1. Lymphopenia-induced spontaneous T-cell proliferation as a cofactor for autoimmune disease development
by Armelle Le Campion, Marie-Claude Gagnerault, Cédric Auffray, Chantal Bécourt, Maud Poitrasson-Rivière, Eliette Lallemand, Boris Bienvenu, Bruno Martin, Françoise Lepault, and Bruno Lucas
Blood
Volume 114(9):
August 27, 2009
©2009 by American Society of Hematology

2. Frequency of CD4+ T cells undergoing spontaneous proliferation in lymphopenic C57BL/6 mice.
Frequency of CD4+ T cells undergoing spontaneous proliferation in lymphopenic C57BL/6 mice. Twenty-five to 1000 CD4+ T cells from pooled superficial cervical, axillary, brachial, inguinal, and mesenteric LNs of CD45.1 C57BL/6 mice were injected into CD45.2 C57BL/6 CD3ϵ−/− mice. One month after transfer, the presence of CD45.1+CD4+CD8− TCR+ cells in the periphery (pooled LN and spleen cells) of recipient mice was individually tested. (A) Absolute numbers of recovered CD45.1+CD4+CD8− TCR+ cells are shown as a function of the number of injected CD4+ T cells. Each point represents an individual mouse, and horizontal lines represent the mean number of CD45.1+CD4+CD8− TCR+ cells recovered from positive mice. (B) Numbers of injected CD4+ T cells are plotted against the log frequency of negative mice (ie, injected mice in which CD4+ T cells were not detected 1 month after transfer). The frequency of C57BL/6 autoreactive CD4+ T cells was estimated at 1/214 (95% confidence limits, 1/163 to 1/281). (C) TCR/forward light scatter (FSC), Vβ8/FSC, and Vβ4/FSC dot plots for gated CD45.1+CD4+CD8− T cells are shown for 1 control C57BL/6 mouse and 3 representative recipient mice injected with 100 CD4+ T cells.
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

3. The spontaneous proliferation of regulatory T cells in a lymphopenic environment requires help from conventional CD4+ T cells.
The spontaneous proliferation of regulatory T cells in a lymphopenic environment requires help from conventional CD4+ T cells. (A) A total of 102 LN CD4+ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ−/− mice. Fluorescence dot plots show TCR, CD44, CD25, and Foxp3 expression on gated CD4+CD8− T cells from a control C57BL/6 mouse and a representative recipient mouse 1 month after transfer. (B) A total of 102 CD4+CD25− or CD4+CD25+ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ−/− mice, alone or together with 5 × 104 CD45.2+CD4+ T cells. One month later, the presence of CD45.1+CD4+CD8− TCR+ cells in the periphery of recipient mice was individually tested. Left: absolute numbers of CD45.1+CD4+CD8− TCR+ cells; right: frequency of positive mice and absolute numbers of CD45.1+CD4+CD8− TCR+ cells in positive mice. Data show means ± SEM of mice for 3 independent experiments. (C) A total of 102 CD4+CD25+ T cells from CD45.1 C57BL/6 mice and 5 × 104 CD8+ T cells from CD45.2 C57BL/6 mice was cotransferred into C57BL/6 CD3ϵ−/− IIKO or C57BL/6 CD3ϵ−/− mice. The absolute numbers of CD45.1+CD4+CD8− TCR+ cells recovered from recipient mice 1 month after transfer are shown. (D) A total of 102 CD45.1+CD4+CD25+ T cells from C57BL/6 mice was coinjected with 5 × 104 CD4+ T cells from C57BL/6 IL-2−/− mice or C57BL/6 control littermates into C57BL/6 CD3ϵ−/− mice. Absolute numbers of CD45.1+CD4+CD8− TCR+ cells recovered from recipient mice 1 month after transfer are shown. Each point represents an individual mouse, and horizontal lines represent the mean number of CD45.1+CD4+CD8− TCR+ cells in positive mice (B left; C-D). ns, not significant; NA, not applicable.
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

