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The Journal of Immunology, 1999, 162: 3905-3914.
Copyright © 1999 by The American Association of Immunologists

Selection of CD8+ T Cells with Highly Focused Specificity During Viral Persistence in the Central Nervous System1

Norman W. Marten*, Stephen A. Stohlman*,{dagger}, Wendy Smith-Begolka{ddagger}, Stephen D. Miller{ddagger}, Emmanuel Dimacali*, Qin Yao*, Sheldon Stohl*, Joan Goverman§ and Cornelia C. Bergmann2,*,{dagger}

Departments of * Neurology and {dagger} Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033; {ddagger} Immunobiology Center, Northwestern University Medical School, Chicago, IL 60601; and § Department of Molecular Biotechnology and Immunology, University of Washington, Seattle, WA 981195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR {alpha}/ß-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-{gamma} enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8+ CTL constitute a potent immune defense mechanism during most primary viral infections. Activation, expansion, and expression of effector function is driven by TCR recognition of viral peptides presented by MHC class I expressed on the surface of infected cells (1, 2). Peptide specificity is conferred by the highly polymorphic complementarity-determining region 3 (CDR3)3 domains of both the TCR {alpha}- and ß-chains comprising the V, D, and J regions (3, 4, 5). The induction of strong polyclonal T cell responses during viral infections involves varied TCR usage correlating to broad specificity for their cognate epitope (6, 7). Although a broad CTL response is generally thought to be superior in containing virus spread and providing subsequent protection, CTL with extremely limited TCR usage can also be effective in controlling viral replication (8). However, the dominance of distinct TCR clonotypes may drastically alter the pathogenesis of disease, supporting a correlation between TCR phenotype, target cell avidity, and disease outcome (8, 9, 10).

Concomitant with a reduction in viral load, the vast majority of activated CTL undergo apoptosis, thereby limiting tissue destruction and potential initiation of autoimmune responses (11, 12). A small proportion of virus-specific CD8+ T cells become memory cells, capable of more rapid activation and expansion than naive CTL precursors (CTLp) upon re-encounter with Ag (12, 13). Little is known about the relationship between T cells activated during acute infection and the memory pool. Both differential activation states and different T cell subsets have been implicated in the selection of CD8+ T cells into memory (12, 14, 15). The deterministic model, one of two basic models proposed by Müllbacher and Flynn (15), predicts that the ratios of CTLp frequencies to distinct epitopes in memory are identical with the primary response. CTLp undergo an initial division, leading to one activated cell that undergoes polyclonal expansion. The other daughter cell, destined to become a memory CTL, remains refractory. By contrast, in the stochastic model some activated CTL from the expanded polyclonal response lose the activated phenotype and become memory CTL (15). In this case the relative frequencies of T cells entering into memory is influenced by TCR and microenvironmental parameters and thus is not necessarily representative of the original CTLp frequencies. Analysis of antitumor and Listeria monocytogenes-specific CD8+ T cells suggest that memory populations maintain diversity similar to that observed in primary CTL (16, 17). By contrast, studies with lymphocytic choriomeningitis virus (LCMV) and EBV indicate that distinct selective pressure is placed on cells that enter into memory (18, 19, 20, 21). One factor potentially affecting the diversity of CD8+ memory T cells is persisting Ag found during many chronic viral infections (12, 20, 21).

The acute and persistent infection caused by the neurotropic JHM strain of the mouse hepatitis virus (JHMV) provides a model to analyze reactivity and selection of CTL populations in the presence of low Ag levels confined to the central nervous system (CNS). During acute JHMV infection, CTL provide a critical component in reducing viral titers as shown by clearance of infectious virus before the appearance of neutralizing Ab (22). Furthermore, cytolytic activity can be detected ex vivo from CNS-derived cells, but not from cervical lymph nodes or splenocytes (23, 24), suggesting that most virus-specific CTL traffick to the CNS during acute infection. Despite this potent local effector function, persistent infection is established in the CNS associated with chronic ongoing primary demyelination (25). Although infectious virus can generally not be isolated from mice following 14 days postinfection (p.i.), viral RNA and in some cases viral Ag can still be detected for at least 1 yr, suggesting low levels of ongoing viral replication (23, 26). Furthermore, adoptive transfer of virus-specific CTL provides protection against lethal JHMV challenge by clearing virus from most CNS cell types with the exception of oligodendrocytes (27). The observation that these CTL-protected recipients do not develop chronic demyelination suggests that the kinetics of CTL effector responses and viral replication play a pivotal role in determining the persistent state and possibly maintaining preferential CD8+ T cell subsets.

The dominant CTL response to JHMV infection in BALB/c (H-2d) mice is specific for an Ld-restricted epitope comprising residues 318–326 of the nucleocapsid (N) protein (28). CD8+ T cell fine specificity was monitored by comparing splenocytes and brain-derived T cells from acutely and persistently infected mice for recognition of variant N epitope peptides containing single amino acid substitutions at the TCR contact residues (29). Increased specificity for wild-type (wt) peptide of splenocytes from persistently infected mice compared with immunized mice that had cleared Ag indicated CD8+ T cell selection at the polyclonal, systemic level. Reactivity patterns of N-specific CTL clones established from the CNS of both acutely (CTLac) and persistently (CTLper) infected mice suggested selection of T cell subsets during persistence at the site of infection. CTLac clones expressed TCRs capable of recognizing a broad range of substitutions within the TCR contact residues. By contrast, CTLper clones exhibited highly focused specificity for the wt N epitope. Sequence analysis of TCR {alpha}- and ß-chain CDR3 regions revealed limited, but diverse, TCR usage correlating with distinct recognition patterns. In vivo focusing of the T cell response was confirmed by ex vivo IFN-{gamma} ELISPOT analysis of CNS mononuclear cells. Although the number of T cells within the CNS capable of secreting IFN-{gamma} in response to the N epitope declined during persistence, the overall Ag specificity of the responding T cell population was considerably more focused compared with that of the population responding during acute disease. These data provide the first evidence for selective survival of CD8+ T cells with highly focused reactivity during a persistent CNS infection derived from an initial population exhibiting diverse cross-reactivities during the primary immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and virus strains

