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The Journal of Immunology, 1998, 161: 1094-1103.
Copyright © 1998 by The American Association of Immunologists

Novel Diversity in Th1, Th2 Type Differentiation of Hemagglutinin-Specific T Cell Clones Elicited by Natural Influenza Virus Infection in Three Major Haplotypes (H-2b,d,k)

Christine M. Graham, Claire A. Smith and D. Brian Thomas1

National Institute for Medical Research, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report novel diversity in the lymphokine (LK) secretion profile of hemagglutinin-specific, CD4+ T cell clones elicited by influenza virus infection in three major haplotypes: I-Ad- or I-Ed-restricted T cell clones obtained from individual BALB/c donors, and specific for three distinct antigenic peptides (p56–76, or p186–205 or p177–199), were uniformly Th1 type, releasing only IFN-{gamma} on activation. In contrast, extensive diversity was evident for the C57BL/10 or CBA/Ca repertoire. Sibling T cell clones, established from the same C57BL/10 donor and expressing identical TCR ß-chains in their recognition of p186–205, released either (IFN-{gamma} and IL-5) or (IFN-{gamma} and IL-4 and IL-5) or (IL-4 and IL-5 and IL-10) following Ag-specific or nonspecific stimulation. Similarly, I-Ak-restricted T cell clones, specific for p120–139 secreted either (IFN-{gamma} only) or (IFN-{gamma} and IL-5) or (IFN-{gamma} and IL-2 and IL-5) on activation. Despite such phenotypic diversity within the individual’s repertoire, all clones had been maintained under identical in vitro culture conditions. Moreover, sequence analyses of TCR ß gene usage indicated that in most instances clones from the same donor expressed identical (VDJ)ß rearrangements, indicative of a common progenitor cell. FACS analysis of cytoplasmic cytokine production confirmed that for the novel phenotype (IFN-{gamma} and IL-5), both LKs were synthesized at the single cell level. Sibling families of T cell clones, established from a common donor following viral infection but differing in LK secretion, may offer a suitable model system for further studies of signal transduction mechanisms that discriminate between Th1- and Th2-specific responses to a well defined protective Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The influence of host genetic background on the in vivo cellular immune response to infectious pathogens provides convincing support for the Th1, Th2 model of CD4+ T cell differentiation as initially indicated by in vitro studies of the lymphokine (LK)2 secretion pattern of Ag-specific CD4+ T cell clones following TCR ligation (1, 2, 3, 4, 5). Th1 cells secrete the inflammatory cytokines IL-2, IFN-{gamma}, and lymphotoxin and elicit delayed type hypersensitivity responses that are essential for combating intracellular pathogens. Th2 cells, on the other hand, secrete IL-4, -5, -10, and -13 and participate in humoral responses and protective immunity to extracellular pathogens. A primary paradigm for genetic regulation of Th1, Th2 development has been experimental infection of inbred and congenic mice with the intracellular protozoan, Leishmania major (6, 7, 8). The extreme susceptibility of BALB/c, or congenic BALB/b, mice to cutaneous leishmaniasis, as compared with most other inbred strains, has been attributed to disregulation of IL-4 production with resultant skewing of T cell responses to Th2 development and disease progression. Linkage studies have indicated segregation of disease susceptibility to a single gene, located on the distal end of chromosome 11 (9), although there is recent evidence for further gene involvement (10). Interestingly, studies by Murphy and colleagues (11), using TCR {alpha}ß transgenic mice on different genetic backgrounds (B10.D2 or BALB/c) and specific for an OVA peptide, have also mapped a single dominant locus controlling Th1, Th2 development to mouse chromosome 11. B10.D2 T cells, maintained in vitro in the absence of exogenous cytokines, had a greater capacity to maintain IL-12 responsiveness and Th1 development, while BALB/c T cells were biased to Th2 development.

