BACKGROUND
in cd <dig> immunity, different contexts of antigen recognition, whether in the ‘natural setting’ of disease or the experimental setting of antigen-priming adjuvant regimens, favor the preferential development of effector populations that belong primarily to th <dig>  th <dig> or th <dig> subsets  <cit> .
in diseases such as allergic asthma, there is strong, local th <dig> immunity, while influenza infection induces a th <dig> profile  <cit> .
there are several mechanisms driving cd <dig> t cells into these alternate differentiation fates, including the local cytokine milieu.
this partly reflects the experimental designs most accessible for reductionist models .
however, it has relevance extending to the mechanisms involved in natural infection.
dendritic cell  programming through il- <dig> or il- <dig> leading to th1-polarization is commonly modeled by toll-like receptor  agonism with bacterial products, the prototypic example being tlr agonism by bacterial lipopolysaccharide  <cit> .
the nature of stimuli driving a th <dig> environment is less clear.
il- <dig> and il- <dig> are candidates for the promotion of an innate th2-priming environment.
however, il- <dig> or il- <dig> knockout dc can drive th <dig> responses  <cit> .
clues as to the supply of a pro-th <dig> dc program have come from looking at th2-associated pathogen responses.
schistosome egg antigen , which induces a robust th <dig> program in dc, capable even of overriding bacterial th <dig> signals  <cit> , depends on a relatively non-activated transcription profile in the dc, somewhat reminiscent of a tolerogenic profile  <cit> .
this is in keeping with the view that activation of th <dig> cells may encompass reduced t cell receptor  avidity and/or co-stimulation and altered signaling, synapse formation and off-rates.
several lines of evidence suggest that th <dig> activation has different avidity requirements for th <dig> activation.
a basic precept is that the tcr reads different potencies of activation to initiate different effector outcomes with the ‘strength of stimulation’ required for a th <dig> response greater than for a th <dig> response  <cit> .
furthermore, more sustained engagement of the tcr by pmhc is required to polarize tcr transgenic lymphocytes into a th <dig> program  <cit> .
the serial triggering model of t-cell activation offers an explanation for these observations; since any given pmhc complex must activate several receptors for cell triggering, the affinity of the interaction must be low and/or the off-rate fast to enable disengagement to occur and the more sustained the necessary interaction, the greater this will be the case  <cit> .
there are many studies on the relative dose of antigen required for a th <dig> or th <dig> response; a common observation is that th <dig> responses require relatively high concentrations, and th <dig> responses much lower  <cit> .
possible mechanisms for a relationship between antigen dose and t cell polarization may be both through a direct effect on differential cd <dig> signaling  <cit>  and through the ability to modulate expression of co-stimulatory molecules expressed by cd <dig> cells; high antigen dose can favor th <dig> development through dose-dependent up-regulation of cd40l  <cit> .
indeed, it has been argued that ca2+ signaling may be reduced in th <dig> response and that the threshold interaction necessary for inducing ca2+ signaling may not be reached by th <dig> cells  <cit> .
in several models, pmhc complexes showing reduced affinity interactions with tcr are associated with preferential skewing to a th <dig> response.
t cells specific for proteolipoprotein  131- <dig> and selected under th2-favoring conditions show a shift in peptide-tcr primary contact residues compared with th <dig> clones  <cit> .
while avidity maturation on a per-cell basis is a property of the b cell and not the t cell repertoire, t cell clonal selection leads to the progressive appearance of selective tcr usage  <cit> .
for example, the repertoire of tcr response to sperm whale myoglobulin 110- <dig> in adjuvant is initially diverse, then progressively losing lower affinity clones and resulting in an oligoclonal population of tcrβ receptors of intermediate affinity  <cit> .
similarly, pigeon cytochrome c peptide specific tcrβ chains show strong selection for cdr <dig> length and residues characteristic of antigen binding, compatible with progressive clonal maturation and the suggestion of population-level affinity maturation  <cit> .
repertoire maturation in development of the cd <dig> response to listeria involves narrowing of the tcr repertoire, associated with increased affinity  <cit> .
knowing that development of a response involves selective fine-tuning from available receptors and that avidity requirements differ for activation of different th subsets, it would be expected that they might selectively expand different tcr repertoires.
we previously showed that selection of favored tcrs from the peptide-specific pool differs under th <dig> or th <dig> conditions  <cit> .
when an initial, mixed pool of primed cells was divided into th <dig> or th <dig> polarized cultures, different tcr sequences were preferentially selected under the two conditions.
across different pmhc combinations, there was no clear pattern or homogeneity evident in the selection for tcrβ usage, but th <dig> conditions preferentially selected elongated cdr3α sequences.
the example of the response to plp 56-70/h-2ag <dig> was studied in detail.
screening of polarized th line libraries suggested that while th <dig> cultures favored these elongated cdr3α loops but also encompassed receptors with shorter loops, the long loops could never be found in th <dig> cultures.
molecular modeling offered a potential explanation, predicting a bulky, obstructive interaction of reduced affinity for the elongated th <dig> receptors.
thus, contrary to experiments with the do <dig>  tcr transgenic mouse, whereby a th1-derived tcr can be skewed to mediate either th <dig> or th <dig> effector functions  <cit> , in a physiological polarizing environment, receptor features may be preferentially selected so as to skew the future memory response for an appropriate cytokine profile  <cit> .
while it had been envisaged that the cytokine program was faithfully transmitted to progeny cells by chromatin remodeling, polarization is now perceived as a plastic event  <cit> .
in this context, there may be evolutionary advantage in building information on the appropriate response into the tcr itself, so that the response cannot be diverted to a pathogen-inappropriate response by the local inflammatory environment.
we here investigate this hypothesis by generating tcr transgenics carrying an elongated cdr <dig>  th2-derived tcrα chain.
we show that cells carrying this receptor, particularly when paired with the appropriate tcrβ partner, facilitate cytokine skewing away from a th <dig> program.
this is the first time to our knowledge that a causal link between tcr repertoire maturation and th effector polarization has been shown.
the findings suggest that the program for recall of a context-appropriate phenotype may be influenced not just by heritable epigenetic changes, but also in the choice of dominant tcrs themselves.
RESULTS
tcr usage and co-stimulatory molecule expression in polarized cd <dig> t cell lines
we previously described the relatively homogeneous tcrα usage associated with a polarized th <dig> response to plp  <dig> to  <dig>  <cit> .
a noteworthy feature of this tcr usage was the choice of cdr3α loops that were elongated, an observation extended across several different pmhc combinations, suggesting that the th <dig> cytokine environment had influenced preferential utilization of tcrs with these structural features.
in order to establish the impact on cytokine polarization of th2-derived receptors with these features, tcrα chain transgenic lines expressing a representative, elongated cdr3α receptor were generated.
in order to select such a tcr sequence, we sequenced tcrα and β chains from polarized th lines specific for this pmhc combination .
primed lymph node cells  from mice primed with peptide in complete freund’s adjuvant  were split into cultures stimulated with antigen in vitro under polarizing th <dig>  th <dig> or th <dig> conditions.
to generate th <dig> lines, cells were cultured in medium containing il- <dig>  il- <dig> and anti-il- <dig>  to generate th <dig> lines, cells were cultured in medium containing il- <dig>  il- <dig> and anti-ifnγ.
for th <dig> lines, cells were initially cultured in anti-ifnγ, anti-il- <dig>  il- <dig> and tgfβ and expanded in medium containing il- <dig> and il- <dig>  each set of conditions yielded a repertoire of tcrs with reproducibly distinct features .
in each case, the distinctive cdr3s were a characteristic of the tcrα and not the tcrβ repertoire.
under th <dig> conditions, cultures predominantly favored a vα15jα <dig>  receptor with a  <dig> amino acid cdr3α, aasregtgskls.
we never found this receptor or ones similar to it in cultures derived from the same lnc pool but polarized under th <dig> or th <dig> conditions.
cells cultured under th <dig> polarizing conditions favored very different tcrα usage, a quarter of the sequences comprising a vα9jα <dig>  receptor with an elongated, 14-amino acid cdr3α loop, alegiasssfsklv.
this was not accompanied by any overt selection for dominant tcrβ chains.
interestingly, cells cultured under th <dig> polarizing conditions were also distinctive, a large proportion of sequences  being vα11jα <dig>  comprising a short,  <dig> amino acid cdr3α loop .
again, there was no preferred, expanded tcrβ chain.
the findings suggested selection from the available pool primarily on the basis of tcrα sequence and permissible pairing of dominant tcrα chains with multiple possible β chain partners.
the th <dig> cells were similar to th1-polarized cultures in being limited to short cdr3α loops.
there was no significant difference in tcrβ chain cdr <dig> length seen in th <dig> , th <dig>  and th <dig>  tcr sequences.
however, tcrα chain cdr <dig> lengths were significantly longer in th <dig>  than in th <dig>  and th <dig>  tcr sequences .
t cell lines were maintained in culture for one round of re-stimulation under highly polarizing conditions before taking rna for tcr sequence analysis.
tcrα and β chain repertoire analysis of an h2-ag7/plp 56- <dig> specific th <dig>  th <dig> and th <dig> cell lines shows expansion of distinct, dominant tcrα chains in the absence of a dominant β chain.
the frequency  of each unique tcr sequence identified is shown.
cdr <dig> length is defined as the number of amino acids between the invariant c residue and the f/w-g-x-g motif.
tcrα and tcrβ cdr <dig> region lengths are shown in each table.
data are representative of  <dig> tcrα and  <dig> tcrβ sequences for each t cell line.
the mean cdr <dig> length for the tcrα chain is  <dig>  ±  <dig>   for th <dig> cultures,  <dig>  ±  <dig>   for th <dig> cultures, and  <dig>  ±  <dig>   for th <dig> cultures.
the most dominant tcr alpha sequence is shown in bold.
mean cdr3α length  <dig>  +  <dig>   .
mean cdr3β length  <dig>  +  <dig>   .
the most dominant tcr alpha sequence is shown in bold.
mean cdr3α length  <dig>  ±  <dig>   .
mean cdr3β length  <dig>  ±  <dig>   .
the most dominant tcr alpha sequence is shown in bold.
