Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes

Abstract

Following proper activation, naïve "CD4lo" T cells differentiate into effector T cells with enhanced expression of CD4 -"CD4hi" effectors. Autoimmune diabetes-prone NOD mice display a unique set of antigen-experienced "CD4lo" T cells that persist after primary stimulation. Here, we report that a population of such cells remained after secondary and tertiary TCR stimulation and produced cytokines upon antigenic challenge. However, when NOD blasts were induced in the presence of rIL-15, the number of antigen-experienced "CD4lo" T cells was significantly reduced. Clonal contraction, mediated in part by CD95-dependent activation-induced cell death (AICD), normally regulates the accumulation of "CD4hi" effectors. Interestingly, CD95 expression was dramatically reduced on the AICD-resistant NOD "CD4lo" T cells. Thus, while autoimmune disease has often been attributed to the engagement of robust autoimmunity, we suggest that the inability to effectively contract the immune response distinguishes benign autoimmunity from progressive autoimmune diseases that are characterized by chronic T cell-mediated inflammation.

Figure 1

(a) Secondary NOD T cell blasts are resistant to AICD upon tertiary restimulation. NOD or BALB/c Con A blasts were restimulated on anti-CD3-coated plates, washed, and cultured in IL-2-containing medium for 3 days then stimulated again on anti-CD3-coated plates (0.003–3.0 μg/mL; 50 μl/well). Tritium-labeled thymidine was added for the last 16 hrs of a 72-hour culture. The data are expressed as percent change versus control wells [(mean cpm anti-CD3 wells/mean cpm IgG wells) × 100]. The standard deviation of triplicate wells was less than 10%, and the results are representative of four experiments. (b) Culture supernatants were collected at various times (24–96 hrs) following primary stimulation (Con A) of NOD or BALB/c spleen cells or secondary and tertiary stimulations of T cells blasts with plate-bound anti-CD3 (1.0 μg/mL). IFN-γ levels were measured by ELISA, and the results are expressed as ng/mL. The results represent the mean and SD of triplicate wells and are representative of 3 independent experiments. Only P-values that are <.05 are shown.

Figure 2

The expression of CD25, CD44, CD69, CD95, and CD178 is similar on NOD and BALB/c CD4+ spleen cells. Naive spleen cells or live Con A blasts (harvested by histopaque separation 72 hrs after stimulation) from groups of 3 NOD or BALB/c mice were examined individually by flow cytometry for the expression of cell surface molecules associated with activation. The data are expressed as the mean percentage of CD4+ cells (gated) that were positive for the respective molecule in groups of 3 mice, and the results are representative of 3 experiments, with at least 20,000 events collected in each experiment.

Figure 3

“CD4lo” T cells are increased among NOD Con A blasts. (a) Spleen cells from naïve NOD or BALB/c mice were loaded with CFSE and stimulated for 72 hrs with Con A. The resulting blasts were stained with anti-CD4-PE and analyzed by flow cytometry. The results are representative of four experiments. The data are expressed as dot plots of ungated cells (100,000 events). The numbers in the plots represent the percentage of total cells in regions R1 (CD4 MFI > 800) and R2 (CD4 MFI < 250). (b) The values in the bar graph reflect the mean percentage of cells in each category, from 3 separate experiments (100,000 events collected for each sample). Student's t-test was used to compare responses between NOD and BALB/c blasts (only P-values reaching significance, <.05, are shown).

Figure 4

CD4 expression and T cell size increase in T cells undergoing cell division. CFSE-loaded NOD or BALB/c spleen cells were stimulated with Con A, harvested at 24 or 72 hrs, and then counterstained with anti-CD4-PE and CD3-PE/Cy5. For FACS analysis, 100,000 events were collected. The plot represents gated CD3+ cells. (a) Temporal analysis of CD4 expression and size (FSC) among Con A blasts. (b) CD4 expression and cell division (CFSE) in Con A blasts. The mean fluorescence intensity (MFI) for CFSE (X) and CD4 (Y) expression is noted for CD4+ cells at 24 and 48 hrs. (c) The pattern of CFSE expression (cell division) amongst CD4hi and CD4lo T cells from NOD or BALB/c Con A blasts 72 hrs after stimulation.

