A novel multimeric form of FasL modulates the ability of diabetogenic T cells to mediate type 1 diabetes in an adoptive transfer model

Abstract

Activation induced cell death (AICD) via Fas/FasL is the primary homeostatic molecular mechanism employed by the immune system to control activated T-cell responses and promote tolerance to self-antigens. We herein investigated the ability of a novel multimeric form of FasL chimeric with streptavidin (SA-FasL) having potent apoptotic activity to induce apoptosis in diabetogenic T cells and modulate insulin-dependent type 1 diabetes (IDDM) in an adoptive transfer model. Diabetogenic splenocytes from NOD/Lt females were co-cultured in vitro with SA-FasL, SA control protein, or alone without protein, and adoptively transferred into NOD/Lt-Rag1(null) recipients for diabetes development. All animals receiving control (Alone: n=16 or SA: n=17) cells developed diabetes on average by 6 weeks, whereas animals receiving SA-FasL-treated (n=25) cells exhibited significantly delayed progression (p<.001) and decreased incidence (70%). This effect was associated with an increase in CD4(+)CD25(+) T cells and correlated with FoxP3 expression in pancreatic lymph nodes. Extracorporeal treatment of peripheral blood lymphocytes using SA-FasL during disease onset represents a novel approach that may alter the ability of pathogenic T cells to mediate diabetes and have therapeutic utility in clinical management of IDDM.

Fig. 1. Effect of SA-FasL on ex vivo treated cells at transfer

Spleens from NOD mice positive for diabetes were harvested, processed, and resuspended at 4×10⁶/mL in complete MLR medium. Splenocytes were bulk cultured and treated with PBS, SA, or SA-FasL for 7.5 hours. Ex vivo treated cultures were analyzed for apoptosis (AnnexinV-FITC positive and 7-AAD negative) by flow cytometry. CD4⁺, CD4⁺CD25⁺, and CD8⁺ T-cell populations were analyzed for apoptosis using AnnexinV-FITC and T-cell antibodies: CD25-PE, CD8-PerCp, and CD4-APC. Effect of SA-FasL on ex vivo treated cells was statistically analyzed by Mann-Whitney U test. (A) Absolute number of live and apoptotic cells transferred. (B) Absolute number of CD4⁺ and CD8⁺ apoptotic cells transferred. (C) Absolute number of CD4⁺, CD4⁺CD25⁺, CD4⁺CD25^-, and CD8⁺ live cells transferred. * P<0.01. †P<0.05.

Fig. 1. Effect of SA-FasL on ex vivo treated cells at transfer

Spleens from NOD mice positive for diabetes were harvested, processed, and resuspended at 4×10⁶/mL in complete MLR medium. Splenocytes were bulk cultured and treated with PBS, SA, or SA-FasL for 7.5 hours. Ex vivo treated cultures were analyzed for apoptosis (AnnexinV-FITC positive and 7-AAD negative) by flow cytometry. CD4⁺, CD4⁺CD25⁺, and CD8⁺ T-cell populations were analyzed for apoptosis using AnnexinV-FITC and T-cell antibodies: CD25-PE, CD8-PerCp, and CD4-APC. Effect of SA-FasL on ex vivo treated cells was statistically analyzed by Mann-Whitney U test. (A) Absolute number of live and apoptotic cells transferred. (B) Absolute number of CD4⁺ and CD8⁺ apoptotic cells transferred. (C) Absolute number of CD4⁺, CD4⁺CD25⁺, CD4⁺CD25^-, and CD8⁺ live cells transferred. * P<0.01. †P<0.05.

Fig. 1. Effect of SA-FasL on ex vivo treated cells at transfer

Spleens from NOD mice positive for diabetes were harvested, processed, and resuspended at 4×10⁶/mL in complete MLR medium. Splenocytes were bulk cultured and treated with PBS, SA, or SA-FasL for 7.5 hours. Ex vivo treated cultures were analyzed for apoptosis (AnnexinV-FITC positive and 7-AAD negative) by flow cytometry. CD4⁺, CD4⁺CD25⁺, and CD8⁺ T-cell populations were analyzed for apoptosis using AnnexinV-FITC and T-cell antibodies: CD25-PE, CD8-PerCp, and CD4-APC. Effect of SA-FasL on ex vivo treated cells was statistically analyzed by Mann-Whitney U test. (A) Absolute number of live and apoptotic cells transferred. (B) Absolute number of CD4⁺ and CD8⁺ apoptotic cells transferred. (C) Absolute number of CD4⁺, CD4⁺CD25⁺, CD4⁺CD25^-, and CD8⁺ live cells transferred. * P<0.01. †P<0.05.

