single immunodominant G epitope use a restricted range of TCR Vss genes and predominantly express Vss14 TCR.     

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.     

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.     

Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using an H-2K(d)-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct (51)Cr release assay and intracellular gamma interferon (IFN-gamma) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-gamma production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8(+) RSV-specific CTL responses in vivo.     

Differential role of gamma interferon in inhibiting pulmonary eosinophilia and exacerbating systemic disease in fusion protein-immunized mice undergoing challenge infection with respiratory syncytial virus

Secondary exposure to respiratory syncytial virus (RSV) can lead to immunopathology and enhanced disease in vaccinated individuals. Vaccination with individual RSV proteins influences the type of secondary RSV-specific immune response that develops upon challenge RSV infection, as well as the extent of immunopathology. RSV-specific memory CD4 T cells can directly contribute to immunopathology through their cytokine production. Immunization of BALB/c mice with a recombinant vaccinia virus (vv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia upon RSV challenge, whereas immunization of mice with a vv expressing the fusion (F) protein does not. We analyzed the CD4 T-cell response to an I-E^d-restricted CD4 T-cell epitope within the F protein of RSV corresponding to amino acids 51 to 66 in an effort to better understand the similarities and differences in the immune response elicited by the G versus the F protein. Vaccination with the G protein induces a mixture of RSV G-specific Th1 and Th2 cells with a restricted T-cell receptor repertoire. In contrast, we demonstrate here that immunization with the F protein elicits a broad repertoire of RSV F-specific CD4 T cells that predominantly exhibit a Th1 phenotype. However, in the absence of gamma interferon (IFN-γ), RSV F_51-66-specific CD4 T cells secreted interleukin-5, and mice developed pulmonary eosinophilia after RSV challenge. IFN-γ-deficient mice exhibited decreased weight loss compared to wild-type controls, suggesting that IFN-γ exacerbates systemic disease. These data demonstrate that IFN-γ can have both beneficial and detrimental effects during a secondary RSV infection.     

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccines.     

Proliferative expansion and acquisition of effector activity by memory CD4+ T cells in the lungs following pulmonary virus infection

The memory CD4+ T cell response to the respiratory syncytial virus (RSV) attachment (G) protein in the lungs of primed BALB/c mice undergoing challenge pulmonary RSV infection is dominated by effector T cells expressing a single Vbeta-chain, Vbeta14. We have used Vbeta14 expression to examine the kinetics of the activation, accumulation, and acquisition of the effector activity of memory CD4+ T cells responding to pulmonary infection. This analysis revealed that proliferative expansion and effector CD4+ T cell differentiation preferentially occur in the respiratory tract following rapid activation within and egress from the lymph nodes draining the respiratory tract. These findings suggest that, in response to natural infection at a peripheral mucosal site such as the lungs, memory CD4+ T cell expansion and differentiation into activated effector T cells may occur predominantly in the peripheral site of infection rather than exclusively in the lymph nodes draining the site of infection.     

Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

CD8⁺ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8⁺ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8⁺ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8⁺ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M2_82-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8⁺ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8⁺ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8⁺ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8⁺ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8⁺ T cells. These memory CD8⁺ T cells had lower cytokine expression than effector CD8⁺ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8⁺ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8⁺ T cell cytokine expression.     

Activation and inactivation of antiviral CD8 T cell responses during murine pneumovirus infection

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261-270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN-gamma and TNF-alpha staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs.     

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.     

Expression of interleukin-4 by recombinant respiratory syncytial virus is associated with accelerated inflammation and a nonfunctional cytotoxic T-lymphocyte response following primary infection but not following challenge with wild-type virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.     

Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus

IFN-gamma-deficient (IFN-gamma(-/-)) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4(+)-depleted mice, especially in mice also deficient in IFN-gamma. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-gamma(-/-) mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-gamma(-/-) hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8(+) effector T cells is one important factor influencing the requirement for T cell help during viral infection.     

Efficient lung recruitment of respiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.     

Respiratory syncytial virus infection in C57BL/6 mice: clearance of virus from the lungs with virus-specific cytotoxic T cells.

