The linear range for accurately quantifying antigen-specific T-cell frequencies by tetramer staining during natural immune responses.

Peptide-MHC Class I tetramer technology has allowed the identification, enumeration and isolation of antigen-specific CD8+ T cells without culture or further stimulation 1. MHC Class II tetramers for similar studies on CD4+ T cells have lagged behind mainly because of the difficulty in generating class II reagents 2. In many cases the abundance of antigen-specific T cells is modest so some form of enrichment by affinity chromatography (e.g. negative selection on CD8) allows practicable quantification on flow cytometry. Recently, positive enrichment by tetramers 3 has allowed quantification of such small numbers of antigen-specific CD8+ T cells that naïve frequencies can now be determined 4, 5. Tetramer staining has thus availed quantifying T cells under natural immune responses e.g. without the need for adoptive transfer of TCR-transgenic T cells. However, the number of naïve CD8+ T-cell precursors in the steady state can range from about 50 to 300 cells 3-6. During acute influenza infection and expansion of T cells, an increased percentage of CD8+ T cells is influenza-specific. This percentage decreases as the infection is being cleared, and a small proportion remains as memory T cells. Hence the number of antigen-specific CD8+ T cells can range dramatically, with a 1000-fold increment 9 days after influenza infection and a 10-fold decrement 7 months later 6, 7. Such a wide range of numbers may be challenging for accurate enumeration and the accuracy of quantification over such a range has hitherto not been validated. We therefore sought to determine the level of reliability of CD8 versus tetramer enrichment methods for quantification. Using the immunodominant PA224-233 epitope for our test tetramer, we simulated a scenario where we had a wide range in the number of antigen-specific CD8+ T cells and tested two different existing enrichment protocols. Using an aliquot of the single-cell suspension from infected spleen samples, the numbers of influenza PA 224-233-specific CD8+ T cells were determined by tetramer staining directly (without enrichment manipulation); the frequency in such samples was so high that this could be performed without excessive hours on flow analysis. The infected spleen sample was serially diluted (four-fold each dilution) with pooled naïve spleen cells. Each diluted sample, including the undiluted infected spleen cells, was then split equally to be processed by either positive selection with tetramer-coated beads or negative selection enrichment for CD8+ T cells. The cells were similarly stained for flow cytometry (Fig. 1) and the determined numbers compared with the values arithmetically predicted by the dilution factor of the original sample (Fig. 2). Gating strategy of the flow cytometry used for the identification of influenza PA-specific CD8+ T cells. Cells were first gated based on (A) their side and forward scatter profiles, followed by (B) selection of single cells based on the area versus height of forward scatter. (C) CD8+ cells were then differentiated by exclusion from the dump gate that included cells positive for CD4, F4/80 and CD45R surface markers. CD8+ cells were further separated into (D) PA-tetramer+CD3+ and (E) CD44high CD62Llow cells. Reliability for enumeration of antigen-specific CD8+ T cells using different enrichment protocols. Activated PA-specific CD8+ T cells in unprocessed samples were enumerated by staining for CD3+CD44high PA-tetramer positive CD8+ T cells without any enrichment. This number was used to calculate the predicted values using the dilution factor (circles). The samples of each dilution were equally divided into two portions; one for CD8 enrichment (squares) and the other for PA enrichment protocols (triangles). (A, B) Each graph presents a comparison of enrichment protocols in an individually infected mouse. Negative enrichment for CD8+ T cells on magnetic beads using a panel of antibodies against CD4+ T cells, B cells and dendritic cells, granulocytes, etc. allowed practicable FACS analysis for moderate numbers of epitope-specific CD8+ T cells by tetramer staining. We found that the enumeration using this method linearly matched the predicted values, when the absolute frequency was 103–105 (Fig. 2A and B show repeat experiments using different mice). At >105, it is better to dilute the sample (and therefore process fewer cells); we opine that the amount of tetramer being used became limiting. At <103, the quantification after CD8 enrichment overestimated the numbers of epitope-specific T cells. This occurrence corresponded with the events acquired on the tetramer channel being so few that the background events became relatively substantial and therefore inaccuracy ensued. Positive enrichment for epitope-specific CD8+ T cells on magnetic beads coated with tetramer greatly reduces the number of cells required for acquisition on FACS to determine the numbers of epitope-specific T cells. For frequencies <103, we found this method allowed for more accurate enumeration than for the CD8 enrichment method. As we performed our dilutions with naïve spleens, the absolute numbers of cells at high dilutions do not fall below the numbers of epitope-specific cells in the naïve mouse (102) 6. At high frequencies the tetramer enrichment method underestimated the absolute frequency. This was probably not due to the limiting capacity of the column or the capacity of staining because in such cases there would have been a plateauing with increasing frequency. This underestimation at frequencies of 103–105 was linear so may not be problematic, if the techniques were used to make relative comparisons between two samples. Our conclusions are that in very high absolute frequencies (>105) no enrichment is required, for frequencies of 103–105 negative selection for CD8 T cells allows for linearly accurate enumeration while for frequencies below that positive tetramer enrichment was more appropriate. We believe that these guidelines would be useful for antigen-specific T-cell quantification, especially as the field moves beyond the use of TCR-transgenic mice. This work was supported by the National Health and Medical Research Council of Australia, DSO National Laboratories of Singapore and Juvenile Diabetes Research Foundation. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. 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