The genetic background is also instrumental in regulating Th1, Th2 responses in allergic or autoimmune conditions. In the human, development of asthma and atopy is linked to a region on the long arm of chromosome 5, syntenic with mouse chromosome 11 (12, 13). Also, genetic differences have been reported in susceptibility to autoimmune disease induction in MHC-congenic strains (14) with a Th1 type response being required for the development of experimental autoimmune encephalomyelitis. Analysis of the I-Ak-restricted T cell responses of MHC-congenic B10.A and B10.BR mice to a peptide of myelin basic protein (15) showed that B10.BR T cells secreted Th1-type cytokines and induced experimental autoimmune encephalomyelitis on adoptive transfer, while B10.A T cells were biased to Th2-type development. Differences between B10.A and B10.BR T cells were attributed to the release of a novel inhibitory cytokine from B10.A APCs that inhibited IFN-{gamma} production.

It is now established that the cytokine environment, following initial activation of naive T cells, determines the Th1, Th2 developmental pathway (16, 17, 18, 19, 20, 21, 22, 23, 24); IL-12 and IFN-{gamma} promote Th1 type development and inhibit Th2 type responses. Conversely, IL-4 and IL-10 are critical for the induction of a Th2 phenotype and inhibit Th1 cytokine production. As a result, it is commonly assumed that IFN-{gamma} or IL-4 and IL-10 production are antithetic following polyclonal T cell activation.

Despite extensive in vivo studies of immunity to viral infection (25, 26, 27, 28, 29), indicative of a predominantly Th1-type response and skewing of the neutralizing Ab repertoire to the IgG2a isotype by IFN-{gamma}, there have been few reports of in vitro CD4+ T cell clonal analysis of LK status following natural viral infection of inbred and/or MHC-congenic mice. This has prompted us to examine the cytokine phenotype of hemagglutinin (HA)-specific CD4+ T cell clones, established from individual donors, either C57BL/10 (H-2b), or BALB/c (H-2d) or CBA/Ca (H-2k) mice several weeks after intranasal infection with X31 virus (H3N2 subtype), and therefore representative of the T cell memory repertoire. Moreover, CBA/Ca mice are particularly susceptible to X31 infection, with associated immunopathology, in contrast to most other inbred and MHC-congenic strains (30), and an initial aim was to determine whether disease susceptibility correlated with Th1, Th2 status.

We have found major differences in the LK secretion phenotype of HA-specific T cell clones established from these three strains of mice. Whereas T cell clones established from BALB/c mice had a typical Th1 phenotype and released exclusively IFN-{gamma} on antigenic stimulation, sibling T cell clones from the same C57BL/10 donor exhibited a complex pattern of LK secretion, either (IFN-{gamma} and IL-5) or (IFN-{gamma} and IL-4 and IL-5) or (IL-4, and IL-5, and IL-10), despite exhibiting identical rearrangements of their TCR (VDJ)ß genes. Interestingly, CBA/Ca mice with marked susceptibility to influenza X31 infection exhibited an intermediate phenotype insomuch as T cell clones from some individuals were uniformly Th1 type, and only released IFN-{gamma}, whereas a more complex phenotype (IFN-{gamma} and IL-2 and IL-5) was evident for others. The genetic background of C57BL/10 mice in particular may be instrumental in the generation of such LK phenotypic diversity from a common progenitor T cell and provide a relevant model for further regulatory studies of memory T cell development following virus infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c or CBA/Ca or C57BL/10 mice were bred under specific pathogen-free conditions at the National Institute for Medical Research and used at 3 mo of age.

Virus

The X31 virus is a recombinant between A/Aichi/2/68 and A/PR/8/34 that expresses surface glycoproteins of the H3N2 subtype and PR8 internal proteins. X31 was grown in the allantoic fluid of embryonated hen eggs and viral titers determined by hemagglutination assays using turkey erythrocytes.

