Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs

Abstract

Safe induction of autoantigen-specific long-term tolerance is the "holy grail" for the treatment of autoimmune diseases. In animal models of type 1 diabetes, oral or i.n. immunization with islet antigens induces Tregs that are capable of bystander suppression. However, such interventions are only effective early in the prediabetic phase. Here, we demonstrate that a novel combination treatment with anti-CD3epsilon-specific antibody and i.n. proinsulin peptide can reverse recent-onset diabetes in 2 murine diabetes models with much higher efficacy than with monotherapy with anti-CD3 or antigen alone. In vivo, expansion of CD25(+)Foxp3(+) and insulin-specific Tregs producing IL-10, TGF-beta, and IL-4 was strongly enhanced. These cells could transfer dominant tolerance to immunocompetent recent-onset diabetic recipients and suppressed heterologous autoaggressive CD8 responses. Thus, combining a systemic immune modulator with antigen-specific Treg induction is more efficacious in reverting diabetes. Since Tregs act site-specifically, this strategy should also be expected to reduce the potential for systemic side effects.

Figure 1. Purity and functionality of the anti-CD3 F(ab′)₂ .

(A) The purity of anti-CD3 was assessed by staining with Coomassie blue brilliant. (B) ELISA experiment to compare the binding capacities of the purified nonmitogenic (NM) F(ab′)₂ and full-length anti-CD3. (C) The half-life of the CD3 was evaluated in vivo. After i.v. injection of 200 μg into mice (n = 6), 50 μl blood was taken daily and the presence of anti-CD3 F(ab′)₂ in the serum assessed by ELISA. NT, nontreated. (D) In vivo activity of the NM anti-CD3 F(ab′)₂ was evaluated in RIP-LCMV mice (n = 6). Blood samples were analyzed days 1, 3, 8, and 15 after treatment with anti-CD3 alone and their contents in CD4⁺, CD8⁺, and CD25⁺ T cells compared with those of untreated and uninfected RIP-LCMV mice (control). (E) The efficacy of our commercial anti-CD3 F(ab′)₂ was controlled by treating NOD mice after new-onset diabetes with 3 Ab doses (10, 50, and 100 μg/d, 5 consecutive days). The percentage of mice showing long-term remission was calculated and is shown in the figure.

Figure 2. Synergy between non–Fc-binding anti-CD3 and hpIIp in treating NOD and RIP-LCMV mice after overt diabetes.

Mice were treated with either the anti-CD3 or peptide alone [suboptimal dose of anti-CD3 F(ab′)₂ i.v. or hpIIp i.n., both 40 μg/injection] or a combination of both (combination therapy [CT]). (A) In the H-2^d RIP-LCMV model (upper panel), the efficacy of the combination treatment reached 50% (n = 34) compared with 20% with anti-CD3 alone (n = 26) or 0% with hpIIp alone (n = 6). In the NOD model (lower panel), the efficacy reached 55% with the combination treatment (n = 31), compared with 37% with anti-CD3 alone (n = 54) or 22% with the hpIIp alone (n = 9). At week 9 after treatment, the statistical significance was evaluated between the anti-CD3 alone and the combination therapy groups (*P < 0.05). (B) Pancreatic islets were scored for the presence of mononuclear infiltration (upper left panel). The average percentage shown was determined from at least 8 mice per group. Shown are histological stainings of pancreata from RIP-LCMV-NP mice (lower panels). At week 5 after treatment with anti-CD3 alone or in conjunction with the proinsulin peptide (CT), pancreata from protected animals were harvested. Six-micrometer tissue sections were cut and collected for immunochemistry. Sections were costained for insulin and NP expression (lower left panel). Sections were probed for cellular infiltration by CD4⁺ and CD8⁺ T cells (lower right panel). Representative sections are shown in each panel. Original magnification, ×20.

Figure 3. Treatment with a combination of anti-CD3 and hpIIp increases the number of CD4⁺ T cells with a regulatory phenotype.

The data are from euglycemic animals treated with anti-CD3 (n = 5 experiments with 3–5 mice per experiment) or with the combination therapy (n = 5 experiments with 3–5 mice per experiment). As control, nontreated animals were used (n = 4 experiments with 4–6 mice per experiment). (A) The percentage of CD4⁺CD25⁺ cells is shown. (B) After intracellular staining, the percentage of CD4⁺Foxp3⁺ cells was evaluated. For each group, the mean ± SD is given, with P = 0.016 for the combination treatment group and P = 0.089 for the NM anti-CD3–treated group as compared with the untreated control group (None). (C) CD4⁺CD25⁺ cells expressed high levels of Foxp3. For each sample, cells were lysed in loading buffer containing SDS and β-mercaptoethanol and used in the Western blotting experiment. 5 × 10⁵ purified CD4⁺CD25⁺ and CD4⁺CD25^– cells were obtained from mice protected by treatment with anti-CD3 (α-CD3) alone or in combination with the hpIIp. As positive and negative controls (control+ and control–, respectively), 10⁵ HeLa cells stably expressing or not, respectively, the mouse Foxp3 protein were loaded. Actin was used to evaluate the amount of loaded proteins. (D–I) Long-term protected RIP-LCMV mice were sacrificed and the percentage of Treg markers in pooled splenocytes and PLNs cells determined. The percentages of GITR⁺ and Foxp3⁺ cells was measured in the CD4⁺ population (D and E). The percentage of GITR⁺, Foxp3⁺, CTLA-4⁺, and CD62L⁺ cells were measured in the CD4⁺CD25⁺ population (F–I).