mean cdr3α length  <dig>  ±  <dig>   .
mean cdr3β length  <dig>  ±  <dig>   .
when assayed for release of ifnγ, il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig>  each of the polarized lines showed the expected profiles .
a caveat here is that, in line with observations from others using in vitro and in vivo models, t cells within th <dig> cultures could flip relatively easily into co-expression of il- <dig> and ifnγ  <cit>  or single expression of ifnγ,  particularly if derived from an ex-vivo inflammatory environment.
in line with the idea that selection from the initial pool under th polarizing conditions acts through selection of clones with differing avidities, th <dig> and th <dig> lines developed with markedly differing profiles of co-stimulatory molecule expression .
th <dig> lines show greater expression of cd40l, in line with the idea that cd40l-blockade preferentially blocks th <dig> responses   <cit> .
expression of ox <dig>  intercellular adhesion molecule- <dig>  and cd <dig> were also greater on th <dig> lines .
conversely, th <dig> inducible t-cell co-stimulator  expression was greater, as predicted by the finding that icos stimulation preferentially triggers th <dig> cytokines   <cit> .
the cd <dig> expression differences are reminiscent of observations in a recent paper by mandl and colleagues, who showed that tcr transgenic cd5hi clones accounted for high tetramer binding  <cit> .
generation of tcrvα chain transgenics
we expressed the immunodominant selected tcrα sequence  receptor with an elongated, 14-amino acid cdr3α loop, alegiasssfsklv) in the ptα expression cassette, and made two independent transgenic founder lines expressing the dominant th2-derived tcrα chain with an elongated cdr <dig> region .
we expressed the immunodominant selected tcrα sequence  receptor with a 12-amino acid cdr3α, aasregtgskls) in the ptα expression cassette, and made one transgenic founder line with the dominant th1-derived tcrα chain with a shorter cdr <dig> region .
all the transgenic lines were then backcrossed onto the original non-obese diabetic, h2-e transgenic  background in which the t cells had been characterized .
transgenic expression of tcrvα chain with an elongated cdr <dig> does not favor th <dig> responses in primed cells
with respect to tcr selection in th <dig> t cell lines, the absence of any dominant tcrβ chain in cultures led us to hypothesize that transgenic expression of the tcrα chain with an elongated cdr <dig> region alone might be sufficient to impact on cytokine phenotype of the t cell response.
we initially assessed the functional impact of the elongated tcrα chain on cd4+ t cell responses by immunizing mice to look both at initial recall responses and subsequently after a second antigen boost in vivo .
the nature of ex-vivo cytokine responses is heavily influenced by the adjuvants used for priming, with cfa giving strong th <dig> polarization through the effect of m. tuberculosis on myd88-dependent signaling  <cit> .
we, therefore, assessed whether this tcr could confer propensity to develop a th <dig> program in the face of a th1-skewing, cfa priming regimen.
mice were immunized on day  <dig> and day  <dig> with plp  <dig> to  <dig> in cfa and incomplete freund’s adjuvant , respectively, and t cell responses sampled at days  <dig>   <dig> and  <dig>  at each timepoint, t cell responses of tcrα chain transgenics were similar to littermate controls as judged by proliferation .
interestingly, however, while the littermate controls generated a strong ifnγ response, this was absent after the initial recall in the tcrα transgenics at day  <dig>  .
this suggested that use of this receptor was less effective at maintaining ifnγ transcription.
when mice were boosted, we observed an ifnγ response in littermates but not in tcrα transgenics .
t cells from the tcrα chain transgenics were harvested at day  <dig>  cultured with peptide for 48 h and cdr <dig> analysis of the cd4+cd69+  and cd4+cd69−  cells was carried out.
the lack of an ifnγ response in tcrα transgenics after boosting correlated with selection of a single shared vβ <dig> partner chain, as demonstrated by tcr sequencing and by spectratyping using v gene specific primers .
all the cd4+cd69+ cells expressed the same tcrβ chain, trbv <dig> trbj2- <dig> with an elongated 13-amino acid cdr <dig> region assqagtgedtqy.
thus, the defective ifnγ recall response was associated with the clonal expansion of a population of cells expressing a single trbv <dig> trbj2- <dig> chain with an elongated cdr <dig>
cytokine polarization evolves in cultured cell lines in line with favored tcrαβ selection
in order to look in more detail at the relationship between progressive polarization and focusing of the tcr repertoire, we set out to look at polarization and tcrβ selection during progressive re-stimulation of t cell lines in vitro.
this was done in the absence of exogenous polarizing factors in the medium.
this allowed us to further explore the possibility that, with progressive re-stimulation and selection, there may be increasing focus on particular tcr pairs, leading to impaired th <dig> responses and gradual promotion of th <dig> responses.
primed draining lnc were used to establish t cell lines in vitro.
elongated cdr <dig> tcrα chain transgenic lines  <dig> and  <dig>  short cdr <dig> tcrα chain transgenic line  <dig> and transgene negative littermate controls were primed with peptide in cfa and, 10 days later, lnc were harvested and t cell lines established.
line  <dig> and line  <dig> tcrα transgenic cells rapidly selected dominant tcrβ chain expansions .
by the fourth re-stimulation, over half of the tcrβ repertoires for the tcrvα transgenic lines comprised a single vβ chain  and by the sixth re-stimulation, the tcrvβ repertoire was composed almost exclusively of this vβ chain.
these studies, along with the studies shown above in figure  <dig>  argue that several different tcrβ chains can pair with the elongated tcrα chain to yield the desired pmhc specificity and that in a given mouse or t cell line, one of several possible sequences may acquire clonal dominance.
this selection of tcrβ chains in the context of pmhc activation was confirmed by spectratyping .
as early as the second re-stimulation, cd <dig> t cell lines from line  <dig> tcrα transgenics show constrained spectra, with a significant contraction of the tcrvβ chain repertoire for several vβ gene families while spectra from littermate control t cell lines show tcrvβ diversity .
we then looked at this tcr focusing during evolution of a given t cell line in relation to skewing of cytokine production.
at the same time as making cdna at each re-stimulation for tcr sequence analysis and spectratyping, cytokine production was analyzed by elisa and rna used for real-time analysis of subset-specific transcription factors.
with progressive re-stimulations in vitro, the reciprocal nature of th <dig> and th <dig> polarization in littermate controls and tcrα transgenic lines with the shorter compared to those with elongated cdr <dig> regions is seen.
tcrα transgenics with the elongated cdr <dig> region  make little or no ifnγ, but, after a lag of one re-stimulation, make large amounts of il- <dig>  il- <dig> and il- <dig> .
tcrα transgenic lines with the shorter cdr <dig> region  and non-transgenic controls make ifnγ, but no il- <dig>  il- <dig> and il- <dig>  in line with this, gata- <dig> transcription is progressively up-regulated in tcrα elongated cdr <dig> transgenic cultures compared with littermate controls .
there is no bias of the tcrvβ chain repertoire in naïve tcrvα chain transgenic splenocytes at day  <dig> , or in a primary response in draining lymph nodes at day  <dig> post-immunization as demonstrated by spectratype analysis .
generation of tcrαβ transgenics
the likely interpretation of the simultaneous appearance of dominant tcrβ chain sequences in the tcrα chain transgenic lines, adoption of a spontaneous th <dig> phenotype and impaired th <dig> program, was thus that features of this preferred tcrαβ pair were incompatible with effective maintenance of th <dig> activation and, therefore, favored emergence of a th <dig> response.
we therefore expressed one of the immunodominant, selected tcrβ sequences  in the ptβ expression cassette , generated tcrβ transgenics, and crossed these to line  <dig> tcrα transgenics on the same nod.e background.
tcrαβ transgene impacts on ex-vivo t cell phenotype
to assess the functional impact of the tcrαβ on primary ex-vivo t cell responses to peptide, tcrαβ transgenics and littermate controls were primed with peptide/cfa, again supplying a maximal th1-skewing environment.
at day  <dig>  t cell responses were analyzed ex-vivo and t cell lines established.
while tcrαβ transgenics, as would be expected, show enhanced t cell proliferation responses to peptide, ifnγ and il- <dig> responses were absent, and they made substantial amounts of il- <dig> .
as before, immediately ex-vivo we could detect no il- <dig>  il- <dig> or il <dig> .
the absent production of ifnγ and il- <dig> and enhanced production of il- <dig> observed in the tcrαβ transgenic line was seen in the context of priming with the th <dig> promoting adjuvant cfa making this result all the more striking.
il- <dig> production was not significantly different between the groups.
the phenotype of the tcrαβ transgenic lines was further confirmed by real time pcr for ifnγ, gata- <dig> and rorγt.
the tcrαβ transgenic lines expressed high levels of gata- <dig>  and low levels of ifnγ and rorγt relative to the littermate controls in keeping with a th <dig> phenotype .
furthermore, gata- <dig> expression increased with successive re-stimulations, while rorγt expression decreased in keeping with the reduction in il- <dig> production .
the level of t-bet transcription, however, showed no obvious pattern through re-stimulations of the tcr transgenic or wt lines .
this is in line with an increasingly complex model for t-bet in the th <dig> program: in several models, th <dig> polarization occurs in the face of t-bet transcription  <cit> .
because our investigations had used cfa priming, it was important to gauge the effect of immunizing tcrαβ double transgenic, tcrα chain single transgenic and littermate control transgenics with peptide in ifa lacking th1-skewing killed mycobacteria .
at day  <dig>  lnc were harvested and 3h-thymidine incorporation and cytokine production determined ex-vivo.
the results are in line with the larger pmhc-specific t cell response in the tcrαβ transgenics, but confirm the th <dig> bias: tcrαβ transgenic cells respond strongly to peptide making il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig>  but no ifnγ or il- <dig> .
tcrα chain transgenics, and littermate controls made il- <dig> and small amounts of il- <dig> only .
this is in keeping with the tcrαβ receptor supporting a th <dig> phenotype.
addressing alternative hypotheses for cytokine-skewing in the tcr transgenics
the tcrαβ transgenic lines make more il- <dig> than littermate controls, indicating t cell survival potential .