T cell clones

BALB/c, CBA/Ca, or C57BL/10 mice were infected intranasally, under volatile anesthesia, with X31 virus (5 hemagglutination U (HAU)), and CD4+ T cell lines were established from individual donors 8 to 12 wk after recovery from primary infection and following rechallenge with purified bromelain-cleaved HA (5 µg). T cell clones were obtained by limiting dilution after 4 to 6 rounds of antigenic stimulation and were maintained in vitro by restimulation with UV light-inactivated X31 virus (100 HAU/ml) and irradiated (30 Gy) syngeneic spleen cells as APCs every 10 to 12 d with the addition of IL-2-containing supernatant from Con A-stimulated rat splenocyte cultures after 3 days of restimulation, as described in previous publications (31, 32, 33).

Cytokine assays

Cytokines were assayed by ELISA capture with the following mAbs: for IFN-{gamma}, a capture mAb R4-6A2 (kindly provided by Dr. J. Langhorne); and biotinylated detecting mAb XMG1.2 (PharMingen, San Diego, CA). All other reagents were from PharMingen: for IL-2 capture JES6-5H4 and detection JES6-IA12; for IL-4 capture BVD4-ID11 and detection BVD6-24G2; for IL-5 capture TRFK5 and detection TRFK4; for IL-10 capture SXC-1 and detection JES5-2A5, as directed by the manufacturer using the relevant recombinant cytokines as standards. Recombinant IFN-{gamma} was a generous gift from Genentech (South San Francisco, CA). T cell clones (105/ml) were cultured for 72 h with HA peptide (2.5 µg/ml), anti-CD3 mAb (2C11; 5 µg/ml), or Con A (2.5 µg/ml) supplemented with irradiated syngeneic splenocytes (2 x 106/ml) as APC. Splenocytes were depleted of T cells before culture by a two-step treatment with anti-Thy-1.2 mAb and guinea pig complement.

RT-PCR sequencing of TCR ß-chains

Total RNA from 5 x 106 cells was incubated with 400 ng p(dT)15, 2 mM deoxynucleotide triphosphate, and 20 U of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Lewes, U.K.) in a 20-µl reaction volume (manufacturer’s buffering system) at 42°C for 1 h. We used 0.5 µl of cDNA per PCR reaction.

TCR ß-chain gene usage was determined by PCR screening with a panel of Vß-specific primers in conjunction with a primer specific for the constant region of the ß-chain. All primers used were as published previously (33). ß-Chain DNA was amplified in a 100-µl volume containing 0.12 µM primers, 250 µM deoxynucleotide triphosphates, 5% glycerol, 2.5 U Taq polymerase (Perkin-Elmer, ABI, Columbia, MD), 100 mM Tris (pH 8.8), 50 mM KCl, 1.5 mM MgCl2. Cycling conditions were 1 times (95°C/3 min, 50°C/1 min, 70°C/1.5 min), 40 times (95°C/1 min, 50°C/1 min, 70°C/1.5 min), 1 time (70°C/5 min).

PCR products were purified (using Wizard PCR Preps; Promega, Southampton, U.K.) and directly sequenced by a modified chain termination method (34). Vß-specific primers used for ß-chain cDNA sequencing have been published elsewhere (35), and full sequence across the Vß-Dß-Jß junctional regions was determined.

Synthetic peptides

Peptides were synthesized according to the HA1 sequence of X31 virus with a 430A peptide synthesizer (Applied Biosystems, Foster City, CA) using FASTmoc chemistry and purified (90%) by reverse-phase HPLC.

FACS analysis

T cell clones were analyzed by FACS for Vß expression using the following mAb specific reagents: Vß4 (KT4.10 (36)), Vß6 (RR4-7 (37)) and Vß8 (F23.1 (38)); and a FITC-conjugated rabbit anti-mouse IgG Ab (Sigma-Aldrich, Poole, U.K.) or FITC-conjugated rabbit (Fab)2 anti-rat IgG (Serotec, Oxford, U.K.).

