Antigen-specific CD8(+) T cells persist in the upper respiratory tract following influenza virus infection.

Because little is known about lymphocyte responses in the nasal mucosa, lymphocyte accumulation in the nasal mucosa, nasal-associated lymphoid tissue (NALT), and cervical lymph nodes (CLN) were determined after primary and heterosubtypic intranasal influenza challenge of mice. T cell accumulation peaked in the nasal mucosa on day 7, but peaked slightly earlier in the CLN (day 5) and later (day 10) in the NALT. Tetrameric staining of nasal mucosal cells revealed a peak accumulation of CD8 T cells specific for either the H-2D(b) influenza nucleoprotein epitope 366-374 (D(b)NP(366)) or the H-2D(b) polymerase 2 protein epitope 224-233 (D(b)PA(224)) at 7 days. By day 13, D(b)PA(224)-specific CD8 T cells were undetectable in the mucosa, whereas D(b)NP(366)-specific CD8 T cells persisted for at least 35 days in the mucosa and spleen. After heterosubtypic virus challenge, the accumulation of CD8 T cells in the nasal mucosa was quicker, more intense, and predominantly D(b)NP(366) specific relative to the primary inoculation. The kinetics and specificity of the CD8 T cell response were similar to those in the CLN, but the responses in the NALT and spleen were again slower and more protracted. These results indicate that similar to what was reported in the lung, D(b)NP(366)-specific CD8 T cells persist in the nasal mucosa after primary influenza infection and predominate in an intensified nasal mucosal response to heterosubtypic challenge. In addition, differences in the kinetics of the CD8 T cell responses in the CLN, NALT, and spleen suggest different roles of these lymphoid tissues in the mucosal response.     

Quantification of repertoire diversity of influenza-specific epitopes with predominant public or private TCR usage

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vbeta8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vbeta4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50-550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.     

Multiple Distinct Forms of CD8+ T Cell Cross-Reactivity and Specificities Revealed after 2009 H1N1 Influenza A Virus Infection in Mice

Influenza primed mice are protected against lethal infection with H1N1 A/CA/04/E3/09 virus, and T depletion and serum transfer studies suggest a T-dependent mechanism. We therefore set out to investigate the quality of the cross-reactive T cell response to CA/E3/09 in mice primed with H3N2 influenza A/Hong Kong/X31 virus. Sequences of the immunodominant nucleoprotein (NP) NP366–374 and acid polymerase (PA) PA224–233 CD8 epitopes from X31 each differ from the CA/E3/09 virus by one amino acid: an M371V substitution at position 6 of the NP peptide, and an S224P substitution at position 1 of the PA peptide, raising questions about the role of these epitopes in protection. PA224–233 peptides from either virus could elicit IFN-γ spot forming cells from mice infected with X31, indicating cross-reactivity of these two peptides. However, no T cell responses to either PA224–233 peptide were detectable after primary CA/E3/09 infection, suggesting it is cryptic in this virus. In contrast, primary responses to the NP366 peptides were detectable after infection with either virus, but did not cross-react in vitro. Similarly, H2-D^b tetramers of each NP epitope stained CD8+ T cells from each respective virus infection, but did not obviously cross-react. Early after lethal CA/E3/09 challenge, X31 primed mice had enhanced IFN-γ responses toward both NP366 peptides, as well as recall responses to a set of subdominant NP and PA peptides not detectable after primary X31 infection alone. Furthermore, dual-tetramer staining revealed an expanded population of CD8 T cells reactive to both NP366 variant peptides also not seen after the priming infection alone. These observations demonstrate unusual CD8+ T cell cross-reactivity and specificity are elicited after primary and secondary CA/E3/09 influenza virus infections.     

Late signals from CD27 prevent Fas-dependent apoptosis of primary CD8+ T cells

The role of costimulation has previously been confined to the very early stages of the CD8+ T cell response. In this study, we demonstrate the requirement for CD27 costimulation during the later phase, but not programming of the primary CD8+ T cell response to influenza virus and reveal a novel mechanism of action for CD27 costimulation. CD27 signals, during the later phase of the primary CD8+ T cell response, prevent apoptosis of Ag-specific CD8+ T cells. Blocking CD27L (CD70) on days 6 and 8 after infection reduces the number of NP(366-374)-specific CD8+ T cells, increases their sensitivity to CD95/Fas-mediated apoptosis, and up-regulates FasL on CD4+ T cells. This reduction of NP(366-374)-specific CD8+ T cells requires the presence of CD4+ T cells and Fas signaling. Lack of CD27 signals also decreases the quality of memory CD8+ T cell responses. Memory CD8+ T cells, which express surface CD27 similar to naive cells, however, do not require CD27 costimulation during a secondary response. Thus, CD27 acts indirectly to regulate primary Ag-specific CD8+ T cell responses by preventing apoptosis of CD8+ T cells during the later phase of the primary response and is required for optimal quality of memory cells, but is not required during normally primed secondary CD8+ T cell responses.     