Figure 5

The “CD4lo” T cells that persist among NOD Con A blasts are antigen-experienced. NOD spleen cells were cultured for 72 hrs with Con A, stained with anti-CD4-PE and anti-CD69-FITC (a), anti-CD62L-FITC (b), or anti-CD25-FITC (c), and analyzed by flow cytometry (20,000 events were collected for each analysis). The selected regions (boxes) in the dot plots mark the “CD4lo” population, and the numbers indicate their percentage in the total population. The underlined numbers indicated the percentage of CD4⁺ cells in the total population. (d) Freshly isolated NOD splenocytes were stained with anti-CD4-PE and IgG-FITC for comparison to baseline CD4 expression on naïve T cells. These data are representative of two experiments.

Figure 6

“CD4lo” T cells are present in Con A blasts produced from the spleen cells of NOD.BDC2.5 TCR transgenic mice. (a) Spleen cells from BDC2.5 TCR transgenic mice were stimulated with Con A for 72 hrs. The cells were then layered onto Ficol-Histopaque and centrifuged to recover an enriched population of the smaller “CD4lo” population. The Con A blasts and the enriched “CD4lo” population were cultured overnight in IL-2-supplemented medium and then stained with anti-CD4-PE and anti-CD69 FITC for FACS analysis. The numbers in the dot plots indicate the percentage of total cells that are within a region. A total of 20,000 events were collected for each plot. (b) 5 × 10⁶ “CD4lo”-enriched or “CD4hi”-enriched T cells were each adoptively transferred into 6 NOD.scid mice. As a control, untreated BDC2.5 TCR Tg spleen cells were also transferred into a control group of NOD.scid mice. Ten days later the pancreas was recovered from at least two mice per group, fixed, embedded, and cut into 5 micron sections that were subsequently stained with hematoxylin and eosin. The sections were viewed and photographed by a blinded observer. At least 50 islets were counted for each group to evaluate the pattern of insulitis. The arrows point to areas of inflammation, peri-insulits, which was predominant in “CD4lo” T cell recipients, and invasive insulitis, which was representative of most islets in recipients of “CD4hi” T cell recipients. The remaining mice were monitored for the development of diabetes as described in the Methods. (c) Spleen cells were recovered from the recipients in (b), stained with anti-CD4-PE and anti-TCR V beta 4-FITC, and then 100,000 events were collected during FACS analysis. The data in the dot plot were gated on viable cells (FSC × SSC). The numbers indicate the percentage of cells in the region. The number in the parentheses for the “CD4lo” recipients indicates the percentage of “CD4lo” cells in the total CD4⁺ population.

Figure 7

Antigen-experienced “CD4lo” T cells give rise to “CD4hi” T cells. “CD4lo” T cells were enriched from the Con A blasts of NOD spleen cells as described in Figure 6. The “CD4lo” T cells or Con A blasts were cultured overnight in IL-2-containing complete medium, then each was labeled with CFSE before incubation on anti-CD3-coated plates. Twenty-four and 48 hrs later the cells were labeled with anti-CD4-PE and analyzed by flow cytometry to reveal cell divisions. Proliferating “CD4hi” T cells arising from the blasts are indicated in the encircled population the numbers indicate the percentage in the total cell population. For each analysis, 100,000 events were collected, and the results are representative of four experiments.

Figure 8

CD95 is poorly expressed on antigen-experienced CD4lo T cells. (a) Naïve and Con A activated spleen cells pooled from two NOD or BALB/c mice were stained with anti-CD4 and CD95. R1 denotes region of double positive cells, CD4hiCD95hi, in Con A blasts. R2 denotes the region of single positive cells, CD4loCD95(−). Twenty-thousand events were collected for the analysis. The numbers indicate the percentage of the total population that resides in R1 or R2. (b) A summary of 3 experiments performed as in (a), representing the mean and SD from 3 individual mice. The P-values are only displayed for those comparisons between NOD and BALB/c that were below  .05.