Single-cell analysis of cytokine synthesis

Clone Bpp-19 (5 x 105/ml) was stimulated with immobilized anti-CD3 (2C11; 10 µg/ml) in 6-well plates for 17 h. Brefeldin A was added for the last 10 h of culture. Cells were washed with PBS, 1% (v/v) FCS and surface stained with anti-CD4 biotin and FITC-streptavidin or anti-CD4 phycoerythrin (PE) (PharMingen) or left unstained. After washing, cells were fixed with 4% (w/v) paraformaldehyde for 20 min at 4°C. After washing with PBS-FCS (1%)-saponin (0.1% w/v), cells were stained with anti-IL-5 biotin and FITC-streptavidin or anti-IFN-{gamma} PE (PharMingen), or both. After washing, cells were analyzed by the FACS using forward scatter/side scatter (FSC/SSC) parameters for gating. The specificity of the anti-IFN-{gamma} PE fluorescence was confirmed by inhibition following preincubation with recombinant IFN-{gamma} (100 ng/ml), and background gating was established with an irrelevant rat IgG1-PE (PharMingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Common T cell cloning protocol

We emphasize that the same procedures of in vivo priming and in vitro culture were rigorously maintained throughout this study so as not to bias the expressed T cell repertoire. CD4+ T cell lines were established from individual donors following intranasal X31 infection and a recovery period of 8 to 12 wk. They were maintained using a "feed-starve-feed" cycle with a constant concentration of Ag (100 HAU/ml of UV-inactivated X31 virus) and irradiated syngeneic spleen cells as APC, and T cell clones were established by limiting dilution after a maximum of 4 to 6 cycles of antigenic stimulation. At no stage was there in vitro selection by either purified Ag (HA or HA peptides) or exogenous cytokines (with the exception of IL-2 addition for cloning), and T cell clones were only exposed to HA peptides in assays to establish their TCR specificity or LK phenotype. These comparative studies between different haplotypes were unlikely to have been influenced, therefore, by selective differences in in vitro culture.

Th1 phenotype of I-Ad- and I-Ed-restricted T cell clones

The LK secretion profile and TCR gene usage of clones established from and representative of four BALB/c donors specific for three distinct antigenic peptides are summarized in Table IGo.


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  		Table I. Th1 phenotype Ag specificity, and TCR usage of I-Ad- or I-Ed-restricted T cell clones

 
T cell supernatants were assayed for LK release, by ELISA, 72 h after activation with HA peptide, anti-CD3, or Con A (supplemented with T cell-depleted, irradiated APC). A consensus phenotype was evident for both Ag-specific and nonspecific stimulation, insomuch as IFN-{gamma} was the only LK detected over a range of Ag concentrations (1–100 µg/ml HA peptide), or after mitogen treatment, and therefore was indicative of a Th1 phenotype, as illustrated in Figure 1Go.



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  		FIGURE 1. Quantitative ELISA of LK release by T cell clones from individual BALB/c donors following stimulation by viral peptide, anti-CD3, or Con A (summarized in Table IGo).

 
All of the T cell clones (a minimum of four clones per donor) that we have characterized from donor BA5T, BA5E, or BA6E expressed identical TCR ß-chains (Table IVGo) and therefore were sibling clones, derived from a common progenitor cell. In contrast, three distinct T cell lineages were represented by donor ML7p and were distinguished by TCR usage (Vß4 or Vß 8.3) and/or class II restriction (Ad or Ed) in their recognition of either p186–205 or p177–199.


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  		Table IV. Nucleotide and deduced amino acid sequence comparison of TCR ß-chain junctional regions for T cell clones from H-2b,d,k mice

 
Novel LK phenotype of I-Ak-restricted T cell clones

Table IIGo summarizes the LK status of I-Ak-restricted T cell clones, representative of three CBA/Ca donors and specific for two distinct antigenic peptides. All clones established from the same donor, CB10T (Vß4Jß2.3) or CB12T (Vß8.2Jß2.1) expressed identical TCR ß-chains (Table IVGo), suggesting recruitment of an oligoclonal response to influenza infection, and were uniformly of the Th1 type, insomuch as IFN-{gamma} was the only LK detected, by ELISA, following either Ag-specific or nonspecific stimulation (Fig. 2Go).