Figure 4. hpIIp–specific T cells from mice treated with anti-CD3 and i.

. proinsulin peptide exhibit a regulatory cytokine profile after in vitro stimulation. (A) Splenocytes and PLNs from animals treated and cured by the anti-CD3 alone or in combination with the hpIIp were used in vitro in a cytokine secretion assay. Cells were stimulated with a mixture of anti-CD3 and anti-CD28 Abs, various peptides (hpIIp, insB9–23, and LCMV-specific GP61–80 and NP118–126) or remained nonstimulated. After 72 hours, the supernatants were analyzed by ELISA as well as using the Luminex 100 LabMAP System. The data represent the levels of cytokine above the background observed without stimulation (NM-anti-CD3 [IL-4: 17 pg/ml, IL-5: 430 pg/ml, IL-10: 10 pg/ml, TGF-β1: 530 pg/ml, and IFN-γ: 2,500 pg/ml] or combination therapy [IL-4: 41.5 pg/ml, IL-5: 380 pg/ml, IL-10: 200 pg/ml, TGF-β1: 580 pg/ml, and IFN-γ: 3,000 pg/ml]). Data for an average of 4 mice per group are shown. *P < 0.05 compared with the nonstimulated control. (B) To confirm the cytokine profile found in the supernatants, intracellular cytokine stainings were performed. The cells were stimulated specifically with the hpIIp or unspecifically with a mixture PMA/ionomycin or remained unstimulated (nonstimulated) for 6 hours in the presence of an inhibitor of intracellular protein transport (monensin). The cells were recovered and stained at the surface for CD4 and intracellularly for Foxp3 and the cytokines TGF-β, IL-10, and IL-4. The cytokine expression was analyzed in the CD4⁺Foxp3⁺ (upper panel) and CD4⁺Foxp3^neg (lower panel) populations.

Figure 5. Reduced autoaggressive CD8⁺ T cell responses after combination treatment.

(A) The percentage of NP118 tetramer–positive CD8⁺ T cells in nondiabetic LCMV-infected RIP-NP mice (5 weeks after infection) or after treatment (anti-CD3 alone or combination therapy). Numbers above the gated cells correspond to the percentage of tetramer-positive CD8⁺ T cells found in the individual mice represented in the figure. Histograms, shown as insets, represent the average of n = 6–9 mice per group. As control, BALB/c mice were used. *P = 0.0036 compared with mice that have been infected but are in the memory phase of viral clearance (memory mice) and **P = 0.0057 compared with mice treated with NM-anti-CD3 alone. (B) The percentage of NRP-V7 tetramer–positive CD8⁺ T cells is shown for prediabetic NOD mice (10-week-old mice), for diabetic NOD mice, and for cured NOD mice after treatment (anti-CD3 alone or combination therapy). Numbers in the upper-right corner correspond to the percentage of tetramer-positive CD8⁺ T cells found in the individual mice shown in the figure. Histograms, shown as insets, represent the average of n = 5 mice per group. (C) The percentage of IFN-γ– and TNF-α–expressing CD8⁺ T cells was measured by intracellular staining in nondiabetic LCMV-infected RIP-NP mice, either nontreated (memory) or treated (anti-CD3 or combination therapy). Rows correspond to 3 different in vitro stimulations. Splenocytes and PLN cells were pooled and stimulated with either PMA/ionomycin or NP118 peptide or remained unstimulated (None). Histograms, shown as insets, correspond to the mean ± SD of IFN-γ– or TNF-α–positive CD8⁺ T cells (n = 3–5 mice per group). *P < 0.05 compared with the LCMV memory control.

Figure 6. CD4⁺ CD25⁺ T lymphocytes generated by combination therapy exert dominant tolerance and block autoimmune diabetes after adoptive transfer.

(A) Adoptive transfer of 5 × 10⁶ CD4⁺ T cells from nondiabetic donors, either nontreated RIP-LCMV mice (control, n = 5 mice) or mice treated with the anti-CD3 alone (n = 4 mice) or in combination with the proinsulin peptide (n = 6 mice). A 3-day antigenic stimulation of splenocytes derived from protected mice was performed for each group (treated as well as control) to expand the proinsulin-specific Treg population prior to adoptive transfers. The putative Tregs were transferred into nonimmunodeficient RIP-LCMV-NP recipient mice day 5 after LCMV infection. (B) CD4⁺CD25⁺ and CD4⁺CD25^– T cells were purified from treated RIP-LCMV mice protected by the anti-CD3 alone (lower panel) or in combination with the proinsulin peptide (upper quadrant). Those cells (10⁶) were transferred into nonimmunodeficient RIP-LCMV-NP recipient mice 5 days after LCMV infection (n = 6 mice per group). (C) Proliferation of CD4⁺CD25⁺ lymphocytes in the spleens and PLNs of RIP-LCMV recipient mice. CD4⁺CD25⁺ T lymphocytes were purified from treated RIP-LCMV mice protected by the anti-CD3 alone (left panels) or in combination with the hpIIp (right panels). These cells were CFSE labeled and transferred into wild-type RIP-LCMV recipient mice day 5 after infection. Spleens and PLNs were removed from recipients 2 days after transfer and stained with a CD4-specific antibody. The data are representative of 2 independent experiments. Numbers correspond to the mean ± SD of CFSE-diluted CD4⁺CD25⁺ lymphocytes (n = 2 mice per group).