Male BALB/c (H-2d) mice were purchased from The Jackson Laboratories (Bar Harbor, ME) at 6 wk of age and certified naive prior to mouse hepatitis virus (MHV) exposure. Mice were housed in an accredited animal facility at the University of Southern California and infected within 1 wk of arrival. Immune mice were produced by i.p. injection of 5 x 106 plaque-forming units of plaque-purified DM isolate of JHMV (30). For analysis of the uncloned CTL population, persistent CNS infections were induced by intracranial injection of 32 µl containing 500 plaque-forming units of the reduced virulence 2.2v-1 virus as previously described (31). Clonal CTL populations were derived from mice persistently infected with the low virulence plaque-purified DS isolate (30) as described below. Sequence analysis of all viruses used show the identical conserved sequence within the immunodominant Ld-restricted epitope. Viruses were propagated and quantitated by plaque assay using the murine DBT astrocytoma cell line as previously described (30).

Isolation of CNS-derived and splenic CD8+ T cells

CTL clones were derived from the CNS of mice at 6 days (acute phase) or 46 days (persistent phase) p.i. as previously described (24). Briefly, brains were removed and pressed through 200-µm Nitex gauze (Teckom, Lancaster, PA). Cells were isolated from the 30–68% interface of Percoll (Pharmacia, Uppsala, Sweden) step gradients, washed twice in RPMI 1640 medium, and resuspended in RPMI 1640 plus 5% FCS. Adherent cells were removed by incubation on plastic petri dishes for 2 h at 37°C. Nonadherent cells were washed and cultured with syngeneic irradiated spleen cells infected with JHMV in Iscove’s modified DMEM supplemented with 10% FCS. CD8+ T cell lines were generated by limiting dilution after three or four cycles of restimulation on JHMV-infected splenic feeder cells following CD4+ T cell depletion (32). The characteristics of the CTLac lines derived from the CNS during acute infection have been previously described (24). Clonal populations derived from a single limiting dilution use the same nomenclature as the original description, i.e., B8 (24), and clones derived from a second limiting dilution were designated by an additional numeric designation, i.e., B8.5. Following expansion, clones were maintained by weekly passage in 24-well plates with RPMI medium supplemented with 2 mM glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M ß2-ME, nonessential amino acids (RPMI complete), 10% FCS, and 10% supernatant from Con A-stimulated rat spleen cells (RCS) with irradiated syngeneic spleen cells and 1 µM N peptide. Clonal CTLper were derived as described above. All CTL clones were tested for TCR usage and specificity following two limiting dilutions.

CNS-derived mononuclear cells used in the IFN-{gamma} ELISPOT assays were isolated from the brains of infected mice as described above for CTL clones with the modification that cells were suspended in 30% Percoll and pelleted onto 1 ml of 70% Percoll. This was followed by two washes in RPMI 1640 supplemented with 2.5 mM HEPES and resuspension in Iscove’s medium supplemented with 2 mM glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M ß2-ME, nonessential amino acids (Iscove’s complete), and 10% FCS.

For bulk CTL assays, splenocytes (8 x 107) taken from immunized mice at least 21 days after peripheral infection (immune mice) or from i.c. infected mice at 30 days p.i. (persistent phase) were stimulated in vitro with 1 µM N peptide in RPMI complete supplemented with 10% FCS and 5% RCS and assayed at 6–7 days poststimulation. No evidence for persistence following i.p. infection was detected by PCR of RNA samples extracted from the liver or CNS of immunized mice.

CTL assays and synthetic peptides

CTL assays were performed as previously described (28). Briefly, syngeneic (H-2d) J774.1 or BC10 ME target cells were labeled with 100 µCi of Na51CrO4 (New England Nuclear, Boston, MA) at 37°C for 1 h and washed three times before use. Peptides were incubated at 37°C with target cells (1 x 104) in 96-well plates at concentrations ranging from 10-13–10-6 M for 30 min before addition of CTL. Bulk CTL were tested at an E:T cell ratio of 25:1, and CTL clones at an E:T cell ratio of 5:1. After 4 h of incubation, 100 µl of supernatant was removed, and specific 51Cr release was determined. Specific lysis was defined as 100 x (experimental release - spontaneous release)/(detergent release - spontaneous release). Maximum spontaneous release values were <25% of the total detergent release values in all experiments.

The 9 mer peptide pN318–326 (APTAGAFFF) as well as variants containing individual substitutions at positions (p) 1, 7, and 8 were purchased from Chiron Mimotopes (Clayton, Australia). Peptides were solubilized at approximately 1 mM in DMSO and diluted in PBS as previously described (29).

