- Email: [email protected]
Limiting dilution analysis of primary cytotoxic T-cell precursors
Journal of Immunological Methods 208 Ž1997. 85–90
Limiting dilution analysis of primary cytotoxic T-cell precursors Maurice Zauderer a
a,)
, Alfred Singer
b
Cancer Center, UniÕersity of Rochester Medical Center, Box 704, Rochester, NY 14642, USA b Experimental Immunology Branch, National Cancer Institute, Bethesda, MD, USA Received 29 May 1997; accepted 18 July 1997
Abstract An efficient colorimetric assay has been adapted for limiting dilution analysis of cytotoxic T-cell precursors. Application of this assay in a suitable experimental model of thymic education could be especially useful in identifying factors that shape the CD8 T-cell repertoire. The essential elements of such a model are described here and elsewhere. q 1997 Elsevier Science B.V. Keywords: Cytotoxic T-cell; XTT assay; Limiting dilution analysis
1. Introduction We have adapted a colorimetric assay for efficient limiting dilution analysis of cytotoxic T-cell precursors in vitro. The number of viable cells remaining following cell mediated lymphocytotoxicity ŽCML. can be determined by the ability of the expanded target cells to convert a tetrazolium salt, XTT, into a chromogenic product ŽRoehm et al., 1991.. Interpolation into a standard growth curve allows the colorimetric reading to be translated into specific lysis. An interesting application of this assay would be the study of factors that shape the cytotoxic T-cell repertoire.
X Abbreviations: XTT, sodium 3 -w1-wŽphenylamino.-carbonylx3,4-tetrazoliumx-bisŽ4-methoxy-6-nitro.benzene-sulfonic acid hydrate; PMS, phenazine methosulfate ) Corresponding author. Tel.: q1-716-2752060; fax: q1-7164619547; e-mail: [email protected]
2. Materials and methods 2.1. Mice C57BLr6NCr mice were obtained from the National Cancer Institute, Frederick Cancer Research Animal Facility and were maintained in compliance with federal statutes and NIH policy. 2.2. XTT assay for Õiable cells Modified from the procedure of Roehm et al. Ž1991.. Briefly, phenazine methosulfate ŽPMS, Sigma. is dissolved in PBS at 125 mM and may be stored at 48C for up to one month. An XTT solution Žsodium 3X-w1-wŽphenylamino.-carbonylx-3,4-tetrazoliumx-bisŽ4-methoxy-6-nitro.benzene-sulfonic acid hydrate, Sigma. at 0.6 mgrml is prepared fresh in complete medium by dissolution at 568C for 5 min. PMS solution is added to 10 ml XTT solution for a
0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 1 3 1 - 2
86
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
final PMS concentration between 7.5 and 25 m M. Cells are seeded in 96 well flat bottom plates in 100 m l complete medium. Medium controls with no cells are included on every plate. After a standardized period of culture optimized for each cell line, 25 m l of XTTrPMS solution is added to each well. PMS incubation is continued at 378C for 2 to 8 h depending on the cell line, the cell number and the final PMS concentration. When an orange–yellow color develops, the plate is assayed for absorbance at 450 nm in an ELISA reader. Higher PMS concentrations give a more rapid readout but toxicity limits the length of the incubation. For greatest sensitivity a low PMS concentration can be employed during a 24 h incubation. A control plate with a titration of known cell concentrations is included in each experiment to construct a standard curve. 2.3. Limiting dilution analysis of primary in Õitro CTL responses Normal spleen cells were distributed to 80 wells of a 96 well plate at either 3 = 10 5 or 5 = 10 4 cells per well in 100 m l complete medium with 100 m M peptide and 15% supernatant of Con A stimulated mouse spleen cells. On day 6 each well was split and transferred to 96 well flat bottom plates seeded 4 h earlier with 300 target cellsrwell pulsed with either no peptide or 100 nM peptide for CML assays. A colorimetric XTT assay for viable cells was carried out after allowing 72 h for expansion of those target cells that survived CML. Two columns of each assay plate are set aside as controls with target cells alone or medium alone.
