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.     

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.     

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.     

Influenza epitope-specific CD8+ T cell avidity, but not cytokine polyfunctionality, can be determined by TCRβ clonotype

Cytokine polyfunctionality has recently emerged as a correlate of effective CTL immunity to viruses and tumors. Although the determinants of polyfunctionality remain unclear, there are published instances of a link between the production of multiple effector molecules and the peptide plus MHC class I molecule avidity of T cell populations. Influenza A virus infection of C57BL/6J mice induces CTL populations specific for multiple viral epitopes, each with varying proportions of monofunctional (IFN-γ(+) only) or polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) CTLs. In this study, we probe the link between TCR avidity and polyfunctionality for two dominant influenza epitopes (D(b)NP(366) and D(b)PA(224)) by sequencing the TCR CDR3β regions of influenza-specific IFN-γ(+) versus IFN-γ(+)IL-2(+) cells, or total tetramer(+) versus high-avidity CTLs (as defined by the peptide plus MHC class I molecule-TCR dissociation rate). Preferential selection for particular clonotypes was evident for the high-avidity D(b)PA(224)-specific set but not for any of the other subsets examined. These data suggest that factors other than TCRβ sequence influence cytokine profiles and demonstrate no link between differential avidity and polyfunctionality.     

CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: functional and structural implications

To investigate protective immunity conferred by CTL against viral pathogens, we have analyzed CD8(+) T cell responses to the immunodominant nucleoprotein epitope (NP(366-374)) of influenza A virus in B6 mice during primary and secondary infections in vivo. Unlike the highly biased TCR Vbeta repertoire, the associated Valpha repertoire specific for the NP(366-374)/D(b) ligand is quite diverse. Nonetheless, certain public and conserved CDR3alpha clonotypes with distinct molecular signatures were identified. Pairing of public Valpha and Vbeta domains creates an alphabeta TCR heterodimer that binds efficiently to the NP(366-374)/D(b) ligand and stimulates T cell activation. In contrast, private TCRs, each comprising a distinct alpha chain paired with the same public beta chain, interact very differently. Molecular dynamics simulation reveals that the conformation and mobility of the shared Vbeta CDR loops are governed largely by the associated Valpha domains. These results provide insight into molecular principles regarding public versus private TCRs linked to immune surveillance after infection with influenza A virus.     

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.     

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.     

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.     

Valpha and Vbeta public repertoires are highly conserved in terminal deoxynucleotidyl transferase-deficient mice

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Valpha-Jalpha and Vbeta-Dbeta-Jbeta rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the alphabeta TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT(o/o) mice, even for the CDR3 loops which are shorter than those found in TdT(+/+) animals. Moreover, the 10- to 20-fold decrease in alphabeta T cell diversity in TdT(o/o) mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3beta in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3beta. We interpret our findings as suggesting that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHCalpha helices are involved in the selection of public repertoires.     

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.     

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 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.     

Consequences of suboptimal priming are apparent for low-avidity T-cell responses

The emergence of the novel reassortant A(H1N1)-2009 influenza virus highlighted the threat to the global population posed by an influenza pandemic. Pre-existing CD8(+) T-cell immunity targeting conserved epitopes provides immune protection against newly emerging strains of influenza virus, when minimal antibody immunity exists. However, the occurrence of mutations within T-cell antigenic peptides that enable the virus to evade T-cell recognition constitutes a substantial issue for virus control and vaccine design. Recent evidence suggests that it might be feasible to elicit CD8(+) T-cell memory pools to common virus mutants by pre-emptive vaccination. However, there is a need for a greater understanding of CD8(+) T-cell immunity towards commonly emerging mutants. The present analysis focuses on novel and immunodominant, although of low pMHC-I avidity, CD8(+) T-cell responses directed at the mutant influenza D(b)NP(366) epitope, D(b)NPM6A, following different routes of infection. We used a C57BL/6J model of influenza to dissect the effectiveness of the natural intranasal (i.n.) versus intraperitoneal (i.p.) priming for generating functional CD8(+) T cells towards the D(b)NPM6A epitope. In contrast to comparable CD8(+) T-cell responses directed at the wild-type epitopes, D(b)NP(366) and D(b)PA(224), we found that the priming route greatly affected the numbers, cytokine profiles and TCR repertoire of the responding CD8(+) T cells directed at the D(b)NPM6A viral mutant. As the magnitude, polyfunctionality, and T-cell repertoire diversity are potential determinants of the protective efficacy of CD8(+) T-cell responses, our data have implications for the development of vaccines to combat virus mutants.     

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.     

Homogenization of TCR repertoires within secondary CD62Lhigh and CD62Llow virus-specific CD8+ T cell populations

Influenza virus-specific CD8(+) T cell clonotypes generated and maintained in C57BL/6J mice after respiratory challenge were found previously to distribute unequally between the CD62L(low) "effector" (T(EM)) and CD62L(high) "central" (T(CM)) memory subsets. Defined by the CDR3beta sequence, most of the prominent TCRs were represented in both the CD62L(high) and CD62L(low) subsets, but there was also a substantial number of diverse, but generally small, CD62L(high)-only clonotypes. The question asked here is how secondary challenge influences both the diversity and the continuity of TCR representation in the T(CM) and T(EM) subsets generated following primary exposure. The experiments use single-cell RT-PCR to correlate clonotypic composition with CD62L phenotype for secondary influenza-specific CD8(+) T cell responses directed at the prominent D(b)NP(366) and D(b)PA(224) epitopes. In both the acute and long-term memory phases of the recall responses to these epitopes, we found evidence of a convergence of TCR repertoire expression for the CD62L(low) and CD62L(high) populations. In fact, unlike the primary response, there were no significant differences in clonotypic diversity between the CD62L(low) and CD62L(high) subsets. This "TCR homogenization" for the CD62L(high) and CD62L(low) CD8(+) populations recalled after secondary challenge indicates common origin, most likely from the high prevalence populations in the CD62L(high) central memory set. Our study thus provides key insights into the TCR diversity spectrum for CD62L(high) and CD62L(low) T cells generated from a normal, unmanipulated T cell repertoire following secondary challenge. A better understanding of TCR selection and maintenance has implications for improved vaccine and immunotherapy protocols.     

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.     

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.     

T cell repertoire diversity is required for relapses in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

Comparison of TCRalphabeta repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT(-/-)) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRalphabeta repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT(+/-) and TdT(-/-) mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Valpha-Jalpha and Vbeta-Jbeta rearrangements in both strains. However, whereas TdT(+/+) and TdT(+/-) mice undergo successive EAE relapses, TdT(-/-) mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT(+/-) mice, new public Valpha-Jalpha and Vbeta-Jbeta rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4(+) T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRalphabeta repertoire diversification contributes to autoimmune progression.