Effect of MHC class I diversification on influenza epitope-specific CD8+ T cell precursor frequency and subsequent effector function

Earlier studies of influenza-specific CD8(+) T cell immunodominance hierarchies indicated that expression of the H2K(k) MHC class I allele greatly diminishes responses to the H2D(b)-restriced D(b)PA(224) epitope (acid polymerase, residues 224-233 complexed with H2D(b)). The results suggested that the presence of H2K(k) during thymic differentiation led to the deletion of a prominent Vβ7(+) subset of D(b)PA(224)-specific TCRs. The more recent definition of D(b)PA(224)-specific TCR CDR3β repertoires in H2(b) mice provides a new baseline for looking again at this possible H2K(k) effect on D(b)PA(224)-specific TCR selection. We found that immune responses to several H2D(b)- and H2K(b)-restricted influenza epitopes were indeed diminished in H2(bxk) F(1) versus homozygous mice. In the case of D(b)PA(224), lower numbers of naive precursors were part of the explanation, though a similar decrease in those specific for the D(b)NP(366) epitope did not affect response magnitude. Changes in precursor frequency were not associated with any major loss of TCR diversity and could not fully account for the diminished D(b)PA(224)-specific response. Further functional and phenotypic characterization of influenza-specific CD8(+) T cells suggested that the expansion and differentiation of the D(b)PA(224)-specific set is impaired in the H2(bxk) F(1) environment. Thus, the D(b)PA(224) response in H2(bxk) F(1) mice is modulated by factors that affect the generation of naive epitope-specific precursors and the expansion and differentiation of these T cells during infection, rather than clonal deletion of a prominent Vβ7(+) subset. Such findings illustrate the difficulties of predicting and defining the effects of MHC class I diversification on epitope-specific responses.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection

In this report, we demonstrate that CD28(-/-) mice are severely impaired in the initial expansion of D(b)/NP366-374-specific CD8 T cells in response to influenza virus infection, whereas 4-1BB ligand (4-1BBL)(-/-) mice show no defect in primary T cell expansion to influenza virus. In contrast, 4-1BBL(-/-) mice show a decrease in D(b)/NP366-374-specific T cells late in the primary response. Upon secondary challenge with influenza virus, 4-1BBL(-/-) mice show a decrease in the number of D(b)/NP366-374-specific T cells compared to wild-type mice such that the level of the CD8 T cell expansion during the in vivo secondary response is reduced to the level of a primary response, with concomitant reduction of CTL effector function. In contrast, Ab responses, as well as secondary CD4 T cell responses, to influenza are unaffected by 4-1BBL deficiency. Thus, CD28 is critical for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the response and is essential for the survival and/or responsiveness of the memory CD8 T cell pool.     

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8(+) T cell responses to H2D(b) complexed with peptides from the viral nucleoprotein (NP(366), ASNENMETM) and acid polymerase (PA(224), SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of D(b)NP(366)- and D(b)PA(224)-specific acute and memory CD8(+) T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8(+) T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8(+)D(b)NP(366)(+) T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.     

A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge

Respiratory challenge of H-2(b) mice with an H3N2 influenza A virus causes an acute, transient pneumonitis characterized by the massive infiltration of CD8(+) T lymphocytes. The inflammatory process monitored by quantitative analysis of lymphocyte populations recovered by bronchoalveolar lavage is greatly enhanced by prior exposure to an H1N1 virus, with the recall of cross-reactive CD8(+)-T-cell memory leading to more rapid clearance of the infection from the lungs. The predominant epitope recognized by the influenza virus-specific CD8(+) set has long been thought to be a nucleoprotein (NP(366-374)) presented by H-2D(b) (D(b)NP(366)). This continues to be true for the secondary H3N2-->H1N1 challenge but can no longer be considered the case for the primary response to either virus. Quantitative analysis based on intracellular staining for gamma interferon has shown that the polymerase 2 protein (PA(224-233)) provides a previously undetected epitope (D(b)PA(224)) that is at least as prominent as D(b)NP(366) during the first 10 days following primary exposure to either the H3N2 or H1N1 virus. The response to D(b)NP(366) seems to continue for longer, even when infectious virus can no longer be detected, but there is no obvious difference in the prevalence of memory T cells specific for D(b)NP(366) and D(b)PA(224). The generalization that the magnitude of the functional memory T-cell pool is a direct consequence of the clonal burst size during the primary response may no longer be useful. Previous CD8(+)-T-cell immunodominance heirarchies defined largely by cytotoxic T-lymphocyte assays may need to be revised.     

Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses

Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.     

A simple mathematical model helps to explain the immunodominance of CD8 T cells in influenza A virus infections

Understanding immunodominance, the phenomenon of epitope-specific T cells expanding in an often distinctly hierarchical fashion, is important for the design of T-cell-based intervention strategies. Several recent studies have investigated immunodominance of H-2D(b)-restricted CD8(+) T cells specific for the nucleoprotein NP366 and acid polymerase PA224 epitopes during influenza A virus infection of C57BL/6 mice. CD8(+) T cells specific for these two epitopes are codominant during primary infection; NP366 dominates during secondary infection. While a number of explanations for this observation have been proposed, none of them can fully account for all the observed data. In this article, we use a simple mathematical model to explain the seemingly inconsistent data. We show that the dynamic interactions between CD8(+) T cells and antigen presentation lead to a situation where CD8(+) T cells are limiting during the initial response whereas antigen is limiting in the secondary response. This "numbers game" between antigen and CD8(+) T cells can reproduce the observed immunodominance of the NP336- and PA224-specific CD8(+) T cells, thereby explaining the reported experimental data.     

Concurrent naive and memory CD8(+) T cell responses to an influenza A virus

Memory Thy-1(+)CD8(+) T cells specific for the influenza A virus nucleoprotein (NP(366-374)) peptide were sorted after staining with the D(b)NP(366) tetramer, labeled with CFSE, and transferred into normal Thy-1.2(+) recipients. The donor D(b)NP(366)(+) T cells recovered 2 days later from the spleens of the Thy-1.2(+) hosts showed the CD62L(low)CD44(high)CD69(low) phenotype, characteristic of the population analyzed before transfer, and were present at frequencies equivalent to those detected previously in mice primed once by a single exposure to an influenza A virus. Analysis of CFSE-staining profiles established that resting tetramer(+) T cells divided slowly over the next 30 days, while the numbers in the spleen decreased about 3-fold. Intranasal infection shortly after cell transfer with a noncross-reactive influenza B virus induced some of the donor D(b)NP(366)(+) T cells to cycle, but there was no increase in the total number of transferred cells. By contrast, comparable challenge with an influenza A virus caused substantial clonal expansion, and loss of the CFSE label. Unexpectedly, the recruitment of naive Thy-1.2(+)CD8(+)D(b)NP(366)(+) host D(b)NP(366)(+) T cells following influenza A challenge was not obviously diminished by the presence of the memory Thy-1.1(+)CD8(+)D(b)NP(366)(+) donor D(b)NP(366)(+) set. Furthermore, the splenic response to an epitope (D(b)PA(224)) derived from the influenza acid polymerase (PA(224-233)) was significantly enhanced in the mice given the donor D(b)NP(366)(+) memory population. These experiments indicate that an apparent recall response may be comprised of both naive and memory CD8(+) T cells.     

Hierarchies in cytokine expression profiles for acute and resolving influenza virus-specific CD8+ T cell responses: correlation of cytokine profile and TCR avidity

The development and resolution phases of influenza-specific CD8(+) T cell cytokine responses to epitopes derived from the viral nucleoprotein (D(b)NP(366)) and acid polymerase (D(b)PA(224)) were characterized in C57BL/6J mice for a range of anatomical compartments in the virus-infected lung and lymphoid tissue. Lymphocyte numbers were measured by IFN-gamma expression following stimulation with peptide, while the quality of the response was determined by the intensity of staining and the distribution of CD8(+) T cells producing TNF-alpha and IL-2. Both the levels of expression and the prevalence of TNF-alpha(+) and IL-2(+) cells reflected the likely Ag load, with clear differences being identified for populations from the alveolar space vs the lung parenchyma. Irrespective of the site or time of T cell recovery, IL-2(+) cells were consistently found to be a subset of the TNF-alpha(+) population which was, in turn, contained within the IFN-gamma(+) set. The capacity to produce IL-2 may thus be considered to reflect maximum functional differentiation. The hierarchy in cytokine expression throughout the acute phase of the primary and secondary response tended to be D(b)PA(224) > D(b)NP(366). Both elution studies with the cognate tetramers and experiments measuring CD8 beta coreceptor dependence for peptide stimulation demonstrated the same D(b)PA(224) > D(b)NP(366) profile for TCR avidity. Overall, the quality of any virus-specific CD8(+) T cell response appears variously determined by the avidity of the TCR-pMHC interaction, the duration and intensity of Ag stimulation characteristic of the particular tissue environment, and the availability of CD4(+) T help.     