Fig. 2. FoxP3 expression in live CD4⁺CD25⁺ T-cells

Spleens from NOD mice positive for diabetes were harvested, processed, and resuspended at 4×10⁶/mL in complete MLR medium. Splenocytes were bulk cultured and treated with PBS, SA, or SA-FasL for 7.5 hours. Ex vivo treated cultures were washed and stained with a saturating amount of AnnexinV-FITC and T-cell antibodies: CD25-PE and CD4-APC. Using a high speed cell sorter, live (AnnexinV negative) CD4⁺CD25⁺ from SA-FasL and control ex vivo treated cultures were sorted and total RNA prepared. CD4⁺CD25⁺ and CD4⁺CD25^- T cells from healthy NOD peripheral lymph node were sorted for FoxP3 mRNA controls. cDNA from sorted cells was prepared and then amplified in duplicate by real-time PCR for GAPDH and FoxP3. FoxP3 mRNA levels were normalized relative to GAPDH mRNA expression and statistically analyzed by Kruskal-Wallace test. Data are presented as the fold-change relative to CD4⁺CD25^- T cells. P>0.05.

Fig. 3. Incidence of diabetes after adoptive transfer of ex vivo treated NOD splenocytes

Diabetogenic splenocytes from NOD/Lt females were bulk cultured in vitro with PBS (Alone), control (SA), and test protein (SA-FasL) for 7.5 hours. After culture, cells were washed and 20×10⁶ex vivo treated cells were adoptively transferred by intravenous tail vein infusion into 4-6 week old male and female NOD/Lt-Rag1^null mice. For 17 weeks all recipients were monitored for diabetes development twice weekly. Diabetes incidence was assessed by Kaplan-Meier life table analysis using the Log-Rank test. All animals receiving untreated (Alone: ∇ white triangle, n=16) or control treated (SA: ▲ black triangle, n=17) cells developed overt diabetes by 73 days (11 weeks). Animals receiving FasL treated (SA-FasL: ■ black square, n=25) cells exhibited significantly delayed onset and decreased incidence. P<0.001.

Fig. 4. Repopulation analyses

Spleen, mesenteric, pancreatic, and peripheral lymph nodes from recipients were harvested, processed, counted, and phenotyped by flow cytometric analysis to determine effective T cell expansion and repopulation. Each cellular compartment was phenotyped using the T cell specific surface marker CD3-FITC and statistically analyzed by Mann-Whitney U test. (A) Cell counts for each compartment and individual animals were obtained and totaled for determination of total cells recovered. (B) Absolute numbers of splenic T cells in individual recipients were calculated based on percent of CD3⁺ cells recovered in spleen. *P<0.01. †P<0.05.

Fig. 4. Repopulation analyses

Spleen, mesenteric, pancreatic, and peripheral lymph nodes from recipients were harvested, processed, counted, and phenotyped by flow cytometric analysis to determine effective T cell expansion and repopulation. Each cellular compartment was phenotyped using the T cell specific surface marker CD3-FITC and statistically analyzed by Mann-Whitney U test. (A) Cell counts for each compartment and individual animals were obtained and totaled for determination of total cells recovered. (B) Absolute numbers of splenic T cells in individual recipients were calculated based on percent of CD3⁺ cells recovered in spleen. *P<0.01. †P<0.05.

Fig. 5. CD4⁺CD25⁺ T cells are increased in SA-FasL treated long-term survivors

Spleens, mesenteric, pancreatic, and peripheral lymph nodes from individual recipients were harvested, processed, counted, and phenotyped by flow cytometric analysis to determine effective CD4⁺CD25⁺ T cell expansion and repopulation. Each cellular compartment was phenotyped using the surface markers specific for CD4⁺CD25⁺ T cells, CD3-FITC, CD25-PE, and CD4-APC and statistically analyzed by Mann-Whitney U test. (A) Absolute numbers of CD4⁺CD25⁺ T cells in individual recipients were calculated for each cellular compartment based on percent of CD3⁺CD4⁺CD25⁺ cells recovered. Absolute numbers of CD4⁺CD25⁺ cells calculated for each compartment were added to determine total cell recovery. Absolute numbers of (B) splenic and (C) pancreatic lymph node CD4⁺CD25⁺ T cells in individual recipients were calculated based on percent of CD3⁺ CD4⁺CD25⁺ cells recovered in spleen and pancreatic lymph nodes, respectively. * P<0.01. †P<0.05.