1983. except that the chromogenic product created from this substrate by dehydrogenases of viable cells is soluble. The class I MHC positive adherent cell line MC57G ŽH-2 b ., previously shown to be a good target for specific cytolytic T-cells, was employed as the target cell for these experiments. In preliminary experiments, it was determined that within the range of 10 to 1000 cells per microwell the XTT assay signal Žoptical density at 450 nm. is linearly related to the number of viable MC57G cells seeded per culture. Table 1 shows the measured OD 450 nm of a 6 h XTT assay 3 to 5 days after cultures were seeded with from 10 to 3,000 viable MC57G adherent cells per microwell. On day 3 cultures which received the highest initial cell number were already confluent and did not give a linear readout. However, cultures initiated at between 100 to 1,000 cells per microwell gave readouts proportional to the initial cell concentration. By day 5 wells initiated at 300 or 1000 cells were also confluent and outside the linear range of readout. At this time point, however, a linear readout was obtained for the cultures initiated at 30 to 100 cells. Even cultures initiated at 10 cells per well gave a linear readout on day 5 although for this data point the OD 450 measured was low and the standard deviation high. To determine whether this reading was indeed significant, the same plates were returned to the incubator to allow the XTT assay to continue for a full 24 h. The results confirmed the linear readout even for cultures initiated at 10 cells per well. This demonstrates a major advantage of the XTT assay. Since the chromogenic product is soluble, cells do not have to be destroyed by solubilization with 10% SDS to measure the optical density. If a more reliable reading is required,
3. Results 3.1. An efficient assay for limiting dilution analysis of specific cytotoxic T-cell precursors In order to analyze the relative frequency of specific cytotoxic T-cell precursors, we sought to develop a simple and rapid assay for cytotoxic function more suitable for extensive limiting dilution analysis than 51 Cr release. We elected to measure the viable cells remaining following CML by their ability to convert the tetrazolium salt XTT into a chromogenic product ŽRoehm et al., 1991.. This is similar to the more familiar MTT assay ŽMosmann,
Table 1 Linearity of XTT viable cell assay Initial OD 450 nm cell No. day 3 day 4 3,000 1,000 300 100 30 10 a
1.96"0.05 1.01"0.11 0.35"0.05 0.1 "0.02
day 5
day 5 Ž24 h.
a
2.45"0.18 a 1.31"0.14 2.47"0.16 a 0.47"0.13 1.09"0.13 2.33"0.2 a 0.18"0.05 0.39"0.09 1.43"0.23 0.11"0.05 0.48"0.11
Outside range of linear OD 450 nm readout.
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90 Table 2 XTT assay of OVA8-specific target cell lysis E:T
45:1 15:1
OD 450 nm Ž% specific lysis. MC57G ŽOVA8.
MC57G ŽN52-59.
MC57G
0.853"0.044 Ž38%. 1.105"0.073 Ž20%.
1.356"0.043 1.432"0.025
1.443"0.041 1.466"0.044
% specific lysiss100=ŽŽ500 Žinitial target. cellsyseeded cells equivalent.r500 cells.. The seeded cells equivalent is derived from a standard curve of OD 450 nm as a function of viable cells seeded three days earlier, as described for Table 1.
plates can be returned to the incubator and will continue to develop the reagent for at least 24 h. An example of the use of this assay to measure specific cytotoxicity is shown in Table 2. A polyclonal line of CD8 q cytotoxic T-cells specific for SIINFEKL ŽOVA8., the immunodominant peptide of ovalbumin Ž257–264. in association with K b , was selected by weekly in vitro stimulation of B6 spleen cells with peptide pulsed syngeneic APC. Specific cytotoxicity was determined at two effector:target cell ratios with addition of 500 target cells per triplicate microwell culture. MC57G cells were sensitized with either 1 m M OVA8, with the unrelated K b-restricted peptide N52–59, or with no peptide. The results demonstrate specific lysis of OVA8 sensitized targets. A parallel titration of the target cells without effectors generates a standard curve for the readout that allows the OD 450 on day 3 to be translated into the equivalent number of viable cells seeded at culture initiation ŽTable 2 legend, seeded cells equivalent.. In this assay, since the target cells are adherent, effector cells could be washed out after 4 to 6 h. Somewhat surprisingly, even at E:T ratios of 60:1, indistinguishable results were obtained whether effectors were washed out or not. In part, this is because the dehydrogenase activity and the corresponding XTT signal from MC57G tumor cells is much larger than that from effector cells. In addition, this observation suggests that the functional half life of cytotoxic T-cells in vitro is limited. 3.2. Limiting dilution analysis of primary peptidespecific responses of CD8 q CTL precursors induced in the presence of 100 m M soluble peptide An example of the use of the XTT assay for limiting dilution analysis of primary peptide-specific