Anti-CD8 Ab-mediated inhibition of target lysis by CTL

A requirement for CD8 costimulation was determined by analyzing target lysis in the presence of anti-CD8 mAb (33). Hybridomas TIB210 and TIB227 secreting mAbs directed against either murine CD8 (2.43) or an isotype control dendritic cell marker (33D1), respectively, were obtained from American Type Culture Collection (Manassas, VA). T cell clones (5 x 104) in 50 µl of RPMI 1640 medium containing 4% FCS were added to 100 µl containing serial three- or fourfold mAb dilutions in RPMI 1640 medium in 96-well plates. Following preincubation for 20 min at room temperature, 1 x 104 51Cr-labeled J774.1 target cells coated with 100 nM pN peptide were added. Supernatants were harvested as described above for CTL assays. The percent inhibition of specific lysis was calculated as 100 x [1 - (specific lysis with mAb/specific lysis without mAb)].

RT-PCR and sequencing of V{alpha}- and Vß-chains

T cell clones were separated from feeder cells by centrifugation using Lympholyte M (Accurate Chemical & Scientific Corp., Westbury, NY). Total cellular RNA from 3 to 4 x 106 cells was prepared as previously described (34) and resuspended in diethylpyrocarbonate-treated water. cDNA was synthesized using oligo(dT) primers and AMV RT (Promega, Madison, WI). V{alpha}- and Vß-chains for each clone were assigned by PCR amplification using a 5' primer specific for each member of the Vß or V{alpha} gene families and a constant antisense 3' primer specific for the Cß or C{alpha} constant regions, respectively (35, 36, 37) (Life Technologies (Gaithersburg, MD) or Integrated DNA Technologies (Coraville, IA)). PCR amplification was conducted in 30–35 cycles as previously described (35, 36, 37). In samples giving rise to more than a single PCR product, PCRs were monitored at limiting reaction cycles, and only prominent products were selected for sequence analysis. Sequence analysis of the CDR3 regions was performed in both directions on PCR products using the SequiTherm system (Epicentre Technologies, Madison, WI) and respective V{alpha}, Vß, or C region primers. Sequence data was then used for assignment of J{alpha} and Jß usage based on analysis of database searches (GenBank).

FACS analysis

Vß-chain assignments for individual clones were confirmed using FITC-conjugated rat anti-TCR mAb specific for Vß6 (RR4-7), Vß7 (TR310), and Vß8.1,8.2 (MR5-2; PharMingen, San Diego, CA). Expression of CD8 was confirmed by staining with rat anti-CD8 mAb 31 M (PharMingen, San Diego, CA). Cells were labeled, washed, resuspended in PBS containing 0.1% FCS, and analyzed by flow cytometry on a FACStar (Becton Dickinson, Mountain View, CA).

IFN-{gamma} ELISPOT assays

ELISPOT assays to measure the frequency of Ag-specific IFN-{gamma}-secreting cells were conducted as previously described (38). Briefly, 96-well plates supporting cellulose ester membranes (MultiScreen HA, Millipore, Bedford, MA) were coated with 10 µg/ml R4–6A2 mAb (PharMingen) in PBS overnight at 4°C. Nonspecific binding was blocked by incubation with RPMI 1640 medium containing 10% FCS. Serial 2.5-fold dilutions of brain mononuclear cells were plated in duplicate and stimulated with irradiated (25 Gy) splenocytes from naive mice (4 x 105/well) in the presence or the absence of 1 µM, 100 nM, and 10 nM peptide. EL-4 supernatant was added as a source of IL-2 to a final 2.5% concentration, and cultures were incubated for 40 h at 37°C. Bound IFN-{gamma} was detected by 8-h incubation at 4°C with biotinylated anti-IFN-{gamma} mAb (5 µg/ml; XMG1.2, PharMingen), followed by consecutive incubations with streptavidin/peroxidase (Sigma, St. Louis, MO) and diaminobenzidene as a substrate (Sigma). Spots from two mononuclear cell dilutions (n = 4) were counted at each peptide concentration.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Splenic CTL exhibit increased specificity for the wt viral epitope during persistence

The dominant CTL response in JHMV-infected BALB/c (H-2d) mice is specific for the highly conserved epitope amino acids (aa) 318–326 (APTAGAFFF) of the viral N protein (28, 29). This response effectively controls viral replication during primary infection, and adoptive transfer of N-specific CTL protects against lethal infection; however, despite the presence of protective CTL, viral RNA and rarely detectable Ag continue to persist (23, 26, 27). To initially determine whether persisting Ag in the CNS influences the peripheral N-specific CD8+ T cell population, CTL reactivities of splenocytes from persistently infected mice were compared with those in mice that had undergone a self-limiting peripheral infection following JHMV administration i.p. Splenocytes from these two groups of mice, either infected i.c. 30 days previously or immunized i.p., were stimulated in vitro with wt N peptide and assayed for recognition of variant peptides as a measure of TCR diversity. Spleens, as opposed to the CNS, were initially chosen as a source of CTL. Chronically infected mice exhibit less CNS T cell infiltration compared with acutely infected animals (39) and are therefore unsuitable for isolating sufficient polyclonal T cells from the CNS for peptide fine specificity by analysis in a 51Cr release assay. Viral infections generally induce polyclonal CTL (6) in which TCR diversity is reflected by distinct fine specificities among CTL clones for variants of the wt peptide. The dominant and subdominant TCR contact residues of the pN 9 mer have previously been mapped to p7 and p8, respectively (29), whereas p2 and p9 were confirmed as Ld binding sites (40). It appears that p1, p3, p4, p5, and p6 do not directly affect N-specific CTL recognition (28, 29). However, p1 is partially exposed to solvent (40, 41) and potentially could be involved in TCR interactions. As peptides with substitutions at subdominant TCR contact sites reveal the greatest differences in TCR reactivity (42, 43), N peptides with substitutions in p8, occupied by a phenylalanine (F) in all MHV strains (28), were chosen to detect differential reactivities at the polyclonal level.