Fig. 5. CD4⁺CD25⁺ T cells are increased in SA-FasL treated long-term survivors

Spleens, mesenteric, pancreatic, and peripheral lymph nodes from individual recipients were harvested, processed, counted, and phenotyped by flow cytometric analysis to determine effective CD4⁺CD25⁺ T cell expansion and repopulation. Each cellular compartment was phenotyped using the surface markers specific for CD4⁺CD25⁺ T cells, CD3-FITC, CD25-PE, and CD4-APC and statistically analyzed by Mann-Whitney U test. (A) Absolute numbers of CD4⁺CD25⁺ T cells in individual recipients were calculated for each cellular compartment based on percent of CD3⁺CD4⁺CD25⁺ cells recovered. Absolute numbers of CD4⁺CD25⁺ cells calculated for each compartment were added to determine total cell recovery. Absolute numbers of (B) splenic and (C) pancreatic lymph node CD4⁺CD25⁺ T cells in individual recipients were calculated based on percent of CD3⁺ CD4⁺CD25⁺ cells recovered in spleen and pancreatic lymph nodes, respectively. * P<0.01. †P<0.05.

Fig. 5. CD4⁺CD25⁺ T cells are increased in SA-FasL treated long-term survivors

Spleens, mesenteric, pancreatic, and peripheral lymph nodes from individual recipients were harvested, processed, counted, and phenotyped by flow cytometric analysis to determine effective CD4⁺CD25⁺ T cell expansion and repopulation. Each cellular compartment was phenotyped using the surface markers specific for CD4⁺CD25⁺ T cells, CD3-FITC, CD25-PE, and CD4-APC and statistically analyzed by Mann-Whitney U test. (A) Absolute numbers of CD4⁺CD25⁺ T cells in individual recipients were calculated for each cellular compartment based on percent of CD3⁺CD4⁺CD25⁺ cells recovered. Absolute numbers of CD4⁺CD25⁺ cells calculated for each compartment were added to determine total cell recovery. Absolute numbers of (B) splenic and (C) pancreatic lymph node CD4⁺CD25⁺ T cells in individual recipients were calculated based on percent of CD3⁺ CD4⁺CD25⁺ cells recovered in spleen and pancreatic lymph nodes, respectively. * P<0.01. †P<0.05.

Fig. 6. Analysis of FoxP3 mRNA expression and suppressive activity of CD4⁺CD25⁺ T cells in lymph node of long-term SA-FasL survivors

(A) Total RNA was extracted from whole pancreatic lymph node. cDNA was amplified in duplicate by real-time PCR for GAPDH and FoxP3. FoxP3 mRNA levels were normalized relative to GAPDH mRNA expression and statistically analyzed by Kruskal-Wallace test. Data are presented as the fold-change relative to control, sorted CD4⁺CD25^- T cells. P>0.05. (B) CD4⁺CD25⁺ cells sorted from healthy naïve NOD peripheral lymph node (control) and long-term SA-FasL survivor pooled peripheral and pancreatic lymph nodes were cultured with control CD4⁺CD25^- T cells at different ratios and evaluated for suppressive function. Data presented is representative of three suppression assays performed evaluating suppressive activity of CD4⁺CD25⁺ from long-term SA-FasL survivors.

Fig. 6. Analysis of FoxP3 mRNA expression and suppressive activity of CD4⁺CD25⁺ T cells in lymph node of long-term SA-FasL survivors

(A) Total RNA was extracted from whole pancreatic lymph node. cDNA was amplified in duplicate by real-time PCR for GAPDH and FoxP3. FoxP3 mRNA levels were normalized relative to GAPDH mRNA expression and statistically analyzed by Kruskal-Wallace test. Data are presented as the fold-change relative to control, sorted CD4⁺CD25^- T cells. P>0.05. (B) CD4⁺CD25⁺ cells sorted from healthy naïve NOD peripheral lymph node (control) and long-term SA-FasL survivor pooled peripheral and pancreatic lymph nodes were cultured with control CD4⁺CD25^- T cells at different ratios and evaluated for suppressive function. Data presented is representative of three suppression assays performed evaluating suppressive activity of CD4⁺CD25⁺ from long-term SA-FasL survivors.