The broad possible pMHC reactivity generated by random gene rearrangements of α and β TCR chains on T cells would seem to ensure reactivity against a diverse array of pathogens, but this broad diversity then raises the question of how many T cells in a host would respond to a specific pMHC complex or against an entire pathogen. Many determinations of CD8+
T-cell precursor frequencies have now been used to calculate the number of pMHC-specific CD8+
T cells within a mouse (Table ), but the determination of the total number of CD8+
T cells that could respond to a viral infection would be possible with these methods only if all of the epitopes of the virus were known. However, the numbers of epitopes found to stimulate T-cell responses are now becoming quite large, as means for detecting them have become more sensitive. A virus like VV is now reported to encode close to 50 H-2Kb
- and H-2Db
-restricted epitopes (20
), a number that would make it very difficult, if not prohibitive, if the precursor frequency for the entire virus was to be determined with the usual methods. Herein, we have described an in vivo LDA that allows an unbiased determination of precursor frequencies for entire viruses without any knowledge of the specificity or number of epitopes.
It is interesting to note the differences calculated for T-cell precursor frequencies depending on the method used (Table ). Extrapolation of Vβ clonotype number per spleen by calculation of a measure of diversity by examination of the CDR3 sequences of pMHC-specific populations as done previously (12
) gives precursor frequencies on the high ends of most estimates, with the highest number of H-2Db
LCMV GP33-specific CD8+
T cells calculated among all methods. This is interesting in that this method is assumed to be an underestimate, because it does not include TCRα diversity, and it also does not account well for redundancy in T-cell populations, i.e., there may be more than one cell of a single clone in a naïve mouse. However, there is some uncertainty in these numbers because they rely on extrapolations of the numbers and diversities of sequenced clones to estimate T-cell precursor frequencies. Compared to our results, those obtained with this extrapolation method would suggest our calculations to be slight underestimates. If there are 1,100 to 1,200 GP33-specific CD8+
T cells per naïve mouse spleen, and the GP33-specific CD8+
T-cell response is approximately 10% of the total LCMV-specific response, we would expect to find about twice as many LCMV-specific precursors. Instead of the 6,760 per mouse as we calculated, we would expect 11,000 to 12,000 LCMV-specific CD8+
If precursor frequencies are instead calculated by transgenic-T-cell competition (2
), where transferred monoclonal transgenic T cells compete against heterogeneous endogenous T-cell populations to determine precursor frequencies, our results look to be overestimates. Assuming a 10% take and 2 × 107
T cells per mouse, this method estimates ~100 H-2Db
LCMV GP33-specific CD8+
T cells per mouse (2
). This result would put our LCMV-specific CD8+
T-cell precursor determination on the high end.
The tetramer-based enrichment assay, which makes use of pMHC tetramers, magnetic-bead enrichment and double-tetramer FACS staining of spleens and lymph nodes to identify pMHC-specific CD8+
T cells (assuming that most naïve T cells reside in lymph organs), seems to yield numbers that are in line with results determined by our in vivo LDA. Depending on the individual determination, there are ~287 (23
) or ~449 (15
LCMV GP33-specific CD8+
T cells in all lymph organs of a naïve mouse, and this result would be on the low end yet still compatible with what our results might predict for a frequency of GP33-specific naïve CD8+
T cell precursors. For VV, the in vivo LDA calculates about 13,850 responsive CD8+
T cells per C57BL/6J mouse. The tetramer-based enrichment assay estimated 1,070 CD8+
T cells specific for the H-2Kb
-restricted VV B8R epitope in the spleen, lymph nodes, and ovaries (10
), and those results would seem consistent with the results that we have described, considering that the B8R peptide response may represent about 10% of the VV-induced response.
About twice as many T cells were responsive to VV than to LCMV (P
< 0.0001). This might in part reflect the observations that the T-cell response to VV peaks earlier than that of LCMV. Having more CD8+
T cells that are specific to VV may increase the likelihood that VV-specific CD8+
T cells interact with stimulating antigen-presenting cells earlier, allowing peak T-cell proliferation to occur earlier. VV also encodes more proteins than LCMV, with almost twice as many VV epitopes described to occur in the C57BL/6J mouse, consistent with the result showing almost twice as many VV-specific precursor T cells than LCMV-specific CD8+
T cells (14
Our results also estimate that 8% of CD8+
T cells in VV-immune mice are VV responsive, and these data are supported by results obtained from VV-immune mice by using peptide stimulations and intracellular cytokine stains that estimate that anywhere from 2 to 11% of CD8+
T cells are specific to the VV-encoded immunodominant B8R epitope in D21 or D40 VV-immune mice (28
), and our own results from intracellular cytokine assays estimate that 0.5 to 2% of CD8+
T cells in VV-immune mice are specific to the VV B8R epitope at 3 to 8 months postinfection (data not shown). This increase in CD8+
T-cell precursor frequency for VV in VV-immune animals by more than 2 orders of magnitude (P
< 0.0001) demonstrates the expected considerable increase of VV-specific memory CD8+
T cells after VV infection. As expected, there was no increase in the number of CD8+
T cells that respond to LCMV in VV-immune mice, and this helps to validate the specificity of our assay. The small but significant decrease in LCMV CD8+
T-cell precursor frequency in VV-immune mice is interesting and may suggest that memory cells may displace some naïve cells in the immune response. We have not systematically addressed changes in VV-specific precursors in LCMV-immune mice because there is a high degree of heterologous immunity in this virus sequence, and the immunity, due to private specificities in the immune repertoire, has such high variability that our in vivo LDA would likely suffer from reproducibility issues (13
It is possible to make an approximation of the number of divisions a CD8+ T cell undergoes after stimulation by examining the burst size or recovered cell number at the limiting dilution. By determining the frequency of CFSElo donor cells among all CD8+ events collected, multiplying that frequency by the total number of CD8+ T cells found in the spleen, and then multiplying that number in accordance with the assumption that 67% of all CD8+ T cells are present in the spleen during infection, we are able to calculate the approximate number of divisions a CD8+ T cell undergoes after virus infection. The numbers of divisions that a VV-specific precursor undergoes by day 6 (~11 divisions) and that an LCMV-specific precursor undergoes by day 7 (~12 or 13 divisions) fall within predicted ranges. However, we approximate that a P14-transgenic T cell undergoes ~14 divisions by day 7 of an LCMV infection, and this is significantly different (P = 0.023) from the number (~12 or 13 divisions) that a naïve CD8+ T cell from a heterogeneous population of T cells undergoes. This may reflect differences in avidity between the transgenic T-cell population and the expected large range of avidities of T cells in a heterogeneous population as a whole or may instead be related to the examination of a monoclonal T-cell population that responds to a highly expressed immunodominant epitope versus a heterogeneous population of CD8+ T cells that contains T cells responding to immunodominant and subdominant epitopes.