Dose-response curves for the wt and each variant peptide were established, and the concentration required to achieve 50% maximal specific lysis was used as an index for CTL recognition. Peptide recognition was tested at concentrations ranging from 10-13 to 10-6 M at a constant E:T cell ratio. Analysis was limited to substitutions predominantly comprising amino acids with relatively conserved, hydrophobic side-chains (Fig. 1Go). CTL derived from immunized mice recognized and lysed target cells coated with all peptides containing p8 substitutions at the same levels as the wt peptide, with the exceptions of slightly weaker recognition of leucine (L) and proline (P; Fig. 1GoA). These data indicate a broad memory response capable of recognizing a wide range of amino acids at the subdominant TCR contact site. By contrast, splenic CTL isolated from persistently infected mice were more restricted in their ability to recognize p8 substitutions (Fig. 1GoB). This is evidenced by the 10- to 1000-fold higher peptide concentrations compared with those of wt peptide required to achieve 50% maximal specific lysis. It is noted that these differences in reactivity are exhibited following expansion of N-specific T cell populations in the presence of wt peptide and IL-2, conditions that minimize a bias toward T cell subsets due to activation state, avidity, or frequencies. Furthermore, a similar analysis of splenocytes from mice infected i.p. 7 days previously revealed no evidence for differential reactivities between primary and memory CTL (data not shown). One hypothesis for the increased specificity of CTLper is that a limited subset derived from a diverse primary CTL population is selected during persistence, possibly based on TCR avidity and/or the differentiation state at the peak of the response.



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  		FIGURE 1. Recognition of N peptides with amino acid substitutions at the subdominant TCR contact residue (p8) by in vitro stimulated splenocytes isolated from either JHMV-immune (A) or persistently infected (B) mice. Splenocytes were assayed at an E:T cell ratio of 25:1 for lysis of BC10 ME target cells coated with peptides comprising either the wt N epitope sequence (wt) or variants containing p8 substitutions. The index amino acids and substitutions are indicated by the single letter code. Each peptide was titrated over a concentration range of 10-6–10-13 M in 10-fold dilutions, and the concentration giving rise to half-maximal lysis (30–40%) achieved for wt peptide was plotted in a bar graph format.

 
CTLper clones express longer CDR3 domains than CTLac clones

Differential fine specificities of polyclonal CD8+ populations from splenocytes during memory and persistence provided evidence for functional maturation of the CTL response at the site of infection. CTL clones established from the CNS during the acute phase (day 6) and during persistence (day 46) were examined to correlate functional reactivity with TCR clonotypes. All lines were initially expanded on JHMV-infected feeders to minimize bias toward the N epitope. The vast majority of CTLac lines (23) and all CTLper lines from infected brains were specific for the wt N epitope (data not shown), consistent with its immunodominant nature. To investigate T cell clonality during both disease phases, TCR {alpha}- and ß-chain CDR3 sequences were determined for seven CTLac and CTLper clones (Table IGo). Both groups displayed limited TCR {alpha}/ß-chain diversity, with a bias toward Vß6 and Vß7 expression. However, four of seven CTLac clones revealed a unique clonotype, indicating a polyclonal response during primary infection. CTLac clones B8.5, B11.4, and B9.5 shared identical TCR {alpha}/ß-chains. By contrast, B6.1 and B21.4 expressed identical ß-chains but different {alpha}-chain J regions, J{alpha}44 and J{alpha}42, respectively. CTLac lines B15 and B17 were double positive for V{alpha} usage (V{alpha}19, V{alpha}8DEG) and therefore were not pursued further. In addition, two other N-specific CTLac lines, B2 and B3, were found to express unique TCR {alpha}-chains (V{alpha}9,J{alpha}41 and V{alpha}8DEG,J{alpha}23, respectively), although we were unable to positively identify their ß-chain usage (data not shown). The CTLper panel exhibited two TCR clonotypes, Vß7, Jß2.1, V{alpha}16, J{alpha}42 and Vß6, Jß1.1, V{alpha}8DEG, J{alpha}44, respectively. The fact that none of the TCR {alpha}/ß-chain combinations identified in the CTLac clones was found in the CTLper clones possibly reflects a selection process or the limited number of clones analyzed. As the clones were isolated from pooled brain homogenates of several mice, these data indicate that the CTL response to N is dominated by TCR chains Vß6 or Vß7 paired with V{alpha}8, V{alpha}9, V{alpha}16, or V{alpha}19. Furthermore, CTLper clones appeared to have longer CDR3 regions in both TCR chains compared with CTLac clones (Table IGo). Whereas the {alpha}- and ß-chains from CTLac clones comprised CDR3 regions of 8–10 aa and 8 or 9 aa, respectively, CTLper clones expressed {alpha}- and ß-chain CDR3 regions of 10 or 11 aa and 9 or 10 aa, respectively. These data support potential differences observed in the polyclonal CTL response over the course of a chronic disease resulting from either selective survival of CTL subsets after viral clearance or selective expansion during persistence.