87
cytotoxic precursor frequency in response to RGYVYQGL ŽN52–59., the immunodominant peptide of VSV nucleoprotein in association with H-2K b , is shown in Table 3. Normal C57BLr6 spleen cells were stimulated in 80 round bottom microwells at 3 = 10 5 cellsr0.1 ml culture with 100 m M N52–59 peptide ŽDe Bruijn et al., 1992. in the presence of 15% con A stimulated spleen cell supernatant ŽTCGF.. On day 6 each well was split three ways for the assay of cytolytic activity on 300 MC57G target cells with either Ž1. no peptide treatment, Ž2. sensitization with 1 m M N52–59, or Ž3. sensitization with 1 m M N52–59 and addition of anti-CD8 to the CML assay. Three days later, the expansion of MC57G cells that survived CML was determined by measuring the OD 450 following 6 h incubation with XTT. The results for non-specific versus peptide-specific killing Žaliquots 1 and 2. are shown in Table 3 and Table 3 Specific lysis of peptide sensitized targets in limiting dilution assay 1
2
3
4
5
6
7
8
9
10
A 1.375 0.774 1.300 1.293 1.339 1.102 1.614 0.895 0.900 1.191 A 0.347 0.088 0.084 0.315 0.230 0.530 0.271 0.192 0.200 0.261 B 0.891 1.344 1.322 1.820 1.099 1.644 0.875 0.863 1.518 1.248 B 0.167 0.262 0.207 0.159 0.346 0.230 0.104 0.260 0.199 0.186 C 1.635 1.353 2.148 1.853 1.195 2.085 1.359 1.477 1.707 0.778 C 0.396 0.182 0.332 0.098 0.093 0.245 0.424 0.106 0.131 0.126 D 1.203 0.905 1.409 1.847 1.681 1.465 1.159 1.737 1.368 1.564 D 0.169 0.074 0.054 0.116 0.050 0.262 0.110 0.160 0.064 0.110 E 1.503 1.337 1.467 1.384 1.501 1.355 1.499 1.679 1.061 0.806 E 0.142 0.115 0.067 0.127 0.070 0.201 0.302 0.163 0.194 0.146 F 1.276 1.601 0.542 1.239 1.138 1.196 0.665 1.713 1.223 1.011 F 0.115 0.207 0.292 0.083 0.131 0.091 0.050 0.409 0.189 0.161 G 1.261 1.605 1.871 1.566 1.515 1.299 0.925 1.436 1.182 0.948 G 0.316 0.332 0.259 0.229 0.316 0.188 0.070 0.165 0.136 0.209 H 1.647 1.150 1.316 1.233 1.150 1.160 0.699 0.896 0.917 0.643 H 0.345 0.124 0.210 0.184 0.144 0.076 0.098 0.188 0.187 0.152 OD 450 readings for each pair of individual assay wells derives from a single primary microculture initiated at 3=10 5 cellsrwell and stimulated with 100 m M N52–59 peptide. The upper reading of each pair is for an aliquot of effector cells added to 300 MC57G target cells in an assay well without peptide sensitization. The lower reading is for a second assay well that received an equivalent aliquot of effectors but in which target cells were first sensitized with N52-59 peptide. XTT assay was carried out 72 h after addition of effectors.
88
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
Fig. 1 is a graphical representation of these results. Each point represents the OD 450 measured for one well following incubation of effector cell aliquots with specifically sensitized versus unsensitized targets. If there were no specific killing, points would
Fig. 1. Scatter plot of OD 450 readings for XTT assays of surviving target cells following N52–59 specific CML in the presence or absence of N52–59 peptide. Effector cells were transferred from primary limiting dilution microcultures initiated at 3=10 5 spleen cellsrwell Žsee Table 3..
Fig. 1. In the matrix of Table 3 the top figure for each of 80 wells is the OD 450 for the aliquot of effector cells incubated with unsensitized targets and the bottom figure is the OD 450 for an identical aliquot of effector cells incubated with specific peptide sensitized targets. Control wells were seeded with the same number of target cells but without effectors and gave an average OD 450 of 1.9 on day 3 Žnot shown.. The difference between this control OD value in the absence of effectors and the OD readings with unsensitized target cells Žtop figure for each well. is the background of non-specific growth inhibition. It can be seen that this non-specific background is variable from well to well. It is, however, readily apparent from scanning the data that each well shows substantially greater specific killing, i.e. significantly lower OD 450 with sensitized target cells Žthe bottom figure. relative to target cells without peptide added Žtop figure.. We have since determined that non-specific growth inhibition is largely associated with using unfractionated splenic effector cells and APC. We have modified the experimental design to minimize this non-specific inhibitory effect by employing effector cells depleted of adherent cells and FcR q cells and reconstituting APC function with smaller numbers of highly enriched dendritic cells.