the high il- <dig> response in the tcrαβ transgenic line, taken together with the high il- <dig> and il- <dig> response, implies that there is no global failure of t cell activation/survival per se, but merely a cytokine-deviated response.
this appears to argue against a possible alternate hypothesis, that increased precursor number may have caused greater competition for ifnγ signals and decreased t cell fitness and/or memory  <cit> .
to further address this issue, we immunized tcrαβ transgenics and littermate controls on day  <dig> and day  <dig> with plp  <dig> to  <dig> in cfa and ifa, respectively, and looked at markers of cell activation and cell survival by real-time pcr and fluorescence activated cell sorter .
real-time pcr analysis of b-cell lymphoma-extra large  expression gave no indication of an enhanced propensity to apoptosis .
anti-apoptotic proteins, such as bclxl, are involved in protecting mitochondrial integrity, for example, in the context of limiting growth factors  <cit> .
bclxl is induced on t cell activation and enhanced by co-stimulation of cd <dig>  <cit> , and it is generally considered that over-expression of b-cell lymphoma  <dig>  or bclxl in t cells prevents death by neglect  <cit> .
furthermore, data from gett and colleagues indicate that induction of bclxl on sustained activation depends on stimulation strength and is associated with resistance to apoptosis, bclxl increase being greatest in t cells that had received prolonged stimulation.
if tcr transgenic cells were incapable of fully functional stimulation and destined for some form of sub-optimal stimulation or death by neglect, one would predict diminished activation of bclxl in the transgenic response.
in fact, we found no difference in bclxl expression.
taken together with the other evidence shown here, including the strong il- <dig> response on activation, we interpret the findings to mean that there is no deficit in activation of the response through the transgenic receptor, but rather, deviation of the transcriptional response to an altered cytokine profile.
furthermore, phenotypic analysis of the primed lymphoid populations at day  <dig> showed no difference in activation/fitness as indicated by staining for cd <dig> .
it has been shown by whitmire and colleagues in a transgenic lymphochoriomeningitis virus  response cell transfer model that only when the number of transferred t cell precursors was low did they generate fully functional memory cells that were cd127/il-7r , with full capacity to produce cytokine and proliferate.
testing therefore whether it was possible that an excess of responder cells here correlated with a response phenotype that was in some way impaired, we analyzed t cell expression of cd <dig>  and found that transgenic cells had enhanced, not reduced expression.
taken together with the other evidence presented here, this makes it unlikely that this was a model of sub-optimal activation.
furthermore, expression of cd62l was similar in the transgenic and littermate responder populations .
thus, the indication from il- <dig> release, as well as apoptosis and activation markers is that the phenotype observed here constitutes an actively skewed functional preference, rather than a failure to trigger properly by the transgenic receptors.
it was hypothetically possible that injection of cognate peptide into a single chain tcrα and tcrαβ transgenic mice had triggered a cytokine storm due to the large number of responding t cells, and this had in turn caused thymic apoptosis leading randomly to a skewed cytokine outcome in surviving cells.
to address this we conducted additional studies.
‘cytokine storm’ is a term derived from the systemic response in toxic or septic shock  <cit> .
here, an acute, excessive spike of systemic pro-inflammatory cytokines leads to downstream events, often including large-scale lymphocyte apoptosis.
in our studies the positive control stimulus of seb led to the predicted spike in systemic pro-inflammatory cytokines, such as ifnγ and tnfα at 2 h, but no systemic change in cytokines was detected after injection of peptide/cfa .
it was nevertheless possible that even in the absence of a systemic response there may have been some acute, storm-driven change in thymic populations accounting in a non-specific way for cytokine skewing.
while seb had a dramatic effect on overall thymocyte numbers and cd4/cd <dig> ratio in littermate control as well as tcrα and tcrαβ transgenics, no such effect was seen with cognate peptide .
none of the peptide-primed non-transgenic controls showed preferred tcrβ chains in sorted cd <dig> sp cells from thymocytes .
however, in one out of four of the mice analyzed, tcrβ chains in sorted cd <dig> sp cells from thymocytes in peptide-primed tcrα transgenics show preferential selection of a preferred tcr heterodimer, 44% of tcrs using a common tcrβ chain .
on the basis of these experiments, the occam’s razor hypothesis, that tcrα chains of differing structures and avidities can influence cytokine program, appears more likely than cytokine preference emerging as a stochastic event in the aftermath of a cytokine storm.
t cell lines with tcrαβ receptor have low binding avidity
we previously predicted from molecular modeling studies that the elongated cdr <dig> region of the tcrα chain, when paired with a preferred tcrβ chain, might result in a lower avidity interaction between the tcr and its pmhc ligand  <cit> .
we therefore started by reappraising functional avidity in a peptide titration of th <dig>  th <dig> and th <dig> polarized, non-transgenic t cell lines cultured for eight days in polarizing medium and looking at ifnγ, il- <dig> and il- <dig> elispots, respectively.
in these short-term lines, to achieve a response of  <dig> sfc/ <dig> cells requires about  <dig> times the peptide concentration in the th <dig> lines compared to th <dig>  and about  <dig> times the peptide concentration in the th <dig> lines compared to th <dig> .
we next looked at tetramer binding characteristics of th <dig>  th <dig> and th <dig> polarized, non-transgenic t cell lines cultured for eight days in polarizing medium and prepared from the same initial pool of primed lnc, using h2-ag <dig> tetramers loaded with either plp <dig> to  <dig> or an irrelevant h2-ag7-binding peptide .
at all concentrations of tetramer tested, a significantly greater proportion of th <dig> and th <dig> polarized cells bound tetramer compared to th <dig> cells despite equivalent levels of cell surface cd <dig> .
similarly, in a peptide titration to examine the functional avidity of short-term t cell lines from tcrαβ transgenics relative to littermates, an equivalent number of il- <dig> spot-forming cells in the tcrαβ transgenics require approximately  <dig> times the peptide concentration required for the ifnγ response in littermates .
we then compared tetramer binding characteristics of t cell lines derived from the th2-type tcrαβ transgenic cells, tcrα chain transgenics with an elongated cdr <dig> and th1-type cells from non-transgenic littermate controls.
as tetramer concentration increased, a higher frequency of th <dig> cells from the control littermate bound tetramer.
however, no tetramer binding was detectable for the tcrαβ transgenic cells, indicating that the avidity of the interaction with the tcrαβ cells was too low for detection .
the tcrα chain transgenics with an elongated cdr <dig> had an intermediate binding avidity.
we demonstrated a functional interaction between the tetramer and tcrαβ transgenic t cells by measuring cytokine production from transgenic cells cultured in the presence of plate-bound tetramer.
in keeping with the th <dig> phenotype, il- <dig>  il- <dig>  il- <dig> and il- <dig> but not ifnγ were detected .
the th <dig> polarized t cell lines produced less il- <dig> with each successive re-stimulation such that by the fourth re-stimulation cytokine production was more in keeping with that of a th <dig> cell line .
we hypothesized that this shift in cytokine production would be accompanied by a reduction in tetramer binding avidity and longer tcrvαcdr <dig> length as t cells with a ifnγ producing phenotype clonally expand.
in keeping with this, we observed a reduction in tetramer binding avidity between the first and fourth re-stimulations of polarized th <dig> lines such that at the fourth re-stimulation, tetramer binding curves for th <dig> and th <dig> lines overlapped .
tcr repertoire analysis at the first and fourth re-stimulations confirmed that the change in cytokine production and tetramer binding avidity occurred alongside an increase in average tcrvαcdr <dig> length .
the mean tcrvαcdr <dig> lengths for the first and fourth re-stimulations are  <dig>  +  <dig>    and  <dig>  +  <dig>   , respectively, .
the difference in mean cdr3α length between the first and fourth re-stimulation shows progression to a tcr repertoire with longer cdr3α regions as cells lose their il- <dig> producing phenotype and become more th1-like in their tetramer binding characteristics and cytokine production.
in light of previous observations in tcr transgenics, notably the early studies by hosken and colleagues  <cit>  using the do <dig>  tcr transgenic line, it might be predicted that the th <dig> tcr transgenic-skewed polarization may be overcome by altering peptide dose.
certainly, among the many factors that can skew cytokine bias would be peptide dose itself.
in the do <dig>  tcr transgenic studies, it was shown that under otherwise equivalent in vitro primary culture of naïve cells, mid-range peptide doses favored the generation of moderate ifnγ responses, while either higher or lower doses favored a switch to development of more th2-like responses, giving the ifnγ response curve the form of a bell-shaped curve  <cit> .
we, therefore, performed a set of experiments in our system to examine the behavior of the th2-derived transgenic tcr across a checker-board of low to high peptide priming dose and a low to high in vitro re-stimulation dose.
our previous experiments had typically utilized a priming and re-stimulation dose of 25 μg/ml, corresponding to 13 μm towards the upper-end of the concentration range that had been tested by hosken and colleagues using naïve t cells in vitro.
we initially primed th <dig> tcrαβ transgenic mice with peptide at  <dig>   <dig> or 125 μg .
popliteal lnc were then challenged in vitro with a titrated dose of peptide from  <dig>  to 100 μg/ml.
as we had seen before, peptide priming with 25 μg/ml did not trigger an ex-vivo ifnγ response, though ifnγ could be elicited at either a higher or lower dose of peptide priming,  <dig> or 125 μg/ml.
thus, these findings are reminiscent of the studies by hosken and colleagues, except that the mid-range peak of ifnγ production that had been seen by them for the do <dig>  tcr is here exchanged, using a th <dig> tcr, for a low/high range peak of ifnγ production.
our curve is the reciprocal of the do <dig>  finding.
furthermore, the mid-range dose of priming with 25 μg/ml peptide was associated with increased transcription of both gata- <dig> and rorγt.
thus, while there is a constraint on ifnγ activation, this is peptide dose dependent and can be over-ridden by either high or low dose peptide priming.
we then used this checker-board titration to analyze cytokine secretion in t cell lines from peptide primed th <dig> tcrαβ transgenic mice, assaying their profile after the third re-stimulation in vitro.
the findings are summarized in figure  <dig>  il- <dig> responses are depicted in blue, il- <dig> in green and ifnγ in red.
