VM cells are a distinct subset of CD8⁺ T cells with memory-like phenotype and function. A) B8R specific T cells were isolated from naive B6, and B6 mice challenged with VV 30 days previously, using magnetic enrichment of tetramer stained cells as described previously () and in the Materials and Methods. B) CD44^hi and ^lo cells, from both naïve and VV-challenged hosts as shown in A, were analyzed by FACS for CD122 and CD49d expression. C, D) Overlay and graph of CD122 expression of the cells from the regions described in A and B. Data is shown as mean fluorescence intensity (MFI) of CD122 staining of each population. E) FACS analysis of spleen CD4 and CD8⁺ spleen cells for the distribution of CD122 and CD49d staining. F) Overlay of CD122 expression from the regions shown in E. G and H) Naïve and VM T cell transfer and immune protection study. CD44^lo and CD44^hiCD49d^lo cells were sorted by FACS. And transferred into separate naïve hosts which were then challenged with LMova. Five days later, the bacterial load in the spleens was determined as previously described. All data are representative of 2–5 experiments performed. Data points are from 3–4 mice per group. Error bars represent standard deviation. All experiments shown were performed 2–5 times. * p value < 0.05, ** p value <0.001, *** p value <0.0001, unpaired t-test.

VM cells are a population independent from Innate Memory cells. CD8⁺ spleen cells from the indicated strains of mice were isolated and stained for CD44, CD122 and CD49d expression. A, B) data shown were gated on all live, B220−, CD8⁺ events. Numbers indicate percent of CD8⁺ T cells in each quadrant. C) Graph of data gated as in A, each data point representing an individual, age matched mouse. Data are shown as percent of VM cells out of total CD8⁺ T cells from each strain. D) Dot plots and histograms of CD8⁺ T cells from WT and id3^−/− mice. Histograms are of all CD44^hi cells shown in the dot plots. Data shown are representative of 4 independent id3^−/− spleens. E) Spleen and thymus from B6 hosts were isolated and CD49d expression was determined on all B220⁻CD4⁻CD8⁺CD44^hiCD122^hi cells in each tissue. Gating strategy is shown in the dot plots and histogram and mean fluorescent intensity of CD49d staining is shown in the graph, each data point representing an individual. All data are representative of 2–5 experiments performed. Error bars represent standard deviation. * p value < 0.05, ** p value <0.001, unpaired t-test.

VM cell development is CD122-dependent. WT × CD122^−/− bone marrow chimeras were made as previously described and as in the Material Methods. Twelve weeks after reconstitution, spleen CD8⁺ T cells were analyzed for CD44 and CD49d expression. B) Percent of VM phenotype cells out of total CD8⁺ T cells from each background, WT or CD122^−/− in the bone marrow chimeric mice shown in A. Each data point represents a separate chimeric host. Data are representative of 3 independent mixed chimera experiments, each with 3–5 chimeras. C) B8R specific T cells were isolated from the chimeric mice using magnetic enrichment of tetramer stained cells as described previously () and in the Materials and Methods. The column bound CD8⁺ cells were gated into CD45.1 (WT) and CD45.2 (CD122^−/−) backgrounds and analyzed for CD44 expression and tetramer staining. Two representative mice are shown (top and bottom plots). D) Peripheral blood from WT×CD122^−/− chimera’s before and after immunization with B8R peptide in conjunction with polyIC and antiCD40 as previously described (ref). Data shown are gated on all B220− CD8⁺CD45.1⁺ (WT) and B220−CD8⁺CD45.2⁺(CD122^−/−) events. Data shown are representative of 4 immunized chimeric mice.

VM cell development is IL-15 dependent. Total CD8⁺ spleen cells (A and B), or B8R-specific spleen cells (C and D) from IL-15Rα^−/− mice were analyzed for the percentage of VM cells. A and B) Total CD8⁺ T cells from WT and IL-15Rα^−/− mice were analyzed for CD44 and CD49d expression (top plots). CD49d^lo cells were gated on and analyzed for the frequency of CD44^hiCD122^hi cells (lower plots and B). C and D) B8R specific T cells were isolated from WT and IL-15Rα^−/− mice as described previously () and in the Materials and Methods. Column bound CD8⁺ T cells were analyzed for CD44 expression of tetramer⁺ cells. Each data point is from an individual mouse. The data shown are representative of 3 independent experiments.