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  		Table II. Lymphokine secretion profile, Ag specificity, and TCR usage of I-Ak-restricted T cell clones

 


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  		FIGURE 2. Quantitative ELISA of LK release by T cell clones from individual CBA/Ca donors following stimulation by viral peptide, anti-CD3, or Con A (summarized in Table IIGo).

 
However, a distinguishing feature of this panel was the novel LK phenotype of clones from donor CB3, and specific for p120–139: Clone CB3-5 secreted IFN-{gamma}, and IL-2, and IL-5 on activation, while clone CB3-9 secreted IFN-{gamma} and IL-5 (Fig. 2Go). FACS analysis established that CB3-5 expressed Vß4, whereas CB3-9 was not recognized by Vß4, or Vß6- or Vß8-specific mAbs and was therefore derived from a distinct progenitor cell. Moreover, differences were noted in the signaling requirements for IL-5 release following stimulation of clone CB3-5 with anti-CD3 (background values) vs HA peptide or Con A.

Diverse LK phenotypes of I-Ab-restricted T cell clones

While the majority of T cell clones, established from the BALB/c (Table IGo) or CBA/Ca donors (Table IIGo) had a common Th1 phenotype, there was extensive diversity in the LK secretion profile of T cell clones from C57BL/10 mice (Table IIIGo).


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  		Table III. Lymphokine secretion profile, Ag specificity, and TCR usage of I-Ab-restricted T cell clones

 
Consider the LK secretion of clones established from donor Bpp that expressed the same TCR ß-chain in their recognition of p186–205 (Table IIIGo). Figure 3Go shows the quantitative ELISA profiles after stimulation with peptide, anti-CD3, or Con A: clone 18 secretes IFN-{gamma} and IL-5 on activation; clones 17 and 19 secrete IFN-{gamma} and IL-5 and IL-4; while clone 9 secretes IL-4 and IL-5 and IL-10. However, each of the clones from donor Bpp had identical (VDJ)ß rearrangements at the amino acid level (Table IVGo). It should be emphasized that significant differences in levels of IFN-{gamma} secretion were seen for individual T cell clones (e.g., Bpp-17 vs Bpp-19; Fig. 3Go). Moreover, although the levels of IL-4 secretion (50–100 pg/ml) are significantly lower than IFN-{gamma} secretion (1–100 ng/ml), they are well within the range of detection for a Th2-type clone.



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  		FIGURE 3. Quantitative ELISA of LK release by T cell clones from individual C57BL/10 donors following stimulation by viral peptide, anti-CD3, or Con A (summarized in Table IIIGo).

 
T cell clones from donor Cpp expressed either TCR Vß8.2 or TCR Vß6 in their recognition of p186–205 and therefore were derived from different progenitors. Clone 6 secreted equivalent levels of IFN-{gamma} and IL-5, while clone 11 was distinguished by the additional secretion of IL-4. Interestingly, there was no evident D gene element in the productively rearranged TCR ß-chain from clone Cpp-6.

It seemed unlikely that threshold differences in TCR ligation-activation could account for this clonal diversity since the same combination of LKs was secreted, by the same clone, over a wide range of Ag concentrations (Fig. 4GoA; 1–100 µg/ml peptide) or following nonspecific stimulation by Con A (Fig. 3Go). However, there were temporal differences in IFN-{gamma} or IL-5 secretion after activation by peptide-pulsed APC (Fig. 4GoB).



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  		FIGURE 4. A, Dose response for IFN-{gamma} or IL-5 secretion by clone Bpp-19 on activation by antigenic peptide, p186–205. B, Kinetics of LK secretion by clone Bpp-19 after activation by APC pulsed for 17 h with p186–205 (10 µg/ml).