Fig. 2. Specific lysis by N52–59 specific CTL induced in primary microculture at 5=10 4 spleen cellsrwell is largely CD8 dependent. ŽA. Scatter plot of OD 450 readings for XTT assays of surviving target cells following N52–59 specific CML in the presence or absence of N52–59 peptide. ŽB. Scatter plot of XTT assay results following N52–59 specific CML in the presence or absence of anti-CD8 blocking antibody. Note: since anti-CD8 inhibition is only relevant in cultures that score positive for killing, only the 46 of 68 cultures that scored positive in CML at this spleen cell concentration are included in the two panels of this figure.
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
center around the diagonal. It is apparent, however, that all points fall to one side of the bissecting line. This demonstrates specific killing. At the spleen cell concentration assayed in the experiment depicted in Table 3 and Fig. 1, 3 = 10 5 cellsrwell, N52–59 specific cytotoxic precursors were not limiting. All wells except one ŽF3. scored positive for killing by the criterion that OD 450 following CML on peptide sensitized targets was more than 3 standard deviations less than the OD 450 on targets without peptide Ždetermined from replicate cultures on a separate plate.. At the next lower spleen cell concentration assayed, 5 = 10 4 cellsrwell, specific precursors were limiting and only 46 of 68 wells scored positive for lysis. By Poisson analysis when F0 s eym s 22r68s 0.32 this corresponds to an average frequency Ž m . of 1.1 peptidespecific precursorsrwell ŽZauderer, 1986.. Antibody inhibition studies have confirmed that these primary peptide-specific cytolytic responses in vitro are largely CD8 dependent. In the above experiment, a third parallel aliquot of effector cells from each well was assayed for CML on specifically sensitized targets Ž1 m M N52–59. in the presence of anti-CD8. Killing of specific targets was inhibited by anti-CD8 in 67 of 79 positive wells Ž85%. at 3 = 10 5 cellsrwell and in 42 of 46 positive wells Ž91%. at 5 = 10 4 cellsrwell. A graphic summary of this result for the 46 wells scored positive for specific killing at 5 = 10 4 spleen cellsrwell is shown in Fig. 2. On the scatter plot for specific versus non-specific killing ŽFig. 2A. all points fall to one side of the diagonal indicating specific killing. In contrast, in the presence of anti-CD8 ŽFig. 2B. the points are generally distributed around the diagonal indicating minimal CD8-independent specific killing.
4. Discussion A simple colorimetric assay for viable cells was adapted to detect cytolytic activity in limiting dilution cultures of normal mouse spleen. Primary peptide-specific responses of CD8 q CTL precursors of normal spleen were efficiently induced in the presence of 100 m M peptide. The study of primary
89
responses in vitro has the advantage that the relative frequency of precursors of diverse specificity can be determined in parallel spleen aliquots without the need for complex immunization protocols with multiple antigens in a single animal. We have separately defined conditions that efficiently support the induction of primary cytolytic responses from the spleen of fetal thymus grafted SCID mice. We anticipate that a particularly informative application of limiting dilution analysis in this SCID model will be to evaluate positive selection of specific T-cells in a normal heterogeneous precursor cell population during fetal thymus organ culture with well defined peptide analogs ŽAshtonRickardt et al., 1994; Hogquist et al., 1994; Sebzda et al., 1994.. The frequency and clonotypes of specific T-cells that subsequently mature in SCID mice engrafted with the treated thymus can be characterized in primary in vitro responses as described here. This would allow a number of issues to be addressed that cannot be clearly addressed in T-cell receptor transgenic fetal thymus organ culture.
Acknowledgements This work was supported by NIH grants AI 37752 and AG 08179.
References Ashton-Rickardt, P.G., Bandeira, A., Delaney, J.R., Van Kaer, L., Pircher, H.-P., Zinkernagel, R.M., Tonegawa, S., 1994. Evidence for a differential avidity model of T-cell selection in the thymus. Cell 76, 651–663. De Bruijn, M.L.H., Nieland, J.D., Schumacher, T.N.M., Ploegh, H.L., Kast, W.M., Melief, C.J.M., 1992. Mechanisms of induction of primary virus-specific cytotoxic T-lymphocyte responses. Eur. J. Immunol. 22, 3013–3020. Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J., Carbone, F.M., 1994. T-cell receptor antagonist peptides induce positive selection. Cell 76, 17–27. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55. Roehm, N.W., Rodgers, G.H., Hatfield, S.M., Glasebrook, A.L., 1991. An improved colorimetric assay for cell proliferation
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M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
and viability utilizing the tetrazolium salt XTT. J. Immunol. Methods 142, 257–265. Sebzda, E., Wallace, V.A., Mayer, J., Yeung, R.S.M., Mak, T.W., Ohashi, P., 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 263, 1615–1618.