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  		Table I. CTL clone TCR {alpha}- and ß-chain usage1

 
CTLac clones display promiscuous recognition of substitutions at the subdominant TCR contact site, whereas CTLper clones accommodate little diversity

To determine whether differential TCR usage correlated with reactivity, N peptide variants with single substitutions at sites exposed to the TCR or solvent were used as probes to analyze CTL reactivity, thereby providing functional TCR fingerprints. Unique CTLac clones recognized a broad array of p8 substitutions (Fig. 2GoA). The greatest negative effects on recognition were exerted by amino acids with small side chains, e.g. glycine (G) or alanine (A), with acidic (glutamic (E) and aspartic acid (D)) or basic (lysine (K) and arginine (R)) residues. Substitutions exerting a notable exception was recognition of the histidine (H)-substituted peptide by one clone. This may be attributed to the aromatic moiety in H, indicating that the overall size of the side chain may be more relevant than charge for proper conformational fit of the CDR3 domain. Despite similar reactivities to the wt epitope, the clones varied significantly in the ability to recognize p8-substituted peptides. Whereas clone B8.5 recognized most substitutions, clone B6.1 was less promiscuous, with an intermediate reactivity. Clone B21.4 exhibited the highest stringency and also required higher peptide concentrations to achieve comparable lysis (e.g., methionine (M) and L substitutions; Fig. 2GoA). The wide range of reactivities to pN variants among the CTLac clones demonstrates that not only is diversity found at the TCR structural level, but it is also distinctly pronounced at the functional level.



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  		FIGURE 2. Effect of amino acid substitutions at the subdominant TCR contact residue (p8) on recognition by CTLac (A) or CTLper clones (B). CTLac clones B6.1, B8.5, or B21.4 (A) or CTLper clones 1.5.1, 2.10.4, or 2.10.8 (B) were assayed at an E:T cell ratio of 5:1 for lysis of J774.1 target cells coated with peptides comprising either wt N epitope or variants containing p8 substitutions as described in Fig. 1Go. The concentration of wt peptide required for half-maximal lysis by CTLac clone B21.4, chosen as a standard for each assay, is indicated by a dashed line. CTLac clone B21.4 was included in assays using CTLper clones to account for variable CTL sensitivities due to culture conditions at any given time. Data shown are from a single assay representative of at least two independent experiments.

 
To determine whether the diversity of substitutions recognized by CTLac is maintained during persistence, clones from persistently infected mice were tested for recognition of the p8-substituted peptides (Fig. 2GoB). Only the tryptophan (W) substitution was recognized at a level comparable to that in wt peptide. Neither the aromatic tyrosine (Y) nor the hydrophobic isoleucine (I), L, or M, all recognized by the CTLac clones (Fig. 2GoA), triggered responsiveness by CTLper clones (Fig. 2GoB). Although CTLper clone 1.5.1 weakly recognized the M substitution, no other substitutions were recognized. Narrowly focused recognition by the CTLper clones compared with that by the CTLac clones suggest that the initial heterogeneous response during primary CTL expansion may undergo subsequent selection in the CNS for TCR usage with higher specificity.

The data in Fig. 2Go are from representative experiments performed independently at differing times for either group. To accommodate fluctuations in the peptide sensitivities of CTLac and CTLper clones, clones from either group were tested in parallel for recognition of target cells coated with increasing concentrations of wt pN peptide (Fig. 3Go). Half-maximal lysis for representative CTLac and CTLper clones was achieved at ~10-11 M. These results were confirmed with all other clones tested, independent of disease status (data not shown), suggesting similar reactivities when clones were grown and assayed simultaneously. CTLac clone B21.4 was therefore included for reference in each assay. The apparently lower reactivity of CTLper clones compared with CTLac clones (Fig. 2Go, A and B) therefore does not reflect lower target cell avidity, but, rather, indicates variabilities in culture conditions during clonal propagation.



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  		FIGURE 3. Ag sensitivity of CTL clones derived from the acute and persistent stages of CNS infection by JHMV. Representative CTL clones derived from either the acute phase (B21.4) or the persistent phase (1.5.1) of infection were tested for recognition of N peptide at concentrations ranging from 10-6 to 10-13 M on J774.1 target cells. Data shown are from a single experiment representative of three or four independent assays.

 
Limited recognition of amino acid substitutions at the dominant TCR contact position

F also occupies the dominant TCR contact site of the N epitope at p7 in all MHV strains (28). As substitutions of main TCR contact residues often result in complete loss of CTL recognition (42, 43), only limited substitutions at this position were anticipated to be permitted. Fig. 4Go confirms a restricted recognition pattern of p7 substitutions for both CTLac and CTLper clones. The only substitution recognized by CTLac clones was Y, while only a single clone also demonstrated a weaker response to the W substitution (Fig. 4GoA). CTLper clones exhibited a similarly restricted recognition pattern (Fig. 4GoB). Only 4 aa could replace F without completely abrogating recognition. Whereas Y or W substitutions were recognized as well as or better than the wt F residue, only weak responses to H and A substitutions at p7 were observed.



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  		FIGURE 4. Limited recognition of substitutions at the dominant TCR contact residue (p7) by either CTLac (A) or CTLper clones (B). CTL recognition of wt and p7-substituted peptides was determined in CTL assays as described in Fig. 2Go.

 
The extremely limited flexibility of both CTLac and CTLper clones with respect to the dominant TCR contact site, allowing only bulky aromatic side chains as replacements for F, confirms a high stringency imposed by F occupancy in this position. This stringent recognition pattern therefore did not reveal significant differences in either TCR specificity between CTLac and CTLper clones or among clones from within one group.