 
Single-cell analysis of IFN-{gamma} and IL-5 synthesis

Although secretion of IFN-{gamma} and IL-5 are considered antithetic within a polyclonal CD4+ T cell population, the TCR junctional region identity of HA-specific T cells that released both IFN-{gamma} and IL-5 had established their clonality (Tables II and III; Figs. 2Go and 3Go). Even so, it could be argued that different T cells, even within a clonal population, released different LKs due to some stochastic mechanism, to variation in activation threshold and TCR signaling, or to temporal differences in transcription-translation of IFN-{gamma} and IL-5 genes. Therefore, we undertook a single-cell analysis, by FACS, of cytoplasmic IFN-{gamma} and IL-5 synthesis following activation by anti-CD3 in the presence of the inhibitor, brefeldin A.

Because IFN-{gamma} and IL-5 secretion exhibit different kinetics following T cell activation (Fig. 4GoB), it was necessary to establish a suitable time interval between TCR ligation, brefeldin A addition, and FACS analysis to ensure optimal cytoplasmic staining of both LKs. Addition of brefeldin A at 0 h or 4 h after activation resulted in the detection of IFN-{gamma} in the majority of T cells (98%) but with no evident IL-5 production. Conversely, if addition of inhibitor was delayed by 12 h, only IL-5 was detected (data not presented). However, on addition of brefeldin A 7 h postactivation, a significant proportion of cells was double positive for both LKs (Fig. 5Go).



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  		FIGURE 5. Single-cell analysis of IFN-{gamma} and IL-5 production by T cell clone Bpp-19. A, anti-CD4- PE followed, after fixation, by anti-IL5 biotin and FITC-strepavidin; B, anti-CD4 biotin and FITC-streptavidin followed, after fixation, by anti-IFN-{gamma} PE; C, fixation followed by anti-IL-5 biotin and FITC-streptavidin and by anti-IFN-{gamma} PE; D, fixation followed by FITC-streptavidin and anti-IFN-{gamma} PE; E, fixation followed by FITC-streptavidin and anti-CD4 PE; F, fixation followed by anti-IFN-{gamma} PE; G, fixation followed by anti-IFN-{gamma} PE, preincubated with recombinant IFN-{gamma} (100 ng/ml); H, fixation followed by anti-IL-5-biotin and FITC-streptavidin; I, fixation followed by anti-IL-5-biotin, preincubated with recombinant IL-5 (50 ng/ml), followed by FITC-streptavidin.

 
Clone Bpp-19 was surface labeled for CD4 followed, after fixation, by cytoplasmic staining for either IFN-{gamma} or IL-5. Alternatively, cells were first fixed and then stained for cytoplasmic IFN-{gamma} and IL-5. The CD4+ gated population contained 29% IL-5+ cells (Fig. 5GoA) and 98% IFN-{gamma}+ cells (Fig. 5GoB). There was a similar numerical distribution of IL-5+ cells (25%; Fig. 5GoC) in the IFN-{gamma}+ gated population that was significantly higher than the conjugate controls (Fig. 5Go, D and E). The specificity of staining was confirmed by substrate inhibition of cytoplasmic staining by recombinant IFN-{gamma} or IL-5 (Fig. 5Go, F–I). We conclude that IL-5 and IFN-{gamma} are synthesized by the same cell, albeit with different temporal rates of expression.

HA-specific T cell lines are oligoclonal

The frequent dominance observed in TCR ß-chain usage by T cells from the same donor (Tables I to III) might have been an artifact of preferential expansion, at limiting dilution, of the most rapidly growing clone. However, a comparison of ß-chain surface expression by T cell line, with the corresponding T cell clone (established after four to six rounds of stimulation with X31 virus and APC) confirmed the oligoclonal nature of the HA-specific response. Figure 6Go shows that the majority of cells within an HA-specific T cell line express the same TCR ß-chain as the corresponding clone. Consequently, the phenotype of such clones can be considered representative of the individual’s expressed T cell repertoire in vitro.