The immunological environment produced by a specific virus infection can have a profound impact on the burst sizes of epitope-specific T cells, as has been demonstrated by experiments examining the T-cell response to recombinant viruses engineered to express the same T-cell epitope (21
). One explanation for this would be the expression of insufficient antigen to engage all of the T-cell precursors, as shown previously (11
). However, we used doses of virus that maximized the burst of the T-cell response, as in Ref (11
), and we feel that virtually all the precursors should have been engaged. Finally, T cells of differing affinities may undergo different numbers of divisions and expand to different peak sizes, as recently demonstrated (39
). Since the in vivo LDA requires extensive proliferation, it is possible that we have missed lower-affinity clones in our assay that would divide fewer than seven or eight times, and this would make our calculated precursor frequencies underestimates. However, analyses of T-cell responses to several epitopes suggest at least 12 to 14 divisions per T cell (23
), which would be detected by our assays. Further, our in vivo LDA seems to be able to detect the bulk of memory T-cell precursors detected by intracellular IFN-γ assays.
Our calculated take of CD8+
T cells is not the normally quoted 10% figure. If we instead use a 10% value for take, the precursor frequencies would be decreased with a naïve mouse having about 1 in 7,805 ± 1,034 CD8+
T cells specific for LCMV and 1 in 3,809 ± 452 CD8+
T cells specific for VV and a VV-immune mouse having about 1 in 34 ± 6 and 1 in 12,036 ± 4,812 CD8 T+
cells specific for VV and LCMV, respectively. Our own rough estimate of total cell numbers (Fig. ) would suggest that slightly less than 67% of CD8+
T cells reside in the spleen, as had been suggested (8
), but given that our attempt to count T cells throughout the body was not exhaustive and that the take would probably only change by at most a 20 to 30% value, we remain confident that our calculations are within a reasonable range of total virus-specific CD8+
T-cell precursor frequencies. Further, our experiments using P14-transgenic T cells also strongly support our estimation of take since our calculations of numbers of precursors equals the number of transgenic T cells obtained using that take value. The efficiencies of T-cell take in our experiments may seem to be in conflict with studies by others using OT-1-transgenic T cells, where 25% of the mice injected with a single OT-1-transgenic T cell had detectable responding donor T cells (31
). However, these single cells were injected intraperitoneally, followed by an immediate intraperitoneal infection. We, instead, chose to allow for a total-body distribution of T cells by way of an intravenous transfer and challenged mice with virus 2 to 5 days later. We therefore remain confident of our take value under the conditions of our system. Further, this in vivo LDA calculated at a 3.8% take gives frequencies with high concordance with the anticipated number of VV-specific memory cells, which can be measured directly by intracellular-cytokine assays. Considering the large amount of data we have in generating the 3.8% figure (Fig. ), we believe that our estimate of take is reasonably accurate in these experiments.
The in vivo LDA could be used to examine virus-specific T-cell precursor frequencies in mice of different ages or physiological states or in mice with histories of different infections. For example, a decline in IAV-specific repertoire diversity leading to epitope-specific holes in the repertoire in aged mice was recently reported (37
). Future experiments may be able to use the in vivo LDA described herein to determine whether whole-virus T-cell precursor frequencies in naïve or immune aged mice are also changed.
Whereas tetramer-based enrichment assays measure the number of cells that are reactive to a particular pMHC complex, the in vivo LDA requires CD8+ T-cell division and proliferation, measuring instead the number of CD8+ T cells that do proliferate in response to a viral infection. It might seem likely that not all virus-specific T cells would react with tetramer and that not all tetramer-specific T cells would be capable of proliferating in response to their cognate antigen. Remarkably, though, while the in vivo LDA described here tells us something different than the tetramer-based enrichment assays described recently, it gives reasonable concordance with those techniques.