A pilot study showing associations between frequency of CD4(+) memory cell subsets at diagnosis and duration of partial remission in type 1 diabetes

Abstract

In some patients with type 1 diabetes the dose of insulin required to achieve euglycemia is substantially reduced soon after diagnosis. This partial remission is associated with β-cell function and good glucose control. The purpose of this study was to assess whether frequencies of CD4(+) T cell subsets in children newly diagnosed with type 1 diabetes are associated with length of partial remission. We found that the frequency of CD4(+) memory cells, activated Treg cells and CD25(+) cells that express a high density of the IL-7 receptor, CD127 (CD127(hi)) are strongly associated with length of partial remission. Prediction of length of remission via Cox regression is significantly enhanced when CD25(+) CD127(hi) cell frequency is combined with either Insulin Dependent Adjusted A1c (IDAA1c), or glycosylated hemoglobin (HbA1c), or C-peptide levels at diagnosis. CD25(+) CD127(hi) cells do not express Foxp3, LAG-3 and CD49b, indicating that they are neither Treg nor Tr1 cells.

Keywords: CD25(+) non-Treg; Partial remission; Regulatory cells; T cell subsets; Type 1 diabetes.

Fig 1. Changes in IDAA1c and C-peptide AUC with time

IDAA1c (A) and C-peptide AUC (B) were calculated for each patient (n = 19) within 3 months of diagnosis (baseline, time 0), and then at 3, 6, 9, 12, 18 and 24 months post-baseline. Each symbol represents a patient. Patients with an IDAA1c above 9 indicated by a horizontal line are not in partial remission at that time point (A). At time points when there are fewer than 19 patients represented, data for either HbA1c (A), or C-peptide (B) was not available.

Fig 2. Immune cell subsets measured in patients newly diagnosed with T1D

PBMC isolated from 19 patients at baseline were labeled for either CD3, CD4, CD25, and CD127, or CD3, CD4, CD45RA, CD45RO and Foxp3. Dot plot A is gated on CD3⁺ CD4⁺ cells and shows co-expression of CD45RO and CD45RA. CD45RO⁺ CD45RA⁻ memory cells are in the upper left quadrant. The plot in panel B is also gated on CD3⁺ CD4⁺ T cells and shows the activated Treg region. The co-expression of CD25 and CD127 on gated CD3⁺ CD4⁺ cells is shown as a dot plot (C). The upper right gate shows the CD25⁺ CD127^hi cells while the lower right gate shows the Treg population. Panels D and E show the relative frequency of all three cell subsets at baseline (base) and at 3 months post-baseline (3 mo) in patients with either good (D) or poor (E) glycemic control during the first three moths post diagnosis. Each symbol at each time point represents an individual patient. The lines link the same patient at the two time points tested. The numbers of patients tested for each cell subset at each time point are shown on the figure.

Fig 3. The relative frequency of CD4⁺ CD45RO⁺ memory cells, activated Treg cells, and CD25⁺ CD127^hi cells correlates with length of remission

The relationship between the relative frequency of CD45RO⁺ cells (A), activated Treg cells (B), CD25⁺ CD127^hi cells (C and D), baseline IDAA1c (E) and baseline C-peptide AUC (F) with length of partial remission. Each symbol represents a patient. Pearson’s Correlation Coefficient (r), R squared (R²) and statistical significance (p) are shown on each panel.

Fig 4. In healthy individuals CD25⁺ CD127^hi cells are central and transitional memory cells

PBMC freshly isolated from healthy adults were co-stained for CD3, CD4, CD25, CD127, CD45RA, CCR7 and CD28 (n = 8). Panel A top dot plot identifies CD4⁺ CD45RA⁻ (RA⁻) and CD4⁺ CD45RA⁺ (RA⁺) cells gated CD4⁺ CD3⁺ cells. The lower left plot is gated on RA⁻ cells and identifies central memory (CM), transitional memory (TM) and effector memory (EM) cells. The lower right plot is gated on RA⁺ cells and identifies naïve and effector memory cells that express RA (EMRA). Panel B shows the mean +/− SD relative frequency of naïve, memory (RA⁻) CM, TM and EM cells in CD25⁺ CD127^hi. No EMRA cells were seen in healthy donors.

Fig 5. CD25⁺ CD127^hi cells are neither Foxp3⁺ Treg nor Tr1 cells

PBMC from healthy donors (n = 5) were thawed and labeled for CD3, CD4, CD25, CD127, and either Foxp3, or LAG-3 and CD49b. (A) A representative example of Foxp3 expression on gated CD4⁺ (pink, CD3⁺ CD4⁺), CD25⁺ CD127^hi (blue, CD3⁺ CD4⁺ CD25⁺ CD127^hi) and Treg (orange, CD3⁺ CD4⁺ CD25⁺ CD127^low) cells, and the percent Foxp3⁺ cells within each subset from all donors is shown in B. The dot plots show a representative example of the co-expression of LAG-3 and CD49b gated on either CD4⁺ (C), or CD25⁺ CD127^hi (D), or Treg (E) cells and the percent single LAG-3⁺ (F), CD49b⁺ (G) and LAG-3/CD49b double positive (H) cells within each cell subset from all donors. The data are shown as mean +/− SD and are pooled from 2 separate experiments. Significant differences shown in panel H were calculated using Mann Whitney, ** p = 0.008, * p = 0.02.

Fig 6. CD25⁺ CD127^hi cells express CD44v6 and a high density of CD44

PBMC freshly isolated from healthy adults were co-stained for CD3, CD4, CD25, CD127, and either CD44v4 and CD44v6 (n = 6) or CD44 (n = 5). Panel A shows a representative example of CD44v6 and CD44v4 expression, as well as isotype control staining, on CD25⁺ CD127^hi cells. The isotype for the CD44v6- and CD44v4-specific antibodies used is the same. The mean +/− SD CD44v6 and CD44v4 expression on CD25⁺ CD127^hi cells is shown in B. Panels C and D show expression of CD44 on total CD4⁺ (pink), CD25⁺ CD127^hi (blue) and Treg (orange) cells (C), and mean fluorescence intensity (MFI) of CD44 on each cell subset (D).

Fig 7. The relative frequency of CD4⁺ CD45RO⁺ and activated Treg cells correlates significantly with frequency of CD25⁺ CD127^hi cells

The relationship between the relative frequency of CD45RO⁺, activated Treg, and CD25⁺ CD127^hi cells in patients with recent onset T1D who have good glycemic control (ages 8–16 years). Each symbol represents a patient. Pearson’s Correlation Coefficient (r), R squared (R²), and statistical significance (p) are shown on each panel.