the key arbiter of cytokine profile in vitro appeared to be the dose of peptide used for in vitro re-stimulation of the lines.
that is, whether initial priming was with  <dig> or 125 μg, the t cells lines produced relatively large amounts of il- <dig> and il- <dig> but little or no ifnγ when re-stimulated with peptide in the range  <dig>  to 10 μg/ml.
however, irrespective of the initial priming dose, the t cell lines showed an ifnγ response when re-stimulated in vitro with 100 μg/ml peptide.figure  <dig> is a schematic summarizing the flow of experiments designed here to test the hypothesis that elongated tcr alpha chain complementarity-determining region  <dig> favors a th2-skewed cd <dig> phenotype.
discussion
there has been considerable effort devoted to elucidating the structural biology of the tcr complex, with 10s of structures solved, yet clear models for the way in which the ligand/receptor interaction between pmhc and tcr may give rise to the very diverse array of t cell effector programs have been elusive  <cit> .
most tcr transgenic lines are generated without regard for specific features of the cytokine profile of the parental cell from which they were derived.
experiments in some autoimmune models bear on this insofar as there are examples in which t cell clones were implicated in an autoimmune effector mechanism and the tcr transgenics derived from them can mediate a spontaneous disease phenotype dependent on preferential use of a similar cytokine profile  <cit> .
candon and colleagues generated transgenics for the tcr of a self-reactive th <dig> clone, showing that a proportion of the mice spontaneously developed autoimmune gastritis characterized by eosinophilic infiltration of the gastric mucosa and th <dig> differentiation of transgenic t cells in the gastric lymph node  <cit> .
appropriate th polarization is most readily attributed to t cells engaging pmhc in the context of an antigen presenting cell  that has been activated by innate signals to provide a particular, polarizing environment  <cit> .
could another aspect of the polarization reside in the tcr sequence itself?
this would have evolutionary value as a contributory mechanism in polarization since, without it, the appropriateness, or otherwise, of the effector response could be at the mercy of influence by any other concurrent processes and/or infections.
it is self-evident that there may be tcr structures that are ‘vanilla’ in nature and capable of being diverted readily to either a th <dig> or th <dig> program; do <dig>  is the prototypic example  <cit> .
knowing that tcrs of cd <dig> cells have been reported across a moderately broad range of affinities and also that affinity/avidity are key determinants of cytokine polarization, it may be predicted that tcrs of different structures would, all else being equal, show inherent bias to different effector profiles.
the principle that tcr sequence correlates with function is accepted in the special case of the treg versus teff tcr repertoires  <cit> , although even in this setting, phenotypic differences have proved difficult to define  <cit> .
we show, using new tcr transgenic models, that an elongated tcrα derived from a strongly th2-skewed t cell line retains imprinting of that effector program to the extent that, even in the face of an overwhelming th1-polarizing environment, such as immunization in cfa containing m. tuberculosis, t cells can proliferate while lacking ifnγ responsiveness.
while the polarization is mediated through the selection of appropriate cdr3α regions and the constraints on tcrβ selection are less apparent, the full th <dig> program is clearly dependent on selecting the correct tcrαβ pair.
a starting point for the studies here was to reappraise more broadly our earlier observations about preferential selection of the tcr repertoire.
this can be thought of as ‘avidity-associated functional maturation’ of the response; while it has long been known from many pmhc examples that the tcr repertoire can focus in on sometimes highly dominant receptor usage, we add the observation that this choice is highly influenced by factors driving th1/th <dig> polarization.
a key observation, reiterated in the current study, had been that th <dig> polarization is associated with cdr3α chains with somewhat elongated loops.
in the present study, we have extended this analysis to investigation of th17-polarized cultures.
once again, tcrβ chains remained relatively heterogeneous while tcrα sequences rapidly attain focused, preferential usage; although selected out of the same starting pool, the th <dig> receptor usage is dissimilar to either th <dig> or th <dig> lines.
like th <dig> lines, tcrα chains are invariably of the th1-like, short, cdr3α type.
we found no examples of elongated cdr3α chains in th <dig> cells.
this may be taken to mean that, like th <dig> cells, th <dig> activation is dependent on ‘high-end’ affinity activation and depends on activation that is incompatible with low affinity tcr activation.
this is in line with the relatively easy plasticity from th <dig> to th <dig> lineages, although it is also known that there are differences between activation signals for th <dig> and th <dig> activation, such as dependence on pkcθ  <cit> .
on the other hand, we found little similarity between the actual cdr3α sequences selected in th <dig> and th <dig> cultures.
to our knowledge this study constitutes the first report of preferential tcr usage associated with the th <dig> phenotype with the associated implications for differential avidity of th <dig> cells.
th <dig> cells are predicted to be at the upper end of the avidity spectrum, with associated implications for differential signaling and synapse formation.
a number of studies have sought to relate changes in the tcr repertoire to the development of the t cell response, although not in the specific context of cytokine programs.
studies of cd <dig> responses have variously shown either a stochastic relationship between the initial response and the expanded repertoire, or substantial focusing with preferential selection of particular clonotypes, sometimes associated with enhanced avidity  <cit> .
those examples of cd <dig> repertoire selection that have been investigated in detail have also tended to focus on the progressive loss of low-affinity clones to achieve the ‘best fit’  <cit> .
what is unclear is how this model could be reconciled with a continuing requirement for differential avidities associated with alternate cytokine programs.
certainly, cells within a th <dig> memory population can make ifnγ if reactivated in the context of innate th <dig> signals  <cit> .
that is, it might not be possible to maintain an overall dominance of th <dig> clones in the face of a tcr repertoire progressively shifting to higher avidity.
for example, in th1/th <dig> dependent experimental autoimmune encephalomyelitis  tcr transgenic models, strength of pmhc-tcr interaction is correlated with more aggressive, spontaneous disease  <cit> .
conversely, decreased cd <dig> expression by polarized th <dig> cells has been shown to contribute to reduced tcr-induced phosphorylation and ca2+ signaling  <cit> .
profiling of co-stimulatory molecules on th <dig> and th <dig> lines showed differences compatible with the notion of enhanced avidity promoting th <dig> differentiation.
up-regulation of cd40l expression in th <dig> cells is a previously well-documented example of this phenomenon, confirmed here  <cit> .
up-regulation of icos was a strong marker of th <dig> differentiation, while cd <dig> up-regulation was a strong marker of th <dig> differentiation.
in line with the notion of cd <dig> up-regulation as a marker of enhanced avidity in th <dig> cells, cd <dig> expression in thymocyte selection is correlated with avidity/signal intensity of the positively selecting tcr-pmhc interaction  <cit> .
for differential engagement between pmhc on the apc and the tcr to lead to these very divergent transcription programs, there must be substantive differences in signaling, from initial formation of the immune synapse.
a number of studies have previously considered differences in immune synapse formation and tcr signaling between th <dig> and th <dig> cells: most of these are compatible with the notion that the synapse leading to th <dig> differentiation comprises a less tightly focused, lower avidity complex.
th <dig>  but not th <dig> cells, fail to cluster tcr at the cell-cell interface due to increased expression of cytotoxic t-lymphocyte antigen  <dig>   <cit> .
in line with this, co-clustering of tcr with cd <dig> in lipid rafts is more common in th <dig> cells  <cit> .
th <dig> immune synapses are morphologically distinct, characterized by the failure to exclude cd <dig> and icam- <dig> from the central zone  <cit> .
these changes are presumed to contribute to diminished strength of signaling.
furthermore, a number of studies have considered mechanisms by which these differences might in turn lead to qualitative signaling differences and alternate transcriptional choices.
strength of signaling has been shown to affect the balance of nfatp and nfatc binding activity, thus directly regulating il- <dig> transcription  <cit> .
we predict from our tcr analysis of th <dig>  th <dig> and th <dig> lines that th <dig> synapse formation and signaling would be distinct from the low avidity interactions characterizing th <dig> activation and more reminiscent of th <dig> activation.
jameson and masopust  <cit>  have summarized a number of studies on the cost to the quality of memory of too much stimulation with the term, ‘everything in moderation: better memory by avoiding over-exertion.’ is it possible that our th <dig> tcrαβ transgenic model in some way elicits a response that is sub-optimal through excessive or inappropriate stimulation?
while our observations here have focused largely on cytokine polarization of the initial response, all of the markers assessed here, including il- <dig> activation, cd <dig> expression and anti-apoptotic markers indicate that responder cells in this transgenic system are fully fit to respond, they merely choose to do it in a different way.
the key evidence that cytokine program can be influenced by the selected tcrαβ sequence itself came with the observation that by transgenic expression of a th <dig> derived receptor, we produced mice that are ‘th1-averse’, even in the face of the most powerful possible stimulus to reverse this; despite showing strong proliferation in response to peptide, as would be expected, considerably higher than wild-type mice, there is no ifnγ or il- <dig> response.
rather, the tcr transgenic mice make a strong th <dig> response, not seen at all in the littermate controls.
why should this be the case?
we speculate that it is an evolutionary failsafe to ensure pathogen-appropriateness of the response.
in experimental immunology, we tend to work with reductionist systems where we can examine polarized th <dig>  th <dig> or th <dig> responses, unimpeded by other influences.
however, in the natural host response to infection there will be many competing influences; thus, th <dig> responses have presumably evolved to provide protection against parasitic infestation, yet in many parts of the world facing a major parasite burden, there is also a high level of infection with m. tuberculosis, itself providing a concomitant drive to th <dig> and th <dig> immunity  <cit> .
indeed, we have previously described another clinical example of this type in respect to the co-existence in the lung of sarcoidosis, which is associated with strong th <dig> responses, and cryptococcus neoformans, for which th <dig> immunity is critical  <cit> .
in complex settings, being able to avoid potentially dangerous reprogramming from pathogen-driven, cytokine milieu and promoting an appropriate effector response, is vital.
cells with lower affinity tcrs adopt a th <dig> phenotype when primed in the absence of competition from cells with higher affinity receptors  <cit> .
evidence that the th2-derived tcr used in these studies are of low affinity comes from molecular modeling studies done in collaboration with yvonne jones’ laboratory  <cit> .
in the present study, we were able to confirm those predictions using direct tetramer binding studies.
we show here that while wt littermate primed cells respond to plate-bound tetramer by making ifnγ, tcrαβ tg cells respond by making th <dig> cytokines, but no ifnγ.
this further emphasizes the fact that the property is purely one of the interaction between pmhc and tcr, uninfluenced by other contributory differences of co-stimulatory molecules.
however, despite the fact that both cell populations can be activated by tetramers, binding to the transgenic t cells is sufficiently low as to be undetectable.
the tetramer binding studies, taken in conjunction with the preferential tcr selection described by us in polarized lines, predict that the preference for tcr avidity will be th17 > th1 > th <dig>
CONCLUSIONS
we propose an additional and previously undefined mechanism for ensuring the cytokine appropriateness of cd <dig> immunity.
tcr preferentially selected under th <dig> conditions tended to use elongated cdr3α loops and when expressed in transgenics, skewed responses away from secretion of ifnγ, favoring th <dig> cytokines.
focusing on the t cell repertoire in antigen specific adaptive immunity can bring with it information not just about pmhc specificity, but also about the qualitative nature of the appropriate cytokine response.