Zauderer, M., 1986. Limiting dilution analysis of effector cells and their precursors in vitro. In: Weir, D., Blackwell, C., Herzenberg, L.A., Herzenberg L.A. ŽEds.., Handbook of Experimental Immunology, 4th ed. Blackwell Scientific, Oxford, pp. 65.1–65.7.
Limiting dilution analysis of primary cytotoxic T-cell precursors Maurice Zauderer a
a,)
, Alfred Singer
b
Cancer Center, UniÕersity of Rochester Medical Center, Box 704, Rochester, NY 14642, USA b Experimental Immunology Branch, National Cancer Institute, Bethesda, MD, USA Received 29 May 1997; accepted 18 July 1997
Abstract An efficient colorimetric assay has been adapted for limiting dilution analysis of cytotoxic T-cell precursors. Application of this assay in a suitable experimental model of thymic education could be especially useful in identifying factors that shape the CD8 T-cell repertoire. The essential elements of such a model are described here and elsewhere. q 1997 Elsevier Science B.V. Keywords: Cytotoxic T-cell; XTT assay; Limiting dilution analysis
1. Introduction We have adapted a colorimetric assay for efficient limiting dilution analysis of cytotoxic T-cell precursors in vitro. The number of viable cells remaining following cell mediated lymphocytotoxicity ŽCML. can be determined by the ability of the expanded target cells to convert a tetrazolium salt, XTT, into a chromogenic product ŽRoehm et al., 1991.. Interpolation into a standard growth curve allows the colorimetric reading to be translated into specific lysis. An interesting application of this assay would be the study of factors that shape the cytotoxic T-cell repertoire.
X Abbreviations: XTT, sodium 3 -w1-wŽphenylamino.-carbonylx3,4-tetrazoliumx-bisŽ4-methoxy-6-nitro.benzene-sulfonic acid hydrate; PMS, phenazine methosulfate ) Corresponding author. Tel.: q1-716-2752060; fax: q1-7164619547; e-mail: [email protected]
2. Materials and methods 2.1. Mice C57BLr6NCr mice were obtained from the National Cancer Institute, Frederick Cancer Research Animal Facility and were maintained in compliance with federal statutes and NIH policy. 2.2. XTT assay for Õiable cells Modified from the procedure of Roehm et al. Ž1991.. Briefly, phenazine methosulfate ŽPMS, Sigma. is dissolved in PBS at 125 mM and may be stored at 48C for up to one month. An XTT solution Žsodium 3X-w1-wŽphenylamino.-carbonylx-3,4-tetrazoliumx-bisŽ4-methoxy-6-nitro.benzene-sulfonic acid hydrate, Sigma. at 0.6 mgrml is prepared fresh in complete medium by dissolution at 568C for 5 min. PMS solution is added to 10 ml XTT solution for a
0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 1 3 1 - 2
86
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
final PMS concentration between 7.5 and 25 m M. Cells are seeded in 96 well flat bottom plates in 100 m l complete medium. Medium controls with no cells are included on every plate. After a standardized period of culture optimized for each cell line, 25 m l of XTTrPMS solution is added to each well. PMS incubation is continued at 378C for 2 to 8 h depending on the cell line, the cell number and the final PMS concentration. When an orange–yellow color develops, the plate is assayed for absorbance at 450 nm in an ELISA reader. Higher PMS concentrations give a more rapid readout but toxicity limits the length of the incubation. For greatest sensitivity a low PMS concentration can be employed during a 24 h incubation. A control plate with a titration of known cell concentrations is included in each experiment to construct a standard curve. 2.3. Limiting dilution analysis of primary in Õitro CTL responses Normal spleen cells were distributed to 80 wells of a 96 well plate at either 3 = 10 5 or 5 = 10 4 cells per well in 100 m l complete medium with 100 m M peptide and 15% supernatant of Con A stimulated mouse spleen cells. On day 6 each well was split and transferred to 96 well flat bottom plates seeded 4 h earlier with 300 target cellsrwell pulsed with either no peptide or 100 nM peptide for CML assays. A colorimetric XTT assay for viable cells was carried out after allowing 72 h for expansion of those target cells that survived CML. Two columns of each assay plate are set aside as controls with target cells alone or medium alone.