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  		FIGURE 6. FACS analysis of TCR Vß expression by HA-specific T cell lines. A, C57BL/10 donor, Bpp, Vß8.2+; B, BALB/c donor, BA5E69, Vß4+; C, CBA/Ca donor, CB10T, Vß4+. Background values are for T cells incubated with the FITC-conjugated Ab alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The contrasting profile of LKs secreted by HA-specific T cell clones, elicited by natural influenza infection in three major haplotypes, may provide a relevant model system with which to study the influence of host genetic background on Th1 vs Th2 responses to a well-defined protective Ag: whereas T cell clones from BALB/c mice were found to be typically Th1 (Table IGo) and released exclusively IFN-{gamma} on activation, novel and diverse LK secretion phenotypes were evident for the C57BL/10 or CBA/Ca repertoire. A frequent finding for these two haplotypes was the secretion of both IFN-{gamma} and IL-5 by the same T cell clone. This could not be attributed to intraclonal heterogeneity since single-cell analysis of cytoplasmic LK synthesis by FACS (Fig. 5Go) established that the same T cell produced both IFN-{gamma} and IL-5 on activation, despite temporal differences in LK production.

A further intriguing aspect of this study was the diversity of LKs produced by sibling T cell clones recognizing the same antigenic peptide, expressing identical (VDJ)ß rearrangements of their TCRs (Table IIIGo; Fig. 3Go) and established from a common C57BL/10 donor. Three distinct LK phenotypes were evident for T cell clones from donor Bpp: IFN-{gamma} and IL-5 (clone 18); or IFN-{gamma} and IL-5 and IL-4 (clone 17, clone 19); or a typical Th2 phenotype of IL-4 and IL-5 and IL-10 secretion (clone 9). Sequence analyses of TCR ß usage indicated that each of these T cell clones was derived from a common progenitor, expressing TCR Vß8.2Jß2.6.

Such diversity in LK phenotype for both the haplotype and the individual’s repertoire is unprecedented, and to our knowledge secretion of IFN-{gamma} and IL-5 and IL-4 by the same T cell clone is a novel finding. Clones obtained from a further C57BL/10 donor, Cpp, that recognized the same peptide, p186–205, originated from different precursor cells as indicated by TCR ß-chain usage (Vß8.2+ or Vß6+). They released IFN-{gamma} and IL-5 (clone 6) or IFN-{gamma} and IL-5 and IL-4 (clone 11).

Phenotypic diversity was also evident in the I-Ak-restricted repertoire (Table IIGo). Clones from donor CB3 secreted either IFN-{gamma} and IL-2 and IL-5 (clone 5) or IFN-{gamma} and IL-5 (clone 9). This was the only occasion on which we had identified an HA-specific T cell clone that secreted detectable levels of IL-2 on activation, not a common phenotype for "mature" effector T cells. T cell clones from two further CBA/Ca mice, CB10T and CB12T, that recognized either p48–68 or p120–139 and expressed different TCR ß-chains (Vß4 or Vß8.2) were all of the Th1 phenotype and released only IFN-{gamma} on activation.

In contrast to this phenotypic diversity, I-Ad- or I-Ed-restricted clones established from four BALB/c mice and that used a variety of TCRs (Vß1 or Vß4 or Vß6 or Vß8.3) in their recognition of three distinct antigenic peptides (p56–76, p177–199, or p186–205) were uniformly Th1 type and released only IFN-{gamma} on activation by either Ag or mitogen (Table IGo).

It might be argued that the LK diversity reported for I-Ab-restricted T cell clones, specific for p186–205, was not representative of the haplotype repertoire, but somehow a (idiosyncratic) feature of one (or few) donor mice. However, we have extended these studies to T cell clones established from further C57BL/10, or CBA/Ca, or MHC-congenic mice and find that the coproduction of IFN-{gamma} and IL-5, and IL-4 is a consistent phenotype of HA-specific T cell clones (unpublished findings). Furthermore, there was no correlation between recognition specificity for p186–205 and TCR Vß gene usage. Whereas all of the T cell clones from donor Bpp (Table IIIGo) were Vß 8.2+, this did not extend to further donors that we have characterized, including donor Cpp (Vß6+; Table IIIGo).