 in cd immunity, different contexts of antigen recognition, whether in the ‘natural setting’ of disease or the experimental setting of antigen-priming adjuvant regimens, favor the preferential development of effector populations that belong primarily to th th or th subsets. in diseases such as allergic asthma, there is strong, local th immunity, while influenza infection induces a th profile. there are several mechanisms driving cd t cells into these alternate differentiation fates, including the local cytokine milieu. this partly reflects the experimental designs most accessible for reductionist models. however, it has relevance extending to the mechanisms involved in natural infection. dendritic cell programming through il- or il- leading to th1-polarization is commonly modeled by toll-like receptor agonism with bacterial products, the prototypic example being tlr agonism by bacterial lipopolysaccharide. the nature of stimuli driving a th environment is less clear. il- and il- are candidates for the promotion of an innate th2-priming environment. however, il- or il- knockout dc can drive th responses. clues as to the supply of a pro-th dc program have come from looking at th2-associated pathogen responses. schistosome egg antigen, which induces a robust th program in dc, capable even of overriding bacterial th signals, depends on a relatively non-activated transcription profile in the dc, somewhat reminiscent of a tolerogenic profile. this is in keeping with the view that activation of th cells may encompass reduced t cell receptor avidity and/or co-stimulation and altered signaling, synapse formation and off-rates. several lines of evidence suggest that th activation has different avidity requirements for th activation. a basic precept is that the tcr reads different potencies of activation to initiate different effector outcomes with the ‘strength of stimulation’ required for a th response greater than for a th response. furthermore, more sustained engagement of the tcr by pmhc is required to polarize tcr transgenic lymphocytes into a th program. the serial triggering model of t-cell activation offers an explanation for these observations; since any given pmhc complex must activate several receptors for cell triggering, the affinity of the interaction must be low and/or the off-rate fast to enable disengagement to occur and the more sustained the necessary interaction, the greater this will be the case. there are many studies on the relative dose of antigen required for a th or th response; a common observation is that th responses require relatively high concentrations, and th responses much lower. possible mechanisms for a relationship between antigen dose and t cell polarization may be both through a direct effect on differential cd signaling and through the ability to modulate expression of co-stimulatory molecules expressed by cd cells; high antigen dose can favor th development through dose-dependent up-regulation of cd40l. indeed, it has been argued that ca2+ signaling may be reduced in th response and that the threshold interaction necessary for inducing ca2+ signaling may not be reached by th cells. in several models, pmhc complexes showing reduced affinity interactions with tcr are associated with preferential skewing to a th response. t cells specific for proteolipoprotein 131- and selected under th2-favoring conditions show a shift in peptide-tcr primary contact residues compared with th clones. while avidity maturation on a per-cell basis is a property of the b cell and not the t cell repertoire, t cell clonal selection leads to the progressive appearance of selective tcr usage. for example, the repertoire of tcr response to sperm whale myoglobulin 110- in adjuvant is initially diverse, then progressively losing lower affinity clones and resulting in an oligoclonal population of tcrβ receptors of intermediate affinity. similarly, pigeon cytochrome c peptide specific tcrβ chains show strong selection for cdr length and residues characteristic of antigen binding, compatible with progressive clonal maturation and the suggestion of population-level affinity maturation. repertoire maturation in development of the cd response to listeria involves narrowing of the tcr repertoire, associated with increased affinity. knowing that development of a response involves selective fine-tuning from available receptors and that avidity requirements differ for activation of different th subsets, it would be expected that they might selectively expand different tcr repertoires. we previously showed that selection of favored tcrs from the peptide-specific pool differs under th or th conditions. when an initial, mixed pool of primed cells was divided into th or th polarized cultures, different tcr sequences were preferentially selected under the two conditions. across different pmhc combinations, there was no clear pattern or homogeneity evident in the selection for tcrβ usage, but th conditions preferentially selected elongated cdr3α sequences. the example of the response to plp 56-70/h-2ag was studied in detail. screening of polarized th line libraries suggested that while th cultures favored these elongated cdr3α loops but also encompassed receptors with shorter loops, the long loops could never be found in th cultures. molecular modeling offered a potential explanation, predicting a bulky, obstructive interaction of reduced affinity for the elongated th receptors. thus, contrary to experiments with the do tcr transgenic mouse, whereby a th1-derived tcr can be skewed to mediate either th or th effector functions, in a physiological polarizing environment, receptor features may be preferentially selected so as to skew the future memory response for an appropriate cytokine profile. while it had been envisaged that the cytokine program was faithfully transmitted to progeny cells by chromatin remodeling, polarization is now perceived as a plastic event. in this context, there may be evolutionary advantage in building information on the appropriate response into the tcr itself, so that the response cannot be diverted to a pathogen-inappropriate response by the local inflammatory environment. we here investigate this hypothesis by generating tcr transgenics carrying an elongated cdr th2-derived tcrα chain. we show that cells carrying this receptor, particularly when paired with the appropriate tcrβ partner, facilitate cytokine skewing away from a th program. this is the first time to our knowledge that a causal link between tcr repertoire maturation and th effector polarization has been shown. the findings suggest that the program for recall of a context-appropriate phenotype may be influenced not just by heritable epigenetic changes, but also in the choice of dominant tcrs themselves.  tcr usage and co-stimulatory molecule expression in polarized cd t cell lines we previously described the relatively homogeneous tcrα usage associated with a polarized th response to plp to. a noteworthy feature of this tcr usage was the choice of cdr3α loops that were elongated, an observation extended across several different pmhc combinations, suggesting that the th cytokine environment had influenced preferential utilization of tcrs with these structural features. in order to establish the impact on cytokine polarization of th2-derived receptors with these features, tcrα chain transgenic lines expressing a representative, elongated cdr3α receptor were generated. in order to select such a tcr sequence, we sequenced tcrα and β chains from polarized th lines specific for this pmhc combination. primed lymph node cells from mice primed with peptide in complete freund’s adjuvant were split into cultures stimulated with antigen in vitro under polarizing th th or th conditions. to generate th lines, cells were cultured in medium containing il- il- and anti-il- to generate th lines, cells were cultured in medium containing il- il- and anti-ifnγ. for th lines, cells were initially cultured in anti-ifnγ, anti-il- il- and tgfβ and expanded in medium containing il- and il- each set of conditions yielded a repertoire of tcrs with reproducibly distinct features. in each case, the distinctive cdr3s were a characteristic of the tcrα and not the tcrβ repertoire. under th conditions, cultures predominantly favored a vα15jα receptor with a amino acid cdr3α, aasregtgskls. we never found this receptor or ones similar to it in cultures derived from the same lnc pool but polarized under th or th conditions. cells cultured under th polarizing conditions favored very different tcrα usage, a quarter of the sequences comprising a vα9jα receptor with an elongated, 14-amino acid cdr3α loop, alegiasssfsklv. this was not accompanied by any overt selection for dominant tcrβ chains. interestingly, cells cultured under th polarizing conditions were also distinctive, a large proportion of sequences being vα11jα comprising a short, amino acid cdr3α loop. again, there was no preferred, expanded tcrβ chain. the findings suggested selection from the available pool primarily on the basis of tcrα sequence and permissible pairing of dominant tcrα chains with multiple possible β chain partners. the th cells were similar to th1-polarized cultures in being limited to short cdr3α loops. there was no significant difference in tcrβ chain cdr length seen in th, th and th tcr sequences. however, tcrα chain cdr lengths were significantly longer in th than in th and th tcr sequences. t cell lines were maintained in culture for one round of re-stimulation under highly polarizing conditions before taking rna for tcr sequence analysis. tcrα and β chain repertoire analysis of an h2-ag7/plp 56- specific th th and th cell lines shows expansion of distinct, dominant tcrα chains in the absence of a dominant β chain. the frequency of each unique tcr sequence identified is shown. cdr length is defined as the number of amino acids between the invariant c residue and the f/w-g-x-g motif. tcrα and tcrβ cdr region lengths are shown in each table. data are representative of tcrα and tcrβ sequences for each t cell line. the mean cdr length for the tcrα chain is  ±  for th cultures,  ±  for th cultures, and  ±  for th cultures. the most dominant tcr alpha sequence is shown in bold. mean cdr3α length  + . mean cdr3β length  + . the most dominant tcr alpha sequence is shown in bold. mean cdr3α length  ± . mean cdr3β length  ± . the most dominant tcr alpha sequence is shown in bold. mean cdr3α length  ± . mean cdr3β length  ± . when assayed for release of ifnγ, il- il- il- il- il- and il- each of the polarized lines showed the expected profiles. a caveat here is that, in line with observations from others using in vitro and in vivo models, t cells within th cultures could flip relatively easily into co-expression of il- and ifnγ or single expression of ifnγ, particularly if derived from an ex-vivo inflammatory environment. in line with the idea that selection from the initial pool under th polarizing conditions acts through selection of clones with differing avidities, th and th lines developed with markedly differing profiles of co-stimulatory molecule expression. th lines show greater expression of cd40l, in line with the idea that cd40l-blockade preferentially blocks th responses. expression of ox intercellular adhesion molecule- and cd were also greater on th lines. conversely, th inducible t-cell co-stimulator expression was greater, as predicted by the finding that icos stimulation preferentially triggers th cytokines. the cd expression differences are reminiscent of observations in a recent paper by mandl and colleagues, who showed that tcr transgenic cd5hi clones accounted for high tetramer binding. generation of tcrvα chain transgenics we expressed the immunodominant selected tcrα sequence receptor with an elongated, 14-amino acid cdr3α loop, alegiasssfsklv) in the ptα expression cassette, and made two independent transgenic founder lines expressing the dominant th2-derived tcrα chain with an elongated cdr region. we expressed the immunodominant selected tcrα sequence receptor with a 12-amino acid cdr3α, aasregtgskls) in