1983. except that the chromogenic product created from this substrate by dehydrogenases of viable cells is soluble. The class I MHC positive adherent cell line MC57G ŽH-2 b ., previously shown to be a good target for specific cytolytic T-cells, was employed as the target cell for these experiments. In preliminary experiments, it was determined that within the range of 10 to 1000 cells per microwell the XTT assay signal Žoptical density at 450 nm. is linearly related to the number of viable MC57G cells seeded per culture. Table 1 shows the measured OD 450 nm of a 6 h XTT assay 3 to 5 days after cultures were seeded with from 10 to 3,000 viable MC57G adherent cells per microwell. On day 3 cultures which received the highest initial cell number were already confluent and did not give a linear readout. However, cultures initiated at between 100 to 1,000 cells per microwell gave readouts proportional to the initial cell concentration. By day 5 wells initiated at 300 or 1000 cells were also confluent and outside the linear range of readout. At this time point, however, a linear readout was obtained for the cultures initiated at 30 to 100 cells. Even cultures initiated at 10 cells per well gave a linear readout on day 5 although for this data point the OD 450 measured was low and the standard deviation high. To determine whether this reading was indeed significant, the same plates were returned to the incubator to allow the XTT assay to continue for a full 24 h. The results confirmed the linear readout even for cultures initiated at 10 cells per well. This demonstrates a major advantage of the XTT assay. Since the chromogenic product is soluble, cells do not have to be destroyed by solubilization with 10% SDS to measure the optical density. If a more reliable reading is required,
3. Results 3.1. An efficient assay for limiting dilution analysis of specific cytotoxic T-cell precursors In order to analyze the relative frequency of specific cytotoxic T-cell precursors, we sought to develop a simple and rapid assay for cytotoxic function more suitable for extensive limiting dilution analysis than 51 Cr release. We elected to measure the viable cells remaining following CML by their ability to convert the tetrazolium salt XTT into a chromogenic product ŽRoehm et al., 1991.. This is similar to the more familiar MTT assay ŽMosmann,
Table 1 Linearity of XTT viable cell assay Initial OD 450 nm cell No. day 3 day 4 3,000 1,000 300 100 30 10 a
1.96"0.05 1.01"0.11 0.35"0.05 0.1 "0.02
day 5
day 5 Ž24 h.
a
2.45"0.18 a 1.31"0.14 2.47"0.16 a 0.47"0.13 1.09"0.13 2.33"0.2 a 0.18"0.05 0.39"0.09 1.43"0.23 0.11"0.05 0.48"0.11
Outside range of linear OD 450 nm readout.
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90 Table 2 XTT assay of OVA8-specific target cell lysis E:T
45:1 15:1
OD 450 nm Ž% specific lysis. MC57G ŽOVA8.
MC57G ŽN52-59.
MC57G
0.853"0.044 Ž38%. 1.105"0.073 Ž20%.
1.356"0.043 1.432"0.025
1.443"0.041 1.466"0.044
% specific lysiss100=ŽŽ500 Žinitial target. cellsyseeded cells equivalent.r500 cells.. The seeded cells equivalent is derived from a standard curve of OD 450 nm as a function of viable cells seeded three days earlier, as described for Table 1.