CTLper exhibit limited reactivity to p1 substitutions

Differential CTL fine specificity as a result of peptide-TCR interactions involving p1, which is exposed to solvent and available for TCR interactions in heterologous Ld binding peptides (40, 41), was also tested. The specificities of CTLac clones in response to p1 substitutions did not vary to the degree seen at p8 (Fig. 5GoA). All substitutions were recognized to some extent by all clones. The greatest negative effects on recognition were imposed by P, acidic (D and E), basic (H), and aromatic (W) amino acids substituting for the neutral A. Six substitutions resulted in less than a 1 log10 change in peptide concentration required for recognition (glutamine (Q), serine (S), valine (V), asparagine (N), M, and T) by any CTLac clone, and three substitutions required less than a 2 log10 change in concentration for 50% maximal lysis (K, R, and I). By contrast, CTLper clones were more confined in the recognition of p1-substituted peptides (Fig. 5GoB). The only efficiently recognized substitution was S, recognized at 2 log10 lower concentrations compared with wt peptide. Acidic residues as well as I, L, and F, all recognized by CTLac clones, completely prevented recognition by CTLper clones. All other substitutions also resulted in partial or complete loss of recognition. Although not readily apparent from examination of CTLac clones, CTLper reactivity revealed that p1 is involved in TCR recognition. Similar to the more focused specificity revealed by substitutions in the subdominant TCR contact site, these data suggest that the N-specific CTL population evolves to be more specific for the wt epitope during viral persistence.



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  		FIGURE 5. Differential effects of p1 substitutions in the N peptide on recognition by CTLac (A) or CTLper (B) clones. CTL recognition of wt and p1-substituted peptides was determined in CTL assays as described in Fig. 2Go.

 
CTL reactivity patterns are not correlated with CD8 dependence

A possible explanation for a more stringent response by CTLper may reside in higher target cell avidity. However, reactivity patterns of CTLper clones were not associated with higher sensitivity to the wt N peptide (see Fig. 3Go). An alternative, indirect assessment of relative TCR-peptide-MHC affinities takes advantage of CTL resistance to lysis inhibition by anti-CD8 mAb (33). CTL expressing low affinity TCRs are more sensitive to the presence of anti-CD8 mAb, resulting in reduced killing compared with CTL with high affinity TCR. This technique was employed to determine whether differences in epitope specificity observed between CTLac and CTLper clones correlated with TCR affinity (Fig. 6Go). Although CTLac clone B6.1 was much more promiscuous than CTLper clone 1.5.1 in its ability to recognize substitutions (compare Figs. 2Go and 5Go), the clones demonstrated similar avidities based on anti-CD8 interference (Fig. 6Go). Furthermore, CTLac clones B8.5 and B21.4 demonstrated marked differences in specificity (Figs. 2Go and 5Go), yet neither clone was significantly affected by anti-CD8 mAb compared with the isotype control (Fig. 6Go). Based on these data, no distinct correlation could be established between peptide fine specificity and TCR affinities.



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  		FIGURE 6. CD8 dependence of cytolytic effector function by CTLac and CTLper clones. Relative TCR affinities for the N epitope of CTLac (A) and CTLper (B) clones were assayed by inhibition of target lysis with serial 4-fold dilutions of either an anti-Lyt-2 specific Ab (filled diamonds) or an isotype control Ab (open diamonds). Data are presented as the percent inhibition of specific lysis in the presence of the indicated Ab compared with that in untreated CTL. The experiment shown is representative of three independent assays.

 
Increased Ag specificity of polyclonal CNS-derived T cells during persistence

Reactivity patterns of clones and restimulated polyclonal CTL may be influenced by preferential expansion of individual clonotypes during culture. The association between persistence and narrowly focused Ag specificity was therefore examined ex vivo. Mononuclear cells freshly isolated from the brain were analyzed for their ability to secrete IFN-{gamma} in response to peptide stimulation using ELISPOT assays (17, 38). To assess the relative frequency of Ag-specific CD8+ T cells in the CNS during acute and persistent infection, CNS-derived populations were initially stimulated with various concentrations of wt peptide (Fig. 7GoA). FACS analysis indicated that the percentage of CD8+ T cells in the CNS mononuclear cell population decreased modestly from approximately 15% during acute disease to approximately 10% during the persistent phase (data not shown). However, the frequency of Ag-responsive, IFN-{gamma}-secreting CD8+ T cells from the acutely infected CNS was almost fivefold higher than that of cells isolated from the persistently infected CNS (Fig. 7GoA). This difference was independent of peptide concentration, suggesting that the reduced number of CD8+ T cells retained following viral clearance demonstrated a similar sensitivity as that of cells isolated during acute infection.



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  		FIGURE 7. Effect of amino acid substitutions at the subdominant TCR contact residue (p8) on recognition by CD8+ T cells isolated from the CNS of mice during acute (day 6 p.i.) or persistent (days 32–35 p.i.) infection with JHMV variant 2.2v-1. Mononuclear cells isolated from brains of JHMV-infected mice during the acute (n = 5) and persistent phases (n = 7) were tested directly ex vivo in IFN-{gamma} ELISPOTs to determine the responsiveness to N peptides. A, Frequency of CD8+ T cells responding to stimulation with wt N-peptide at the indicated concentrations. The percentage of CD8+ brain T cells was determined by staining with mAb to CD8 followed by FACS analysis. B, Relative frequencies of brain mononuclear cells responding to peptide variants containing p8 substitutions compared with wt N epitope (wt)-responding cells. The index amino acid sequence and substitutions are indicated by the single letter amino acid code. Each peptide was titrated over a concentration range of 10-6–10-8 M. The number of cells secreting IFN-{gamma} in response to the altered peptides was normalized to the response to wt N peptide, which was set at 100%. Data shown are from a single assay representative of at least two independent experiments performed with mononuclear cells stimulated in the presence of 10-6 M peptides. Error bars indicate the SE determined for each dataset.