Vaccination with an acidic polymerase epitope of influenza virus elicits a potent antiviral T cell response but delayed clearance of an influenza virus challenge

The mechanisms underlying epitope selection and the potential impact of immunodominance hierarchies on peptide-based vaccines are not well understood. Recently, we have shown that two immunodominant MHC class I-restricted epitopes, NP(366-374)/D(b) (nucleoprotein (NP)) and PA(224-233)/D(b) (acidic polymerase (PA)), which drive the CD8(+) T cell response to influenza virus infection in C57BL/6 mice, are differentially expressed on infected cells. Whereas NP appears to be strongly expressed on all infected cells, PA appears to be strongly expressed on dendritic cells but only weakly expressed on nondendritic cells. Thus, the immune response to influenza virus may involve T cells specific for epitopes, such as PA, that are poorly expressed at the site of infection. To examine the consequences of differential Ag presentation on peptide vaccination, we compared the kinetics of the T cell response and influenza virus clearance in mice vaccinated with the NP or PA peptide. Vaccination with either the NP or PA peptide resulted in accelerated and enhanced Ag-specific T cell responses at the site of infection following influenza virus challenge. These T cells were fully functional in terms of their ability to produce IFN-gamma and TNF-alpha and to mediate cytolytic activity. Despite this enhancement of the Ag-specific T cell response, PA vaccination had a detrimental effect on the clearance of influenza virus compared with unvaccinated or NP-vaccinated mice. These data suggest that differential Ag presentation impacts the efficacy of T cell responses to specific epitopes and that this needs to be considered for the development of peptide-based vaccination strategies.     

Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.     

The context of epitope presentation can influence functional quality of recalled influenza A virus-specific memory CD8+ T cells

Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8(+) T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA(224)) in B6 mice. Vaccination with the PA(224) lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary D(b)PA(224) responses in the lung. Lipopeptide recalled D(b)PA(224)-specific CTL produced lower levels of IFN-gamma and TNF-alpha, but produced similar levels of IL-2 when compared with D(b)PA(224)-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of D(b)PA(224)-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.     

Crystal structures of murine MHC Class I H-2 D(b) and K(b) molecules in complex with CTL epitopes from influenza A virus: implications for TCR repertoire selection and immunodominance

Cytotoxic T lymphocyte (CTL) responses against influenza A virus in C57BL/6 mice are dominated by a small number of viral peptides among many that are capable of binding to major histocompatibility complex (MHC) class I molecules. The basis of this limited immune recognition is unknown. Here, we present X-ray structures of MHC class I molecules in complex with two immunodominant epitopes (PA(224-233)/D(b) and PB1(703-711)/K(b)) and one non-immunogenic epitope (HA(468-477)/D(b)) of the influenza A virus. The immunodominant peptides are each characterized by a bulge at the C terminus, lifting P6 and P7 residues out of the MHC groove, presenting featured structural elements to T-cell receptors (TCRs). Immune recognition of PA(224-233)/D(b) will focus largely on the exposed P7 arginine residue. In contrast, the non-immunogenic HA(468-477) peptide lacks prominent features in this C-terminal bulge. In the K(b)-bound PB1(703-711) epitope, the bulge results from a non-canonical binding motif, such that the mode of presentation of this peptide strongly resembles that of D(b)-bound peptides. Given that PA(224-233)/D(b), PB1(703-711)/K(b) and the previously defined NP(366-374)/D(b) epitopes dominate the primary response to influenza A virus in C57BL/6 mice, our findings indicate that residues of the C-terminal bulge are important in selection of the immunodominant CTL repertoire.     

Virus-specific CD8+ T cells in the liver: armed and ready to kill

Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2D(b)NP(366-374) (D(b)NP(366)) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-gamma secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a "graveyard" for influenza virus-specific CD8+ T cells.     

Immature and mature CD8alpha+ dendritic cells prolong the survival of vascularized heart allografts.

CD8alpha+ and CD8alpha- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8alpha-(myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8alpha+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2(b)) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2(k)) recipients for Th1 responses. Paradoxically and in contrast to their CD8alpha- counterparts, mature CD8alpha+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8alpha+ and CD8alpha- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8alpha+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.