plates can be returned to the incubator and will continue to develop the reagent for at least 24 h. An example of the use of this assay to measure specific cytotoxicity is shown in Table 2. A polyclonal line of CD8 q cytotoxic T-cells specific for SIINFEKL ŽOVA8., the immunodominant peptide of ovalbumin Ž257–264. in association with K b , was selected by weekly in vitro stimulation of B6 spleen cells with peptide pulsed syngeneic APC. Specific cytotoxicity was determined at two effector:target cell ratios with addition of 500 target cells per triplicate microwell culture. MC57G cells were sensitized with either 1 m M OVA8, with the unrelated K b-restricted peptide N52–59, or with no peptide. The results demonstrate specific lysis of OVA8 sensitized targets. A parallel titration of the target cells without effectors generates a standard curve for the readout that allows the OD 450 on day 3 to be translated into the equivalent number of viable cells seeded at culture initiation ŽTable 2 legend, seeded cells equivalent.. In this assay, since the target cells are adherent, effector cells could be washed out after 4 to 6 h. Somewhat surprisingly, even at E:T ratios of 60:1, indistinguishable results were obtained whether effectors were washed out or not. In part, this is because the dehydrogenase activity and the corresponding XTT signal from MC57G tumor cells is much larger than that from effector cells. In addition, this observation suggests that the functional half life of cytotoxic T-cells in vitro is limited. 3.2. Limiting dilution analysis of primary peptidespecific responses of CD8 q CTL precursors induced in the presence of 100 m M soluble peptide An example of the use of the XTT assay for limiting dilution analysis of primary peptide-specific
87
cytotoxic precursor frequency in response to RGYVYQGL ŽN52–59., the immunodominant peptide of VSV nucleoprotein in association with H-2K b , is shown in Table 3. Normal C57BLr6 spleen cells were stimulated in 80 round bottom microwells at 3 = 10 5 cellsr0.1 ml culture with 100 m M N52–59 peptide ŽDe Bruijn et al., 1992. in the presence of 15% con A stimulated spleen cell supernatant ŽTCGF.. On day 6 each well was split three ways for the assay of cytolytic activity on 300 MC57G target cells with either Ž1. no peptide treatment, Ž2. sensitization with 1 m M N52–59, or Ž3. sensitization with 1 m M N52–59 and addition of anti-CD8 to the CML assay. Three days later, the expansion of MC57G cells that survived CML was determined by measuring the OD 450 following 6 h incubation with XTT. The results for non-specific versus peptide-specific killing Žaliquots 1 and 2. are shown in Table 3 and Table 3 Specific lysis of peptide sensitized targets in limiting dilution assay 1
2
3
4
5
6
7
8
9
10
A 1.375 0.774 1.300 1.293 1.339 1.102 1.614 0.895 0.900 1.191 A 0.347 0.088 0.084 0.315 0.230 0.530 0.271 0.192 0.200 0.261 B 0.891 1.344 1.322 1.820 1.099 1.644 0.875 0.863 1.518 1.248 B 0.167 0.262 0.207 0.159 0.346 0.230 0.104 0.260 0.199 0.186 C 1.635 1.353 2.148 1.853 1.195 2.085 1.359 1.477 1.707 0.778 C 0.396 0.182 0.332 0.098 0.093 0.245 0.424 0.106 0.131 0.126 D 1.203 0.905 1.409 1.847 1.681 1.465 1.159 1.737 1.368 1.564 D 0.169 0.074 0.054 0.116 0.050 0.262 0.110 0.160 0.064 0.110 E 1.503 1.337 1.467 1.384 1.501 1.355 1.499 1.679 1.061 0.806 E 0.142 0.115 0.067 0.127 0.070 0.201 0.302 0.163 0.194 0.146 F 1.276 1.601 0.542 1.239 1.138 1.196 0.665 1.713 1.223 1.011 F 0.115 0.207 0.292 0.083 0.131 0.091 0.050 0.409 0.189 0.161 G 1.261 1.605 1.871 1.566 1.515 1.299 0.925 1.436 1.182 0.948 G 0.316 0.332 0.259 0.229 0.316 0.188 0.070 0.165 0.136 0.209 H 1.647 1.150 1.316 1.233 1.150 1.160 0.699 0.896 0.917 0.643 H 0.345 0.124 0.210 0.184 0.144 0.076 0.098 0.188 0.187 0.152 OD 450 readings for each pair of individual assay wells derives from a single primary microculture initiated at 3=10 5 cellsrwell and stimulated with 100 m M N52–59 peptide. The upper reading of each pair is for an aliquot of effector cells added to 300 MC57G target cells in an assay well without peptide sensitization. The lower reading is for a second assay well that received an equivalent aliquot of effectors but in which target cells were first sensitized with N52-59 peptide. XTT assay was carried out 72 h after addition of effectors.
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M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
Fig. 1 is a graphical representation of these results. Each point represents the OD 450 measured for one well following incubation of effector cell aliquots with specifically sensitized versus unsensitized targets. If there were no specific killing, points would
Fig. 1. Scatter plot of OD 450 readings for XTT assays of surviving target cells following N52–59 specific CML in the presence or absence of N52–59 peptide. Effector cells were transferred from primary limiting dilution microcultures initiated at 3=10 5 spleen cellsrwell Žsee Table 3..