We describe respiratory syncytial virus (RSV)-specific cytotoxic T-cell (CTL) lines and clones developed from the spleens of C57BL/6 and BALB/c mice. Line 7 and clones derived from it were H-2Kb restricted, whereas line 12 had both Kb and Db components. Both lines, and all the clones except one, could lyse targets infected with either strain A or strain B RSV. Line 7 or 7-11E1 cells (8 x 10(6) to 10 x 10(6) given intravenously cleared RSV from the lungs of infected mice. There was no morbidity or mortality in any of the infected mice whether or not they received T cells. The C57BL/6 mouse is a useful model system in which to study the role of the CTL response in protective immunity to RSV. CTL lines and clones can mediate clearance of RSV from the lungs of normal mice without producing any associated morbidity.     

Aged Mice Exhibit a Severely Diminished CD8 T Cell Response following Respiratory Syncytial Virus Infection

Respiratory virus infections in the elderly result in increased rates of hospitalization and death. Respiratory syncytial virus (RSV) is a leading cause of severe virus-induced respiratory disease in individuals over the age of 65. CD8 T cells play a critical role in mediating RSV clearance. While it is clear that T cell immunity declines with age, it is not clear to what extent the CD8 T cell response to RSV is altered. Using aged BALB/c mice, we demonstrated that RSV-specific CD8 T cell responses were significantly reduced in the lungs of aged mice at the peak of the T cell response and that this decrease correlated with delayed viral clearance. Despite a decrease in the overall numbers of RSV-specific CD8 T cells during acute infection, their capacity to produce effector cytokines was not impaired. Following viral clearance, the RSV-specific memory CD8 T cells were similar in total number and phenotype in young and aged mice. Furthermore, following infection with a heterologous pathogen expressing an RSV epitope, RSV-specific memory CD8 T cells exhibited similar activation and ability to provide early control of the infection in young and aged mice. These data demonstrate a decrease in the capacity of aged mice to induce a high-magnitude acute CD8 T cell response, leading to prolonged viral replication, which may contribute to the increased disease severity of RSV infection observed for aged individuals.     

Induction of Th-1 and Th-2 Responses by Respiratory Syncytial Virus Attachment Glycoprotein Is Epitope and Major Histocompatibility Complex Independent

In BALB/c mice, sensitization to respiratory syncytial virus (RSV) attachment (G) glycoprotein leads to the development of lung eosinophilia upon challenge infection with RSV, a pathology indicative of a strong in vivo induction of a Th-2-type response. In this study, we found that a strong, RSV G-specific, Th-1-type cytokine response occurred simultaneously with a Th-2-type response in G-primed mice after RSV challenge. Both Th-1 and Th-2 effector CD4(+) T cells recognized a single immunodominant site on this protein, implying that the differentiation of memory CD4(+) T cells along the Th-1 or Th-2 effector pathway was independent of the epitope specificity of the T cells. A similar observation was made in G-primed H-2(b) haplotype mice after RSV challenge, further suggesting that this process is not dependent on the peptide epitope presented. On the other hand, genes mapping to loci outside of the major histocompatibility complex region are crucial regulators of the development of a Th-2-type response and lung eosinophilia. The implication of these findings for the immune mechanisms underlying the pathogenesis of RSV is discussed.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.     

Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection

Whether true memory T cells develop in the face of chronic infection such as tuberculosis remains controversial. To address this question, we studied CD8+ T cells specific for the Mycobacterium tuberculosis ESAT6-related Ags TB10.3 and TB10.4. The shared epitope TB10.3/10.4(20-28) is presented by H-2 K(d), and 20-30% of the CD8+ T cells in the lungs of chronically infected mice are specific for this Ag following respiratory infection with M. tuberculosis. These TB10.3/10.4(20-28)-specific CD8+ T cells produce IFN-gamma and TNF and express CD107 on their cell surface, which indicates their likely role as CTL in vivo. Nearly all of the Ag-specific CD8+ T cells in the lungs of chronically infected mice had a T effector cell phenotype based on their low expression of CD62L and CD45RB. In contrast, a population of TB10.3/10.4(20-28)-specific CD8+ T cells was identified in the lymphoid organs that express high levels of CD62L and CD45RB. Antibiotic treatment to resolve the infection led to a contraction of the Ag-specific CD8+ T cell population and was accompanied by an increase in the proportion of CD8+ T cells with a central memory phenotype. Finally, challenge of memory-immune mice with M. tuberculosis was accompanied by significant expansion of TB10.3/10.4(20-28)-specific CD8+ T cells, which suggests that these cells are in fact functional memory T cells.     

CD8 T cells inhibit respiratory syncytial virus (RSV) vaccine-enhanced disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-gamma-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.     

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.