the ptα expression cassette, and made one transgenic founder line with the dominant th1-derived tcrα chain with a shorter cdr region. all the transgenic lines were then backcrossed onto the original non-obese diabetic, h2-e transgenic background in which the t cells had been characterized. transgenic expression of tcrvα chain with an elongated cdr does not favor th responses in primed cells with respect to tcr selection in th t cell lines, the absence of any dominant tcrβ chain in cultures led us to hypothesize that transgenic expression of the tcrα chain with an elongated cdr region alone might be sufficient to impact on cytokine phenotype of the t cell response. we initially assessed the functional impact of the elongated tcrα chain on cd4+ t cell responses by immunizing mice to look both at initial recall responses and subsequently after a second antigen boost in vivo. the nature of ex-vivo cytokine responses is heavily influenced by the adjuvants used for priming, with cfa giving strong th polarization through the effect of m. tuberculosis on myd88-dependent signaling. we, therefore, assessed whether this tcr could confer propensity to develop a th program in the face of a th1-skewing, cfa priming regimen. mice were immunized on day and day with plp to in cfa and incomplete freund’s adjuvant, respectively, and t cell responses sampled at days and at each timepoint, t cell responses of tcrα chain transgenics were similar to littermate controls as judged by proliferation. interestingly, however, while the littermate controls generated a strong ifnγ response, this was absent after the initial recall in the tcrα transgenics at day. this suggested that use of this receptor was less effective at maintaining ifnγ transcription. when mice were boosted, we observed an ifnγ response in littermates but not in tcrα transgenics. t cells from the tcrα chain transgenics were harvested at day cultured with peptide for 48 h and cdr analysis of the cd4+cd69+ and cd4+cd69− cells was carried out. the lack of an ifnγ response in tcrα transgenics after boosting correlated with selection of a single shared vβ partner chain, as demonstrated by tcr sequencing and by spectratyping using v gene specific primers. all the cd4+cd69+ cells expressed the same tcrβ chain, trbv trbj2- with an elongated 13-amino acid cdr region assqagtgedtqy. thus, the defective ifnγ recall response was associated with the clonal expansion of a population of cells expressing a single trbv trbj2- chain with an elongated cdr cytokine polarization evolves in cultured cell lines in line with favored tcrαβ selection in order to look in more detail at the relationship between progressive polarization and focusing of the tcr repertoire, we set out to look at polarization and tcrβ selection during progressive re-stimulation of t cell lines in vitro. this was done in the absence of exogenous polarizing factors in the medium. this allowed us to further explore the possibility that, with progressive re-stimulation and selection, there may be increasing focus on particular tcr pairs, leading to impaired th responses and gradual promotion of th responses. primed draining lnc were used to establish t cell lines in vitro. elongated cdr tcrα chain transgenic lines and short cdr tcrα chain transgenic line and transgene negative littermate controls were primed with peptide in cfa and, 10 days later, lnc were harvested and t cell lines established. line and line tcrα transgenic cells rapidly selected dominant tcrβ chain expansions. by the fourth re-stimulation, over half of the tcrβ repertoires for the tcrvα transgenic lines comprised a single vβ chain and by the sixth re-stimulation, the tcrvβ repertoire was composed almost exclusively of this vβ chain. these studies, along with the studies shown above in figure  argue that several different tcrβ chains can pair with the elongated tcrα chain to yield the desired pmhc specificity and that in a given mouse or t cell line, one of several possible sequences may acquire clonal dominance. this selection of tcrβ chains in the context of pmhc activation was confirmed by spectratyping. as early as the second re-stimulation, cd t cell lines from line tcrα transgenics show constrained spectra, with a significant contraction of the tcrvβ chain repertoire for several vβ gene families while spectra from littermate control t cell lines show tcrvβ diversity. we then looked at this tcr focusing during evolution of a given t cell line in relation to skewing of cytokine production. at the same time as making cdna at each re-stimulation for tcr sequence analysis and spectratyping, cytokine production was analyzed by elisa and rna used for real-time analysis of subset-specific transcription factors. with progressive re-stimulations in vitro, the reciprocal nature of th and th polarization in littermate controls and tcrα transgenic lines with the shorter compared to those with elongated cdr regions is seen. tcrα transgenics with the elongated cdr region make little or no ifnγ, but, after a lag of one re-stimulation, make large amounts of il- il- and il-. tcrα transgenic lines with the shorter cdr region and non-transgenic controls make ifnγ, but no il- il- and il- in line with this, gata- transcription is progressively up-regulated in tcrα elongated cdr transgenic cultures compared with littermate controls. there is no bias of the tcrvβ chain repertoire in naïve tcrvα chain transgenic splenocytes at day, or in a primary response in draining lymph nodes at day post-immunization as demonstrated by spectratype analysis. generation of tcrαβ transgenics the likely interpretation of the simultaneous appearance of dominant tcrβ chain sequences in the tcrα chain transgenic lines, adoption of a spontaneous th phenotype and impaired th program, was thus that features of this preferred tcrαβ pair were incompatible with effective maintenance of th activation and, therefore, favored emergence of a th response. we therefore expressed one of the immunodominant, selected tcrβ sequences in the ptβ expression cassette, generated tcrβ transgenics, and crossed these to line tcrα transgenics on the same nod.e background. tcrαβ transgene impacts on ex-vivo t cell phenotype to assess the functional impact of the tcrαβ on primary ex-vivo t cell responses to peptide, tcrαβ transgenics and littermate controls were primed with peptide/cfa, again supplying a maximal th1-skewing environment. at day t cell responses were analyzed ex-vivo and t cell lines established. while tcrαβ transgenics, as would be expected, show enhanced t cell proliferation responses to peptide, ifnγ and il- responses were absent, and they made substantial amounts of il-. as before, immediately ex-vivo we could detect no il- il- or il. the absent production of ifnγ and il- and enhanced production of il- observed in the tcrαβ transgenic line was seen in the context of priming with the th promoting adjuvant cfa making this result all the more striking. il- production was not significantly different between the groups. the phenotype of the tcrαβ transgenic lines was further confirmed by real time pcr for ifnγ, gata- and rorγt. the tcrαβ transgenic lines expressed high levels of gata- and low levels of ifnγ and rorγt relative to the littermate controls in keeping with a th phenotype. furthermore, gata- expression increased with successive re-stimulations, while rorγt expression decreased in keeping with the reduction in il- production. the level of t-bet transcription, however, showed no obvious pattern through re-stimulations of the tcr transgenic or wt lines. this is in line with an increasingly complex model for t-bet in the th program: in several models, th polarization occurs in the face of t-bet transcription. because our investigations had used cfa priming, it was important to gauge the effect of immunizing tcrαβ double transgenic, tcrα chain single transgenic and littermate control transgenics with peptide in ifa lacking th1-skewing killed mycobacteria. at day lnc were harvested and 3h-thymidine incorporation and cytokine production determined ex-vivo. the results are in line with the larger pmhc-specific t cell response in the tcrαβ transgenics, but confirm the th bias: tcrαβ transgenic cells respond strongly to peptide making il- il- il- il- and il- but no ifnγ or il-. tcrα chain transgenics, and littermate controls made il- and small amounts of il- only. this is in keeping with the tcrαβ receptor supporting a th phenotype. addressing alternative hypotheses for cytokine-skewing in the tcr transgenics the tcrαβ transgenic lines make more il- than littermate controls, indicating t cell survival potential. the high il- response in the tcrαβ transgenic line, taken together with the high il- and il- response, implies that there is no global failure of t cell activation/survival per se, but merely a cytokine-deviated response. this appears to argue against a possible alternate hypothesis, that increased precursor number may have caused greater competition for ifnγ signals and decreased t cell fitness and/or memory. to further address this issue, we immunized tcrαβ transgenics and littermate controls on day and day with plp to in cfa and ifa, respectively, and looked at markers of cell activation and cell survival by real-time pcr and fluorescence activated cell sorter. real-time pcr analysis of b-cell lymphoma-extra large expression gave no indication of an enhanced propensity to apoptosis. anti-apoptotic proteins, such as bclxl, are involved in protecting mitochondrial integrity, for example, in the context of limiting growth factors. bclxl is induced on t cell activation and enhanced by co-stimulation of cd, and it is generally considered that over-expression of b-cell lymphoma or bclxl in t cells prevents death by neglect. furthermore, data from gett and colleagues indicate that induction of bclxl on sustained activation depends on stimulation strength and is associated with resistance to apoptosis, bclxl increase being greatest in t cells that had received prolonged stimulation. if tcr transgenic cells were incapable of fully functional stimulation and destined for some form of sub-optimal stimulation or death by neglect, one would predict diminished activation of bclxl in the transgenic response. in fact, we found no difference in bclxl expression. taken together with the other evidence shown here, including the strong il- response on activation, we interpret the findings to mean that there is no deficit in activation of the response through the transgenic receptor, but rather, deviation of the transcriptional response to an altered cytokine profile. furthermore, phenotypic analysis of the primed lymphoid populations at day showed no difference in activation/fitness as indicated by staining for cd. it has been shown by whitmire and colleagues in a transgenic lymphochoriomeningitis virus response cell transfer model that only when the number of transferred t cell precursors was low did they generate fully functional memory cells that were cd127/il-7r, with full capacity to produce cytokine and proliferate. testing therefore whether it was possible that an excess of responder cells here correlated with a response phenotype that was in some way impaired, we analyzed t cell expression of cd and found that transgenic cells had enhanced, not reduced expression. taken together with the other evidence presented here, this makes it unlikely that this was a model of sub-optimal activation. furthermore, expression of cd62l was similar in the transgenic and littermate responder populations. thus, the indication from il- release, as well as apoptosis and activation markers is that the phenotype observed here constitutes an actively skewed functional preference, rather than a failure to trigger properly by the transgenic receptors. it was hypothetically possible that injection of cognate peptide into a single chain tcrα and tcrαβ transgenic mice had triggered a cytokine storm due to the large number of responding t cells, and this had in turn caused thymic apoptosis leading randomly to a skewed cytokine outcome in surviving cells. to address