Fig. 1. In the matrix of Table 3 the top figure for each of 80 wells is the OD 450 for the aliquot of effector cells incubated with unsensitized targets and the bottom figure is the OD 450 for an identical aliquot of effector cells incubated with specific peptide sensitized targets. Control wells were seeded with the same number of target cells but without effectors and gave an average OD 450 of 1.9 on day 3 Žnot shown.. The difference between this control OD value in the absence of effectors and the OD readings with unsensitized target cells Žtop figure for each well. is the background of non-specific growth inhibition. It can be seen that this non-specific background is variable from well to well. It is, however, readily apparent from scanning the data that each well shows substantially greater specific killing, i.e. significantly lower OD 450 with sensitized target cells Žthe bottom figure. relative to target cells without peptide added Žtop figure.. We have since determined that non-specific growth inhibition is largely associated with using unfractionated splenic effector cells and APC. We have modified the experimental design to minimize this non-specific inhibitory effect by employing effector cells depleted of adherent cells and FcR q cells and reconstituting APC function with smaller numbers of highly enriched dendritic cells.
Fig. 2. Specific lysis by N52–59 specific CTL induced in primary microculture at 5=10 4 spleen cellsrwell is largely CD8 dependent. ŽA. Scatter plot of OD 450 readings for XTT assays of surviving target cells following N52–59 specific CML in the presence or absence of N52–59 peptide. ŽB. Scatter plot of XTT assay results following N52–59 specific CML in the presence or absence of anti-CD8 blocking antibody. Note: since anti-CD8 inhibition is only relevant in cultures that score positive for killing, only the 46 of 68 cultures that scored positive in CML at this spleen cell concentration are included in the two panels of this figure.
M. Zauderer, A. Singerr Journal of Immunological Methods 208 (1997) 85–90
center around the diagonal. It is apparent, however, that all points fall to one side of the bissecting line. This demonstrates specific killing. At the spleen cell concentration assayed in the experiment depicted in Table 3 and Fig. 1, 3 = 10 5 cellsrwell, N52–59 specific cytotoxic precursors were not limiting. All wells except one ŽF3. scored positive for killing by the criterion that OD 450 following CML on peptide sensitized targets was more than 3 standard deviations less than the OD 450 on targets without peptide Ždetermined from replicate cultures on a separate plate.. At the next lower spleen cell concentration assayed, 5 = 10 4 cellsrwell, specific precursors were limiting and only 46 of 68 wells scored positive for lysis. By Poisson analysis when F0 s eym s 22r68s 0.32 this corresponds to an average frequency Ž m . of 1.1 peptidespecific precursorsrwell ŽZauderer, 1986.. Antibody inhibition studies have confirmed that these primary peptide-specific cytolytic responses in vitro are largely CD8 dependent. In the above experiment, a third parallel aliquot of effector cells from each well was assayed for CML on specifically sensitized targets Ž1 m M N52–59. in the presence of anti-CD8. Killing of specific targets was inhibited by anti-CD8 in 67 of 79 positive wells Ž85%. at 3 = 10 5 cellsrwell and in 42 of 46 positive wells Ž91%. at 5 = 10 4 cellsrwell. A graphic summary of this result for the 46 wells scored positive for specific killing at 5 = 10 4 spleen cellsrwell is shown in Fig. 2. On the scatter plot for specific versus non-specific killing ŽFig. 2A. all points fall to one side of the diagonal indicating specific killing. In contrast, in the presence of anti-CD8 ŽFig. 2B. the points are generally distributed around the diagonal indicating minimal CD8-independent specific killing.
4. Discussion A simple colorimetric assay for viable cells was adapted to detect cytolytic activity in limiting dilution cultures of normal mouse spleen. Primary peptide-specific responses of CD8 q CTL precursors of normal spleen were efficiently induced in the presence of 100 m M peptide. The study of primary
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responses in vitro has the advantage that the relative frequency of precursors of diverse specificity can be determined in parallel spleen aliquots without the need for complex immunization protocols with multiple antigens in a single animal. We have separately defined conditions that efficiently support the induction of primary cytolytic responses from the spleen of fetal thymus grafted SCID mice. We anticipate that a particularly informative application of limiting dilution analysis in this SCID model will be to evaluate positive selection of specific T-cells in a normal heterogeneous precursor cell population during fetal thymus organ culture with well defined peptide analogs ŽAshtonRickardt et al., 1994; Hogquist et al., 1994; Sebzda et al., 1994.. The frequency and clonotypes of specific T-cells that subsequently mature in SCID mice engrafted with the treated thymus can be characterized in primary in vitro responses as described here. This would allow a number of issues to be addressed that cannot be clearly addressed in T-cell receptor transgenic fetal thymus organ culture.
Acknowledgements This work was supported by NIH grants AI 37752 and AG 08179.
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