 
Provided persistence selects for CD8+ T cells with narrower specificity, it would be expected that the frequencies of T cells responding to mutant peptides are reduced compared with that of T cells responding to wt peptide during persistence. N peptides containing amino acid substitutions at p8 were therefore used in ELISPOT assays to assess differential reactivities ex vivo (Fig. 7GoB). During acute infection, all amino acid substitutions at p8 reduced the relative percentage of cells capable of responding compared with the response to wt N peptide. The frequencies of polyclonal responses to L-, M-, Y-, and W-substituted peptides were between 20–50% of that observed for wt peptide. More severe declines in the frequency of responding T cells from acutely infected animals occurred when the wt F at p8 was substituted with I or P (Fig. 7GoB). By contrast, T cells from the persistently infected animals revealed a notable loss in the percentage of cells responding to altered peptides containing the I, L, M, and W substitutions compared with the response of T cells isolated during acute disease. These results demonstrate that despite an overall decrease in CD8+ T cells responding to viral Ag within the brain during persistence (Fig. 7GoA), the ability to respond to Ag was preferentially retained in a population exhibiting a more restricted Ag specificity compared with that in T cells isolated during the primary response (Fig. 7GoB). The increased Ag specificity of polyclonal T cells isolated during persistent disease was readily apparent and was consistent with results obtained with cloned CTL (Fig. 2Go). Minor differences were observed comparing polyclonal CD8+ T cells and cloned CTL. Although the Y substitution was recognized by CTLac clones but not by CTLper clones, similar frequencies of ex vivo responding cells were observed for the p8 Y substitution during both phases of disease. These data clearly suggest that persistence, whether analyzed at the clonal or the polyclonal level is associated with selection of T cells with highly focused specificity in vivo.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of viruses establish persistent infections despite strong primary CD8+ CTL responses, e.g., EBV, measles virus, herpes simplex virus, and HIV in humans and Theiler’s murine encephalomyelitis virus, LCMV, and JHMV in mice (46). However, little is known about the relationship among viral replication, CTL recruitment, and activation states for resolution of virus infection during the acute phase and control of persistence. Similarly, the effects of residual Ag on T cells following viral clearance are poorly understood. Persisting viral genomes, even at barely detectable levels, have recently been demonstrated to result in the retention and/or expansion of activated CD8+ T cells in a tissue-specific manner (47). The extent to which the Ag-experienced T cells induced during primary infection undergo a transition to quiescent memory cells and/or chronically activated cells with effector function may thus be critical for maintaining a persistent state.

Persistent CNS infection by JHMV was used as a model to analyze whether persisting viral RNA, in the absence of detectable infectious virus, provides a sufficient stimulus to drive T cell selection/specificity. Increased specificity for the dominant wt epitope exhibited by bulk splenocytes from mice undergoing persistent CNS infection compared with immune mice provides evidence that low Ag levels, at least within a confined anatomical site, may constitute a selective force in limiting memory CTL diversity. Whereas splenic CTL from immune mice recognized most substitutions at the subdominant TCR contact residue at a level equal to wt peptide, CTL from persistently infected mice required much higher concentrations of variant peptides to achieve similar lysis. A selection process was more readily indicated by differential peptide fine specificities of CTL clones isolated from the CNS during either acute or persistent infection. CTLac clones exhibited broad flexibility with respect to recognition of peptide variants. By contrast, CTLper clones expressed TCRs with limited plasticity, as shown by a highly focused specificity for the wt epitope. Similar results were obtained by IFN-{gamma} ELISPOT assays using freshly isolated CNS mononuclear cells; reduced frequencies of cells responding to altered peptides compared with wt peptide indicated preferential retention of T cells with restricted specificity during viral persistence in vivo. Selection of CTL with increased specificity for the wt epitope was therefore evident not only at the clonal level at the site of infection, but also at the population level both at the site of infection as well as in peripheral lymphoid organs.

TCR analysis confirmed that differential fine specificities reside within the diverse TCR {alpha}/ß-chains expressed by CTLper and CTLac clones. Limited TCR flexibility appears to correlate with longer CDR3 domains preferentially interacting with large amino acid side chains at TCR contact residues. TCR recognition of aromatic Y or W and, to a lesser degree, H substitutions for F at p7 indicated a minimal size requirement for amino acid side chains at the dominant TCR contact position. A similar bias for large amino acid side chains was evident for the subdominant TCR contact residue at p8; a W substitution was the only p8 variation recognized by CTLper clones and was one of the few recognized by CTLac clone B21.4. In fact, clone B21.4, containing the longest CDR3 domains among CTLac clones, displayed the most limited recognition pattern. By contrast, CTLac clone B8.5, with the shortest CDR3 domains, exhibited the greatest promiscuity. These data may be consistent with a deep TCR pocket surrounding p8, as revealed by recent structural analysis of two distinct human TCRs (5). The pocket size determined by CDR3 length may thus dictate the loss or gain of contact sites for heterologous substitutions. Highly focused CTL reactivity to the wt N-epitope during persistence appears to be determined by preferential selection/survival of populations characterized by the distinct sizes of the TCR CDR3 domains. This phenomenon may, however, be unique to individual TCR/pep-MHC interactions as indicated by the CDR3 length-independent peptide promiscuity exhibited by the TCR repertoire specific for the Db-restricted influenza virus epitope (48).