this we conducted additional studies. ‘cytokine storm’ is a term derived from the systemic response in toxic or septic shock. here, an acute, excessive spike of systemic pro-inflammatory cytokines leads to downstream events, often including large-scale lymphocyte apoptosis. in our studies the positive control stimulus of seb led to the predicted spike in systemic pro-inflammatory cytokines, such as ifnγ and tnfα at 2 h, but no systemic change in cytokines was detected after injection of peptide/cfa. it was nevertheless possible that even in the absence of a systemic response there may have been some acute, storm-driven change in thymic populations accounting in a non-specific way for cytokine skewing. while seb had a dramatic effect on overall thymocyte numbers and cd4/cd ratio in littermate control as well as tcrα and tcrαβ transgenics, no such effect was seen with cognate peptide. none of the peptide-primed non-transgenic controls showed preferred tcrβ chains in sorted cd sp cells from thymocytes. however, in one out of four of the mice analyzed, tcrβ chains in sorted cd sp cells from thymocytes in peptide-primed tcrα transgenics show preferential selection of a preferred tcr heterodimer, 44% of tcrs using a common tcrβ chain. on the basis of these experiments, the occam’s razor hypothesis, that tcrα chains of differing structures and avidities can influence cytokine program, appears more likely than cytokine preference emerging as a stochastic event in the aftermath of a cytokine storm. t cell lines with tcrαβ receptor have low binding avidity we previously predicted from molecular modeling studies that the elongated cdr region of the tcrα chain, when paired with a preferred tcrβ chain, might result in a lower avidity interaction between the tcr and its pmhc ligand. we therefore started by reappraising functional avidity in a peptide titration of th th and th polarized, non-transgenic t cell lines cultured for eight days in polarizing medium and looking at ifnγ, il- and il- elispots, respectively. in these short-term lines, to achieve a response of sfc/ cells requires about times the peptide concentration in the th lines compared to th and about times the peptide concentration in the th lines compared to th. we next looked at tetramer binding characteristics of th th and th polarized, non-transgenic t cell lines cultured for eight days in polarizing medium and prepared from the same initial pool of primed lnc, using h2-ag tetramers loaded with either plp to or an irrelevant h2-ag7-binding peptide. at all concentrations of tetramer tested, a significantly greater proportion of th and th polarized cells bound tetramer compared to th cells despite equivalent levels of cell surface cd. similarly, in a peptide titration to examine the functional avidity of short-term t cell lines from tcrαβ transgenics relative to littermates, an equivalent number of il- spot-forming cells in the tcrαβ transgenics require approximately times the peptide concentration required for the ifnγ response in littermates. we then compared tetramer binding characteristics of t cell lines derived from the th2-type tcrαβ transgenic cells, tcrα chain transgenics with an elongated cdr and th1-type cells from non-transgenic littermate controls. as tetramer concentration increased, a higher frequency of th cells from the control littermate bound tetramer. however, no tetramer binding was detectable for the tcrαβ transgenic cells, indicating that the avidity of the interaction with the tcrαβ cells was too low for detection. the tcrα chain transgenics with an elongated cdr had an intermediate binding avidity. we demonstrated a functional interaction between the tetramer and tcrαβ transgenic t cells by measuring cytokine production from transgenic cells cultured in the presence of plate-bound tetramer. in keeping with the th phenotype, il- il- il- and il- but not ifnγ were detected. the th polarized t cell lines produced less il- with each successive re-stimulation such that by the fourth re-stimulation cytokine production was more in keeping with that of a th cell line. we hypothesized that this shift in cytokine production would be accompanied by a reduction in tetramer binding avidity and longer tcrvαcdr length as t cells with a ifnγ producing phenotype clonally expand. in keeping with this, we observed a reduction in tetramer binding avidity between the first and fourth re-stimulations of polarized th lines such that at the fourth re-stimulation, tetramer binding curves for th and th lines overlapped. tcr repertoire analysis at the first and fourth re-stimulations confirmed that the change in cytokine production and tetramer binding avidity occurred alongside an increase in average tcrvαcdr length. the mean tcrvαcdr lengths for the first and fourth re-stimulations are  +  and  + , respectively,. the difference in mean cdr3α length between the first and fourth re-stimulation shows progression to a tcr repertoire with longer cdr3α regions as cells lose their il- producing phenotype and become more th1-like in their tetramer binding characteristics and cytokine production. in light of previous observations in tcr transgenics, notably the early studies by hosken and colleagues using the do tcr transgenic line, it might be predicted that the th tcr transgenic-skewed polarization may be overcome by altering peptide dose. certainly, among the many factors that can skew cytokine bias would be peptide dose itself. in the do tcr transgenic studies, it was shown that under otherwise equivalent in vitro primary culture of naïve cells, mid-range peptide doses favored the generation of moderate ifnγ responses, while either higher or lower doses favored a switch to development of more th2-like responses, giving the ifnγ response curve the form of a bell-shaped curve. we, therefore, performed a set of experiments in our system to examine the behavior of the th2-derived transgenic tcr across a checker-board of low to high peptide priming dose and a low to high in vitro re-stimulation dose. our previous experiments had typically utilized a priming and re-stimulation dose of 25 μg/ml, corresponding to 13 μm towards the upper-end of the concentration range that had been tested by hosken and colleagues using naïve t cells in vitro. we initially primed th tcrαβ transgenic mice with peptide at or 125 μg. popliteal lnc were then challenged in vitro with a titrated dose of peptide from to 100 μg/ml. as we had seen before, peptide priming with 25 μg/ml did not trigger an ex-vivo ifnγ response, though ifnγ could be elicited at either a higher or lower dose of peptide priming, or 125 μg/ml. thus, these findings are reminiscent of the studies by hosken and colleagues, except that the mid-range peak of ifnγ production that had been seen by them for the do tcr is here exchanged, using a th tcr, for a low/high range peak of ifnγ production. our curve is the reciprocal of the do finding. furthermore, the mid-range dose of priming with 25 μg/ml peptide was associated with increased transcription of both gata- and rorγt. thus, while there is a constraint on ifnγ activation, this is peptide dose dependent and can be over-ridden by either high or low dose peptide priming. we then used this checker-board titration to analyze cytokine secretion in t cell lines from peptide primed th tcrαβ transgenic mice, assaying their profile after the third re-stimulation in vitro. the findings are summarized in figure  il- responses are depicted in blue, il- in green and ifnγ in red. the key arbiter of cytokine profile in vitro appeared to be the dose of peptide used for in vitro re-stimulation of the lines. that is, whether initial priming was with or 125 μg, the t cells lines produced relatively large amounts of il- and il- but little or no ifnγ when re-stimulated with peptide in the range to 10 μg/ml. however, irrespective of the initial priming dose, the t cell lines showed an ifnγ response when re-stimulated in vitro with 100 μg/ml peptide.figure  is a schematic summarizing the flow of experiments designed here to test the hypothesis that elongated tcr alpha chain complementarity-determining region favors a th2-skewed cd phenotype. discussion there has been considerable effort devoted to elucidating the structural biology of the tcr complex, with 10s of structures solved, yet clear models for the way in which the ligand/receptor interaction between pmhc and tcr may give rise to the very diverse array of t cell effector programs have been elusive. most tcr transgenic lines are generated without regard for specific features of the cytokine profile of the parental cell from which they were derived. experiments in some autoimmune models bear on this insofar as there are examples in which t cell clones were implicated in an autoimmune effector mechanism and the tcr transgenics derived from them can mediate a spontaneous disease phenotype dependent on preferential use of a similar cytokine profile. candon and colleagues generated transgenics for the tcr of a self-reactive th clone, showing that a proportion of the mice spontaneously developed autoimmune gastritis characterized by eosinophilic infiltration of the gastric mucosa and th differentiation of transgenic t cells in the gastric lymph node. appropriate th polarization is most readily attributed to t cells engaging pmhc in the context of an antigen presenting cell that has been activated by innate signals to provide a particular, polarizing environment. could another aspect of the polarization reside in the tcr sequence itself? this would have evolutionary value as a contributory mechanism in polarization since, without it, the appropriateness, or otherwise, of the effector response could be at the mercy of influence by any other concurrent processes and/or infections. it is self-evident that there may be tcr structures that are ‘vanilla’ in nature and capable of being diverted readily to either a th or th program; do is the prototypic example. knowing that tcrs of cd cells have been reported across a moderately broad range of affinities and also that affinity/avidity are key determinants of cytokine polarization, it may be predicted that tcrs of different structures would, all else being equal, show inherent bias to different effector profiles. the principle that tcr sequence correlates with function is accepted in the special case of the treg versus teff tcr repertoires, although even in this setting, phenotypic differences have proved difficult to define. we show, using new tcr transgenic models, that an elongated tcrα derived from a strongly th2-skewed t cell line retains imprinting of that effector program to the extent that, even in the face of an overwhelming th1-polarizing environment, such as immunization in cfa containing m. tuberculosis, t cells can proliferate while lacking ifnγ responsiveness. while the polarization is mediated through the selection of appropriate cdr3α regions and the constraints on tcrβ selection are less apparent, the full th program is clearly dependent on selecting the correct tcrαβ pair. a starting point for the studies here was to reappraise more broadly our earlier observations about preferential selection of the tcr repertoire. this can be thought of as ‘avidity-associated functional maturation’ of the response; while it has long been known from many pmhc examples that the tcr repertoire can focus in on sometimes highly dominant receptor usage, we add the observation that this choice is highly influenced by factors driving th1/th polarization. a key observation, reiterated in the current study, had been that th polarization is associated with cdr3α chains with somewhat elongated loops. in the present study, we have extended this analysis to investigation of th17-polarized cultures. once again, tcrβ chains remained relatively heterogeneous while tcrα sequences rapidly attain focused, preferential usage; although selected out of the same starting pool, the th receptor usage is dissimilar to either th or th lines. like th lines, tcrα chains are invariably of the th1-like, short, cdr3α type. we found no examples of elongated cdr3α chains