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. 2009 Apr 13;206(4):849-66.
doi: 10.1084/jem.20081382. Epub 2009 Mar 30.

Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response

Gabrielle A Rizzuto  1 , Taha Merghoub, Daniel Hirschhorn-Cymerman, Cailian Liu, Alexander M Lesokhin, Diana Sahawneh, Hong Zhong, Katherine S Panageas, Miguel-Angel Perales, Grégoire Altan-Bonnet, Jedd D Wolchok, Alan N Houghton
Affiliations

Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response

Gabrielle A Rizzuto et al. J Exp Med. .

Abstract

A primary goal of cancer immunotherapy is to improve the naturally occurring, but weak, immune response to tumors. Ineffective responses to cancer vaccines may be caused, in part, by low numbers of self-reactive lymphocytes surviving negative selection. Here, we estimated the frequency of CD8(+) T cells recognizing a self-antigen to be <0.0001% ( approximately 1 in 1 million CD8(+) T cells), which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity, but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit, most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more, acquire polyfunctionality, and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8(+) T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells, followed by in vivo activation, is a new approach that can be applied to human cancer immunotherapy. Further, precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8(+) T cells.

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Figures

Figure 1.
Figure 1.
The precursor frequency of CD8+ T cells recognizing gp10025-33 in the naive repertoire is estimated to be ∼1 in 1 million CD8+ T cells; vaccination induces an oligoclonal, private response. (A and B) Sublethally irradiated animals received adoptive transfer with 30 million splenocytes containing titrating doses (0, 101, 102, and 103) of pmel-1 CD8+Thy1.1+ cells. Three weekly vaccinations with hgp100-encoding plasmid began the following day, and DLNs were harvested 5 d after the last vaccination. Cells were stained and analyzed by flow cytometry to determine frequency and absolute number of hgp100-tetramer+ cells, as well as the relative contributions of Thy1.1+ (transgenic) cells and Thy1.2+ (endogenous) to this response. In A, representative contour plots are shown for mice in each pmel-1 dose group. (B) Mean absolute number of CD8+tetramer+ cells in DLN (n = 3 mice/group; error bars represent the SD). (C) Larger cohorts (n = 10 mice/group) were prepared as in A, with animals receiving either 0 or 101 initial pmel-1 cells. The absolute number of Thy1.1+ (transgenic) or Thy1.2+ (endogenous) CD8+tetramer+ cells in DLNs of those animals that responded above background to vaccine is shown. Flow cytometric contour plots are displayed as supplemental data (Fig. S1). (D) Spleens and DLNs of responders were stained for TCR Vβ gene segment expression. Usage profiles for individual mice, as well as for CD8+ cells from a naive nonvaccinated control, are shown as pie graphs. Roman numerals indicate samples that were subjected to cloning of TCR Vβ CDR3 regions, as reported in Table I. Data shown in A–C are representative of at least three experiments performed under similar conditions. D is representative of nine individual mice from two independent experiments. Fig. S1 is available at http://www.jem.org/cgi/content/full/jem.20081382/DC1.
Figure 2.
Figure 2.
Tumor growth and autoimmune depigmentation elicited by adoptive transfer and vaccination is inversely correlated with CD8+ cell precursor frequency. (A) Experimental scheme. Thy1.2+ mice (15–16 mice/group) were intradermally inoculated with B16 cells and either left untreated (naive) or began treatment on day 4 consisting of sublethal irradiation and adoptive transfer of donor cells. Each experimental group received adoptive transfer of 30 million Thy1.1+ splenocytes, containing varying numbers of purified CD8+Thy1.1+ pmel-1 cells. All treated mice received three weekly vaccinations with hgp100-encoding plasmid, beginning on day 5. Animals were monitored every 3–5 d for tumor growth. (B) Incidence of progressively growing B16 tumors. (C) Tumor growth curves for individual mice. (D) At day 104 after tumor challenge, surviving mice were anaesthetized and imaged using a flatbed scanner. Representative ventral images from 1 animal/group are shown. Depigmentation was quantified, and mean pixel number and SEM is plotted in E. For groups receiving 103, 105, 106, and 6 × 106 pmel-1 cells, P < 0.001 for an overall association between the number of pixels and the initial pmel-1 dose. Pairwise comparisons for differences yield P < 0.001 for 103 versus 106 and P < 0.01 for 103 versus 6 × 106. Data are representative of two experiments.
Figure 3.
Figure 3.
Higher pmel-1 fold expansion at lower initial precursor frequency. (A) Experimental scheme. Thy1.2+ mice were challenged with a supra-lethal dose of B16-matrigel 4 d before treatment. Animals were treated with sublethal irradiation and adoptive transfer of 30 million Thy1.2+ splenocytes, with groups (n = 6 mice/group) receiving different precursor frequencies of CD8+Thy1.1+ pmel-1 cells. One day later all were vaccinated with hgp100-encoding plasmid. (B) At time points shown, tumor, spleen, and DLN from two mice per group were harvested, and the frequency of pmel-1 CD8+ cells was determined by flow cytometry. Fold expansion was calculated by dividing Thy1.1+ pmel-1 frequency among all CD8+ at each time point by initial frequency. The mean of two individual mice is shown. Representative of three individual experiments performed under similar conditions. (C) In separate experiments, nontumor-bearing animals were treated as described above with pmel-1 doses of 103, 105, 106, or 6 × 106 cells, vaccinated 1 d after adoptive transfer and 1 wk later. 5 d after the second vaccination, DLNs were harvested and stained for intracellular expression of Ki67. Flow cytometric overlaid histograms of Ki67 and isotype control (shaded) staining for the different pmel-1 dose groups are shown. Representative plots of nongp100-specific, CD8+ cells (CD8+Thy1.1−) in the lymphopenic host, as well as naive CD8+ cells are shown. Similar pattern of Ki67 reactivity observed in three individual experiments.