Although increased TCR specificity implies a correlation with higher class I/peptide affinity, such a relationship could not be confirmed by either peptide sensitization studies or CD8 dependence. However, in the absence of more direct affinity measurements, the present data cannot rule out that T cell subsets with higher target avidity are maintained by low level Ag stimulation during persistence. Indeed, selective expansion of high vs low avidity CTL by in vitro stimulation with minimal or abundant peptide, respectively, supports this concept (9, 10). Alternatively, lower avidity T cells may be more likely to survive a decline of the acute response, as highly activated T cells are more prone to apoptosis (11). Whether selection takes place during the apoptotic phase or during Ag-driven restimulation of distinct memory cell populations is unclear, as memory T cell subsets can generally only be characterized following Ag restimulation. However, the involvement of Ag as a driving force for tuning memory T cell reactivity is implicated by monitoring T cell responses following in vivo restimulation as well as during persistent infections (18, 19, 20, 21, 49). Enrichment of T cell subsets following secondary Ag exposure in vivo has been demonstrated in both the CD4+ and CD8+ T cell compartments (18, 20, 49). For example, mice immunized with pigeon cytochrome c mount a CD4+ T cell response limited to a single V{alpha}11/Vß3+ TCR (49). Following primary exposure to Ag, activated V{alpha}11/Vß3+ T cells exhibit a distinct preference for a single CDR3 length and for specific amino acids within the J and D regions. However, a secondary Ag exposure resulted in exclusive activation of this distinct CDR3 T cell subset. Similarly, enrichment of CD8+ T cell subsets was observed in mice immunized and subsequently challenged with LCMV (18). Consistent with our observations, CTL responding to the initial LCMV immunization expressed broad reactivity for altered peptides, whereas memory T cells demonstrated significantly higher specificity for the wt epitope (18). Furthermore, T cell selection in the presence of persisting Ag is suggested by restricted amino acid usage within the CDR3 region of TCR derived from CTLp cells with diverse cDNA sequences (21) as well as changes in the ratios of distinct EBV-specific CTL subsets during acute infectious mononucleosis and persistence (20). In this model as well as ours, selected T cells may be preferentially expanded in vivo due to chronic Ag stimulation. Highly focused specificity associated with the selection of CTL with distinct TCR CDR3 regions during persistent JHMV infection link the previously independent observations of a more focused response (18) with selective TCR enrichment (49).

One interpretation of the highly focused response during persistence is that high local Ag present in the CNS during the acute phase allows expansion of CTL expressing diverse TCRs with a broad range of fine specificities, independent of affinity. However, as viral load decreases, only CTL with higher specificity/affinity may be able to engage the limited supply of MHC-bound peptide, thus avoiding apoptosis. This is supported by the observation that CNS infection with Theiler’s virus leads to nonspecific T cell trafficking into the brain and subsequent expansion of reactive T cells at the site of infection (50). During acute JHMV infection, local expansion/activation are also implicated by highly activated CTL with ex vivo cytolytic activity in the CNS, but not in peripheral lymphoid organs (24, 51). Similarly, CNS inflammation during experimental allergic encephalomyelitis is associated with the initial infiltration of both Ag-specific and nonspecific T cells, followed by preferential retention of Ag-specific T cells (52). T cell retention at the site of Ag further infers that selection of T cells with enhanced specificity may be more evident in the CNS as a sequestered site of infection (47). Indeed, chronic Ag stimulation in the CNS below conventional detection thresholds is associated with increased levels of CD8+ T cell infiltration/retention for up to 1 yr (47, 53).

Among the mechanisms implicated in contributing to JHMV persistence is the refractory nature of infected oligodendrocytes to CTL-mediated lysis in vivo (27). Furthermore, virus evolution may lead to attenuated phenotypes or escape from T cell recognition (54). However, survivors of acutely infected adult mice show no evidence for mutations within the N epitope (55), implying that persisting viral Ag may play a role in maintaining a subset of activated Ag-specific CD8+ T cells following viral clearance, as recently demonstrated for a secondary influenza virus CNS infection (47). Our data indicate that Ag-experienced T cells during CNS persistence represent a restricted subset of primary CTL, distinguished by more focused epitope specificity. Whereas initial broad CTL reactivity provides a mechanism to prevent viral CTL escape mutants during acute infection (54, 55), the biological significance of the apparent selection of CTL with high specificity during persistence remains to be investigated. Highly focused "memory" CTL may provide for a rapid response upon reinfection or re-emergence of the infectious agent, while reducing the chances of inducing cross-reactive and possibly self-reactive CTL. More provocative is the idea that persisting RNA may provide sufficient stimulation to activate selected CD8+ T cell effector functions (47), thus not only keeping virus replication at undetectable levels within the CNS but also contributing to chronic inflammation.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS18146, AI33314, NS07149, NS30571, NS23349, and NS535126. Back

2 Address correspondence and reprint requests to Dr. Cornelia C. Bergmann, 1333 San Pablo St., MCH 142, Los Angeles, CA 90033. E-mail address: Back

3 Abbreviations used in this paper: CDR3, complementarity-determining region 3; CTLp, cytotoxic T lymphocyte precursor; LCMV, lymphocytic choriomeningitis virus; CNS, central nervous system; JHMV, JHM strain of mouse hepatitis virus; p.i., postinfection; N, nucleocapsid protein; aa, amino acid; wt, wild type; CTLac, cytotoxic T lymphocytes isolated during acute infection; CTLper, cytotoxic T lymphocytes isolated during persistent infection; ELISPOT, enzyme-linked immunospot; MHV, mouse hepatitis virus; i.c. intracranial; RCS, rat concanavalin A supernatant; p, amino acid position within peptide. Back

Received for publication July 10, 1998. Accepted for publication January 4, 1999.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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