IL-15 stimulation induces the expansion of VM cells in vivo. A) B6 mice were injected with 5ug of IL-15/Rα complexes i.p.. Spleen CD8⁺ T cells were analyzed 5 days later for the percent of cells with naïve (lower left quadrant), VM (upper left quadrant) and antigen memory (upper right quadrant) phenotypes. B) B8R specific T cells were isolated as described above from mice injected with IL-15/Rα complexes as in A. Column-bound cells were analyzed for the percent of CD44^hi tetramer⁺ cells. C) Tetramer⁺ cells from B were further analyzed for their expression of CD49d. D) The absolute number of CD8+ T cells within the naïve (CD44^lo), memory (CD44^hi, CD49d^hi) and VM (CD44^hi, CD49d^lo) CD8+ T cell pools were determined in control and IL-15/Rα complex injected mice. The fold expansion of cells in each subset was then calculated by dividing the cell number in the IL-15/Rα complex injected hosts by the number of cells (derived by averaging the number from 3 independent mice) in the controls. E) B6 mice were injected with increasing doses of IL-15/Rα complexes and 5 days later were analyzed as in A for the percent CD44^hi cells out of the total CD4 and CD8 T cells (CD4s, CD8s) and as in B for the percent B8R⁺CD44^hi cells out of the total B8R tetramer⁺ cells (tetramer⁺). F) IL-15/Rα complexes were injected as in A into the indicated strains. Five days later, the percent CD44^hiCD49d^loCD122^hi cells out of total CD8⁺ T cells was determined as described above. Uninjected controls for each strain are the same data as shown in , shown here for the purposes of comparison to the IL-15/Rα complex injected hosts.

VM cells express high levels of Eomes and their development is Eomes-dependent. A) naïve, VM and antigen memory CD8⁺ T cells were analyzed for Eomes and Tbet expression as described above and in the Materials and Methods. Histograms are from CD8⁺ splenic T cells gated on CD44^lo (naïve), CD44^hiCD49d^lo (VM) or CD44^hiCD49d^hi (antigen memory) cells. B and C) spleen CD8⁺ cells from WT and conditional Eomes^fl/flCD4-cre knockout mice, gated on all CD49d^lo events, were analyzed for CD44 and CD122 expression. C) Data is shown as both the percentage and total number of splenic VM cells from each host. Each data point in C represents an individual mouse. Data shown are representative of 3 experiments performed. ** p value <0.001, ***p value <0.0001, unpaired t-test.

IL-15 is produced by tissue-derived and CD8⁺ lymphoid derived DC subsets which mediate VM cell formation in the periphery. IL-15 BAC transgenic mice expressing GFP under the control of the IL-15 locus were made as described in the Materials and Methods. The lymphoid tissues were isolated, collagenase digested as previously described, and the indicted DC subsets analyzed for their expression of IL-15 (GFP). A and B) DCs isolated from inguinal, axillary and brachial lymph nodes. Left most dot plot was gated on all B220−CD11c⁺ClassII⁺ events. The cells in the regions, representing the indicated DC subsets, were assessed for their expression of GFP. C) DCs isolated from the spleen and analyzed as in A and B. D and E) Analysis of spleen CD8⁺ T cells from B6/129 BatF3^−/− mice and wild type littermate controls. The percent of CD44^hiCD49d^loCD122^hi cells out of total CD8⁺ cells is shown in the upper right quadrant (dot plots) and in the graph. Each data point represents an individual mouse. *** p value <0.0001, unpaired t-test.

B6 BatF3^−/− mice recover both CD8⁺DC cells and VM cells. A and B) Analysis of spleen CD8⁺ T cells from B6 WT, BatF3^−/− crossed only 1–2 generations onto the B6 background (B6/129 BatF3^−/−) and BatF3^−/− mice crossed at least 10 generations onto the B6 background (B6 BatF3^−/−). The percent of CD44^hiCD49d^lo cells out of total CD8⁺ cells is shown in the upper right quadrant (dot plots) and in the graph. Each data point represents an age matched, individual mouse. C) Analysis of lymphoid resident CD8⁺ and CD11b⁺ DCs from LN and spleens of the WT and Batf3^−/− on the indicated backgrounds. DCs were isolated and analyzed as described in . Numbers indicate the percent of DCs in that quadrant. Data are representative of 5–7 mice from 2 independent experiments. *** p value <0.0001, unpaired t-test