in th cells. this may be taken to mean that, like th cells, th activation is dependent on ‘high-end’ affinity activation and depends on activation that is incompatible with low affinity tcr activation. this is in line with the relatively easy plasticity from th to th lineages, although it is also known that there are differences between activation signals for th and th activation, such as dependence on pkcθ. on the other hand, we found little similarity between the actual cdr3α sequences selected in th and th cultures. to our knowledge this study constitutes the first report of preferential tcr usage associated with the th phenotype with the associated implications for differential avidity of th cells. th cells are predicted to be at the upper end of the avidity spectrum, with associated implications for differential signaling and synapse formation. a number of studies have sought to relate changes in the tcr repertoire to the development of the t cell response, although not in the specific context of cytokine programs. studies of cd responses have variously shown either a stochastic relationship between the initial response and the expanded repertoire, or substantial focusing with preferential selection of particular clonotypes, sometimes associated with enhanced avidity. those examples of cd repertoire selection that have been investigated in detail have also tended to focus on the progressive loss of low-affinity clones to achieve the ‘best fit’. what is unclear is how this model could be reconciled with a continuing requirement for differential avidities associated with alternate cytokine programs. certainly, cells within a th memory population can make ifnγ if reactivated in the context of innate th signals. that is, it might not be possible to maintain an overall dominance of th clones in the face of a tcr repertoire progressively shifting to higher avidity. for example, in th1/th dependent experimental autoimmune encephalomyelitis tcr transgenic models, strength of pmhc-tcr interaction is correlated with more aggressive, spontaneous disease. conversely, decreased cd expression by polarized th cells has been shown to contribute to reduced tcr-induced phosphorylation and ca2+ signaling. profiling of co-stimulatory molecules on th and th lines showed differences compatible with the notion of enhanced avidity promoting th differentiation. up-regulation of cd40l expression in th cells is a previously well-documented example of this phenomenon, confirmed here. up-regulation of icos was a strong marker of th differentiation, while cd up-regulation was a strong marker of th differentiation. in line with the notion of cd up-regulation as a marker of enhanced avidity in th cells, cd expression in thymocyte selection is correlated with avidity/signal intensity of the positively selecting tcr-pmhc interaction. for differential engagement between pmhc on the apc and the tcr to lead to these very divergent transcription programs, there must be substantive differences in signaling, from initial formation of the immune synapse. a number of studies have previously considered differences in immune synapse formation and tcr signaling between th and th cells: most of these are compatible with the notion that the synapse leading to th differentiation comprises a less tightly focused, lower avidity complex. th but not th cells, fail to cluster tcr at the cell-cell interface due to increased expression of cytotoxic t-lymphocyte antigen. in line with this, co-clustering of tcr with cd in lipid rafts is more common in th cells. th immune synapses are morphologically distinct, characterized by the failure to exclude cd and icam- from the central zone. these changes are presumed to contribute to diminished strength of signaling. furthermore, a number of studies have considered mechanisms by which these differences might in turn lead to qualitative signaling differences and alternate transcriptional choices. strength of signaling has been shown to affect the balance of nfatp and nfatc binding activity, thus directly regulating il- transcription. we predict from our tcr analysis of th th and th lines that th synapse formation and signaling would be distinct from the low avidity interactions characterizing th activation and more reminiscent of th activation. jameson and masopust have summarized a number of studies on the cost to the quality of memory of too much stimulation with the term, ‘everything in moderation: better memory by avoiding over-exertion.’ is it possible that our th tcrαβ transgenic model in some way elicits a response that is sub-optimal through excessive or inappropriate stimulation? while our observations here have focused largely on cytokine polarization of the initial response, all of the markers assessed here, including il- activation, cd expression and anti-apoptotic markers indicate that responder cells in this transgenic system are fully fit to respond, they merely choose to do it in a different way. the key evidence that cytokine program can be influenced by the selected tcrαβ sequence itself came with the observation that by transgenic expression of a th derived receptor, we produced mice that are ‘th1-averse’, even in the face of the most powerful possible stimulus to reverse this; despite showing strong proliferation in response to peptide, as would be expected, considerably higher than wild-type mice, there is no ifnγ or il- response. rather, the tcr transgenic mice make a strong th response, not seen at all in the littermate controls. why should this be the case? we speculate that it is an evolutionary failsafe to ensure pathogen-appropriateness of the response. in experimental immunology, we tend to work with reductionist systems where we can examine polarized th th or th responses, unimpeded by other influences. however, in the natural host response to infection there will be many competing influences; thus, th responses have presumably evolved to provide protection against parasitic infestation, yet in many parts of the world facing a major parasite burden, there is also a high level of infection with m. tuberculosis, itself providing a concomitant drive to th and th immunity. indeed, we have previously described another clinical example of this type in respect to the co-existence in the lung of sarcoidosis, which is associated with strong th responses, and cryptococcus neoformans, for which th immunity is critical. in complex settings, being able to avoid potentially dangerous reprogramming from pathogen-driven, cytokine milieu and promoting an appropriate effector response, is vital. cells with lower affinity tcrs adopt a th phenotype when primed in the absence of competition from cells with higher affinity receptors. evidence that the th2-derived tcr used in these studies are of low affinity comes from molecular modeling studies done in collaboration with yvonne jones’ laboratory. in the present study, we were able to confirm those predictions using direct tetramer binding studies. we show here that while wt littermate primed cells respond to plate-bound tetramer by making ifnγ, tcrαβ tg cells respond by making th cytokines, but no ifnγ. this further emphasizes the fact that the property is purely one of the interaction between pmhc and tcr, uninfluenced by other contributory differences of co-stimulatory molecules. however, despite the fact that both cell populations can be activated by tetramers, binding to the transgenic t cells is sufficiently low as to be undetectable. the tetramer binding studies, taken in conjunction with the preferential tcr selection described by us in polarized lines, predict that the preference for tcr avidity will be th17 > th1 > th  we propose an additional and previously undefined mechanism for ensuring the cytokine appropriateness of cd immunity. tcr preferentially selected under th conditions tended to use elongated cdr3α loops and when expressed in transgenics, skewed responses away from secretion of ifnγ, favoring th cytokines. focusing on the t cell repertoire in antigen specific adaptive immunity can bring with it information not just about pmhc specificity, but also about the qualitative nature of the appropriate cytokine response. 
in tetramer-binding studies, this transgenic receptor showed low binding avidity for pmhc and polarized t cell lines show tcr avidity for th17 > th1 > th while transgenic expression of a th2-derived, ‘elongated’ tcr-cdr3α and the tcrαβ pair, clearly generated a program shifted away from th immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose. the dominant th2- and th1-derived tcrα sequences with amino acid cdr loops and amino acid cdr loops, respectively, were expressed in tcr transgenics. we propose that selection from responding clones with distinctive tcrs on the basis of functional avidity can direct a preference away from th effector responses, favoring th cytokines. we show that cd polarization involves distinct cdr3α usage: th and th cells favored short tcr cdr3α sequences of and amino acids, respectively, while th cells favored elongated cdr3α loops of amino acids, with lower predicted affinity. the functional impact of these tcrα transgenes was assessed after in vivo priming with a peptide/adjuvant. the short, th1-derived receptor transgenic t cell lines made ifnγ, but not il- or while the elongated, th2-derived receptor transgenic t cell lines made little or no ifnγ, but increased il- and with progressive re-stimulations, mirrored by gata- up-regulation. t cells from primed th tcrα transgenics selected dominant tcr vβ expansions, allowing us to generate tcrαβ transgenics carrying the favored, th2-derived receptor heterodimer. primed t cells from tcrαβ transgenics made little or no il- or ifnγ, but favored il- after priming with complete freund’s adjuvant and il- and after priming with incomplete freund’s. during any immune response, tuning of tcr usage through progressive clonal selection occurs.
 cd t lymphocyte activation requires t cell receptor engagement by peptide/mhc. the tcr complementarity-determining region contains variable α and β loops critical for pmhc recognition. during any immune response, tuning of tcr usage through progressive clonal selection occurs. th and th cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. we therefore hypothesized that cdr3αβ sequence features may intrinsically influence cd phenotype during progression of a response.  we show that cd polarization involves distinct cdr3α usage: th and th cells favored short tcr cdr3α sequences of and amino acids, respectively, while th cells favored elongated cdr3α loops of amino acids, with lower predicted affinity. the dominant th2- and th1-derived tcrα sequences with amino acid cdr loops and amino acid cdr loops, respectively, were expressed in tcr transgenics. the functional impact of these tcrα transgenes was assessed after in vivo priming with a peptide/adjuvant. the short, th1-derived receptor transgenic t cell lines made ifnγ, but not il- or while the elongated, th2-derived receptor transgenic t cell lines made little or no ifnγ, but increased il- and with progressive re-stimulations, mirrored by gata- up-regulation. t cells from primed th tcrα transgenics selected dominant tcr vβ expansions, allowing us to generate tcrαβ transgenics carrying the favored, th2-derived receptor heterodimer. primed t cells from tcrαβ transgenics made little or no il- or ifnγ, but favored il- after priming with complete freund’s adjuvant and il- and after priming with incomplete freund’s. in tetramer-binding studies, this transgenic receptor showed low binding avidity for pmhc and polarized t cell lines show tcr avidity for th17 > th1 > th while transgenic expression of a th2-derived, ‘elongated’ tcr-cdr3α and the tcrαβ pair, clearly generated a program shifted away from th immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose.  we propose that selection from responding clones with distinctive tcrs on the basis of functional avidity can direct a preference away from th effector responses, favoring th cytokines. cd t cellt cell receptorcytokinetransgenicmouseth17th1th2 