4. Frequency of regulatory CD4+ T cells recovered after spontaneous proliferation in a lymphopenic environment.
Frequency of regulatory CD4+ T cells recovered after spontaneous proliferation in a lymphopenic environment. (A) A total of 5 × 106 LN CD4+ T cells from CD45.1 C57BL/6 mice was purified, labeled with CFSE, and transferred into C57BL/6 CD3ϵ−/− hosts. Two and 28 days after transfer, pooled LNs and spleen were analyzed for Foxp3 expression. Fluorescence Foxp3/CFSE dot plots are shown for CD45.1+ CD4+ T cells 2 and 28 days after transfer. (B) A total of 5 × 103 to 5 × 106 CFSE-labeled T cells from CD45.1 C57BL/6 mice was transferred into C57BL/6 CD3ϵ−/− mice and analyzed 1 month after transfer. Proportion of CD45.1+ CFSE− CD4+ T cells expressing Foxp3 is shown as means ± SEM for 3 independent experiments. The gray area indicates the mean ± SEM proportion of Foxp3-expressing cells among peripheral CD4+ T cells from control C57BL/6 mice. ***P < .001; *P < .05; ns, not significant. (C) A total of 106 CD4+CD25+ T cells from CD45.1 C57BL/6 mice and 106 CD4+CD25− T cells from CD45.2 C57BL/6 mice was labeled with CFSE and coinjected into C57BL/6 CD3ϵ−/− mice. Representative Foxp3 fluorescence histograms of gated CD45.1+ or CD45.1− CFSE− CD4+ T cells 1 month after transfer. Percentages of Foxp3+ cells are indicated.
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Increased frequency of peripheral CD4+ T cells undergoing spontaneous proliferation in NOD mice compared with C57BL/6 mice.
Increased frequency of peripheral CD4+ T cells undergoing spontaneous proliferation in NOD mice compared with C57BL/6 mice. (A) A total of 10 to 5 × 104 LN CD4+ T cells from CD45.2 NOD mice was transferred into CD45.1 NOD Cα−/− mice. A total of 25 to 5 × 104 CD4+ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ−/− mice. Absolute numbers of CD45.2+CD4+CD8− TCR+ cells in NOD Cα−/− recipient mice (circle) and of CD45.1+CD4+CD8− TCR+ cells in C57BL/6 CD3ϵ−/− recipient mice (diamond) are shown. (B) Absolute numbers of CD45.1+CD4+CD8− TCR+ cells in C57BL/6 CD3ϵ−/− mice or C57BL/6 Cα−/− mice receiving 5 × 103 CD4+ T cells. (C) CD25, CD44, CD45RB, and CD69 expression as a function of Foxp3 expression on gated LN CD4+CD8− T cells was compared between C57BL/6 and NOD mice. The dot plots shown were generated from the data for 1 mouse, but are representative of 3 individual experiments with at least 2 mice per group. (D) LN CD4+ T cells from C57BL/6 and NOD mice were purified, and naive CD4+ T cells were electronically sorted on the basis of their nonexpression of CD25 and their low or absent expression of CD44. A total of 5 × 103 purified naive CD4+ T cells (CD4+CD25−CD44−/low) from CD45.1 C57BL/6 mice and CD45.2 NOD mice was injected into CD45.2 C57BL/6 CD3ϵ−/− mice and CD45.1 NOD Cα−/− mice, respectively. Absolute numbers of recovered CD45.1+CD4+CD8− TCR+ cells in C57BL/6 CD3ϵ−/− recipient mice and of CD45.2+CD4+CD8− TCR+ cells in NOD Cα−/− recipient mice are shown 1 month after transfer. ***P < .001; **P < .01; ns, not significant. Each point represents an individual mouse, and horizontal lines represent the mean number of CD4+ T cells recovered from positive mice (A-B, D).
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

6. Frequencies of conventional and regulatory T cells undergoing spontaneous proliferation in lymphopenic NOD mice 1 month after reconstitution.
Frequencies of conventional and regulatory T cells undergoing spontaneous proliferation in lymphopenic NOD mice 1 month after reconstitution. (A) Twenty-five CD4+CD25− or CD4+CD25+ LN T cells from CD45.2 NOD mice and 2 × 103 CD4+ T cells from CD45.1 NOD mice were cotransferred into CD45.1 NOD Cα−/− recipients. Left: absolute numbers of recovered CD45.2+CD4+CD8− TCR+ cells. Each point represents an individual mouse, and horizontal lines represent the mean number of CD4+ T cells recovered from positive mice; right: frequency of positive mice and numbers of CD45.2+CD4+CD8− TCR+ cells recovered from positive mice. Data show means ± SEM from 3 independent experiments. (B) A total of 5 × 102 to 5 × 106 CD4+ T cells from CD45.2 NOD mice was labeled with CFSE and transferred into CD45.1 NOD Cα−/− mice. Proportion of CD45.2+ CFSE− CD4+ T cells expressing Foxp3. Results are from 2 independent experiments. The gray area represents the mean ± SEM proportion of Foxp3-expressing cells among CD4+ T cells from control NOD mice. ***P < .001; **P < .01; *P < .05.
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Reduced proportion of regulatory CD4+ T cells and development of autoimmune disorders in reconstituted lymphopenic NOD mice.
Reduced proportion of regulatory CD4+ T cells and development of autoimmune disorders in reconstituted lymphopenic NOD mice. (A) A total of 5 × 102 to 5 × 106 CD4+ T cells from C57BL/6 and NOD mice was transferred into C57BL/6 CD3ϵ−/− and NOD Cα−/− mice, respectively. The number of initially injected CD4+ T cells expanding strongly in response to lymphopenia was evaluated by dividing the total number of CD4+ T cells injected into mice by the frequency of CD4+ T cells, as determined in limiting dilution analyses (1/214 for C57BL/6 mice, and 1/25 for NOD mice). The numbers obtained were plotted against the frequency of Foxp3+ cells among the CD4+ T cells recovered 1 month after transfer. (B) A total of 5 × 103 or 5 × 104 CD4+ T cells from C57BL/6 mice and from NOD mice was transferred into C57BL/6 CD3ϵ−/− mice and NOD Cα−/− mice, respectively. One month after transfer, mice were killed and paraffin-embedded sections of various organs were stained with hematoxylin and eosin. The presence of mononuclear cell infiltrates in the indicated organs is recorded. Results are expressed as the number of infiltrated organs among the total number of organs examined.
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology

8. The initial degree of host lymphopenia governs the expansion of regulatory T cells and, consequently, the development of adoptively transferred diabetes.
The initial degree of host lymphopenia governs the expansion of regulatory T cells and, consequently, the development of adoptively transferred diabetes. (A) The prevalence of diabetes was determined in NOD SCID mice injected with 5 × 104 (plain line) or 10 × 106 (dotted line) CD4+ T cells from BDC2.5 NOD mice (n = 6). (B) Frequency of Foxp3-expressing cells among CD4+ T cells from the pancreatic LNs of NOD SCID mice injected with 5 × 104 or 10 × 106 BDC2.5 NOD CD4+ T cells. ***P < (C) NOD SCID mice received 5 × 104 (left) or 10 × 106 (right) CD4+ or CD4+CD25− T cells from BDC2.5 NOD mice (n = 4/group).
Armelle Le Campion et al. Blood 2009;114:
©2009 by American Society of Hematology