Figure 4.
Figure 4.
At higher precursor frequency, intraclonal competition limits the initial proliferative burst and acquisition of effector phenotype. (A) Experimental scheme. Mice (n = 3–5/group) underwent sublethal irradiation and adoptive transfer of splenocytes that included 105 CFSE-labeled CD8+Thy1.1+ pmel-1 cells. Some groups received competing CD8+Thy1.1+Thy1.2+ pmel-1 cells (0.9 × 106 or 5.9 × 106) or competing CD8+Thy1.2+ OT1 cells (5.9 × 106). 1 d later, mice were vaccinated with hgp100-encoding plasmid, except for a control group that received no vaccination. Vaccine DLNs were harvested from individual mice 5 d after vaccination and proliferation and phenotype of CFSE-labeled CD8+Thy1.1+ pmel-1 cells was assessed. (B) Combined flow cytometric plots of DLNs, gated on CD8+Thy1.1+ pmel-1 cells are shown. Proliferation and phenotypic data for DLNs of individual animals is shown graphically in C and D, respectively. Horizontal line represents mean. P = 0.03 for comparison of vaccinated animals receiving no competitors versus vaccinated animals receiving either high- or low-dose pmel-1 competitors. Representative of two experiments performed under similar conditions.
Figure 5.
Figure 5.
Initial precursor frequency dictates acquisition of vaccine-induced effector phenotype on tumor antigen-specific CD8+ T cells. (A) Mice (n = 3/group) were inoculated subcutaneously with B16-matrigel tumors, underwent sublethal irradiation and adoptive transfer 4 d later, and vaccination with hgp100-encoding plasmid the following day. 5 d after vaccination, DLNs and spleens were harvested. Expression of CD44, CD62L, and LFA-1 are shown by flow cytometric contour and histogram plots of pooled DLNs (n = 3 mice/group), and are gated on CD8+Thy1.1+ pmel-1 cells. Representative of three experiments. (B) Data shown are from spleens of individual mice in three pooled experiments. P < 0.001 for an overall inverse association between the percentage of CD44hiCD62Llo of pmel-1 cells and initial pmel-1 dose. (C) Data shown are from individual spleens of one representative experiment out of three experiments. P = 0.024 for an overall inverse association between MFI of LFA-1 on pmel-1 cells and initial pmel-1 dose.
Figure 6.
Figure 6.
Polyfunctional antigen-specific effector CD8+ cells are generated at lower, but not higher, precursor frequencies. (A) Mice (n = 4–6/group) were inoculated subcutaneously with B16-matrigel tumors, and underwent sublethal irradiation and adoptive transfer 4 d later and vaccination with hgp100-encoding plasmid the following day. 5 d after vaccination, tumors, DLNs, and spleens were harvested. Cells were stimulated for 6 h with EL4 cells and either mgp10025-33 or irrelevant peptide, and then stained for cytokine production (TNF-α and IFN-γ) and CD107a mobilization. TNF-α and IFN-γ production by gated CD8+Thy1.1+ pmel-1 cells is shown for pooled tumors and pooled DLNs. (B) Cytokine+ and CD107a+ cells from A were subject to Boolean gate analysis, and functionality profiles are shown as pie graphs. (C) Graph showing the mean fluorescence intensity (MFI) of IFN-γ for IFN-γ+CD8+Thy1.1+ pmel-1 cells from individual spleens stimulated with mgp10025-33. P < 0.001 for an overall inverse association between MFI of IFN-γ and initial pmel-1 dose. Shown is one representative experiment out of three experiments (CD107a, IFN-γ) and two experiments (CD107a, IFN-γ, and TNF-α).
Figure 7.
Figure 7.
Precursor frequency impacts phenotype of CD8+ “memory” populations. Phenotype of pmel-1 CD8+ cells was analyzed by flow cytometry ∼6 wk after last vaccination. (A) Surviving animals from tumor treatment experiment shown were killed 85 d after the last vaccination. (B) Individual spleens were harvested, stained, and analyzed by flow cytometry for CD44 and CD62L expression on CD8+Thy1.1+ pmel-1 cells. Dot plots and histograms are shown for representative spleens, and individual values are plotted on graphs. P < 0.001 for an inverse overall association between the percentage of CD44hiCD62Llo and initial pmel-1 dose. (C) Naive mice (n = 2–3/group) underwent irradiation and adoptive transfer with 105 or 6 × 106 pmel-1 cells, received three weekly vaccinations, and were rested for 72 d. DLNs were harvested and cells were stimulated for 6 h with EL4 cells and either mgp10025-33 or irrelevant peptide and stained for cytokine production. TNF-α and IFN-γ staining by gated CD8+Thy1.1+ pmel-1 cells are shown for representative mice. Data are from at least two independent experiments that were performed under similar conditions.
Figure 8.
Figure 8.
A model for the impact of antigen-specific precursor frequency on the quality of antitumor responses. (A) The mean values for parameters shown were calculated for experimentally tested pmel-1 dose groups (0, 103, 105, 106, and 6 × 106). For each parameter, the maximal response was set at 100%, and responses for other pmel-1 dose groups were calculated as a percentage of this maximum. Values from spleen, DLN, and tumor are shown graphically. (B) Model based on A. The x axis shows the number of naive tumor antigen–specific CD8+ T cells present at the time of vaccine administration. The y axis is the quality of the antitumor immune response. The curve demonstrates that there is an optimal precursor frequency for generating the strongest antitumor response.
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