We reiterate that all of the HA-specific T cell lines were established, from these three strains of mice, under the same conditions of natural infection and in vitro stimulation (inactivated virus and APC) and in the absence of exogenous cytokines or purified Ag (HA or HA peptides). Moreover, each panel of T cell clones was established from a T cell line by limiting dilution at the same time. Thereafter, clones were maintained under identical culture conditions and clonality was assured by 1) their initial low frequency at limiting dilution (<15 of 96 wells positive for cell growth), 2) recloning at limiting dilution (0.3 cell/well), 3) single productive rearrangements of their TCR ß-chains, and 4) single-cell analysis of cytoplasmic LK synthesis.

The analysis of TCR gene usage had indicated that, with few exceptions (e.g., Table IGo, donor ML7p; Table IIIGo, donor Cpp), a majority of T cell clones, established from the same individual, expressed identical TCR ß-chains, as did the initial T cell line (Fig. 6Go). This suggested an oligoclonal T cell response to influenza infection in vivo. Given the caveat that in vitro culture might skew the expressed repertoire to T cells with a proliferative advantage, how do we account for the observed immunodominance in TCR usage but with, paradoxically, diverse LK secretion phenotypes for I-Ab- or I-Ak-restricted T cell clones?

A natural route of intranasal influenza infection in the absence of adjuvant is likely to have been a major determinant of the expressed T cell repertoire. Influenza virus does not establish systemic infection, or viremia, due to a requirement in its infectious cycle for apical budding from host bronchial epithelial cells into the respiratory tract (39, 40). Virus is cleared 4 to 6 days postinfection by the concerted action of cell-mediated immunity and the neutralizing Ab response. As a result, exposure of the immune system to virus is transient and may provide a temporal restriction to Th recruitment. Moreover, HA-specific T cell lines were established in vitro 8 to 12 wk postinfection and were therefore representative of a Th memory population. There may have been a broader repertoire of effector T cells at earlier times.

The observed differences in LK secretion phenotype between these three strains of mice are unlikely to be due to epitope specificity or the HA peptide recognized in association with a particular class II restriction element: 1) I-Ad- or I-Ed-restricted T cell clones, specific for three distinct peptides, had a common Th1 phenotype (Table IGo); 2) whereas the I-Ad-restricted T cell clone, ML7p-66, which recognized p186–205 had a typical Th1 phenotype (Table IGo), all of the I-Ab-restricted T cell clones thus far characterized (Table IIIGo), and which recognized this same antigenic peptide, were distinguished by their diverse LK secretion profiles. There may have been significant differences, however, in TCR affinity between I-Ab- and I-Ad-restricted T cell clones in their recognition of p186–205. Indeed, Ag concentration or the degree of TCR ligation and signaling has been implicated in the selective production of different LKs (41, 42).

In conclusion, the contrasting Th1 phenotype of HA-specific T cell clones established from BALB/c mice following influenza infection, with the diversity of Th1 or Th2 type cytokines secreted by T cell clones from C57BL/10 or CBA/Ca mice offers a suitable model system for further studies on the molecular basis of LK gene regulation. Since sibling T cell clones from the same C57BL/10 donor express identical TCR ß-chains in their recognition of p186–205, but differ in LK secretion profile, they provide suitable candidates for future subtractive hybridization studies to define Th1- vs Th2-regulatory gene elements.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. D. B. Thomas, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA U.K. Back

2 Abbreviations used in this paper: LK, lymphokine; HA, hemagglutinin; HAU, hemagglutination unit; PE, phycoerythrin. Back

Received for publication January 8, 1998. Accepted for publication March 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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