Distinct requirements for achievement of allotolerance versus reversal of autoimmunity via nonmyeloablative mixed chimerism induction in NOD mice

Abstract

Objectives: Mixed hematopoietic chimerism is associated with islet allograft tolerance and may reverse autoimmunity. We developed low intensity regimens for the induction of mixed chimerism and examined the effects on autoimmunity in prediabetic nonobese diabetic (NOD) mice.

Research design and methods: NOD mice received various combinations of total body irradiation, anti-CD154, anti-CD8alpha, anti-CD4, and anti-Thy1.2 monoclonal antibodies, with or without transplantation of C57BL/6 bone marrow cells and were followed up for development of diabetes, chimerism, and donor skin graft survival. Autoimmunity was assessed by histologic examination of salivary glands and pancreata.

Results: Although conditioning alone prevented or delayed the onset of diabetes, stable mixed chimerism was required for the reversal of isletitis. Mixed chimerism and skin graft tolerance were achieved in NOD mice receiving anti-CD154 with bone marrow transplantation as the means of tolerizing peripheral CD4 T cells to alloantigens. However, isletitis was not reversed in allotolerant mixed chimeras prepared with this regimen.

Conclusions: Partial depletion of peripheral autoreactive NOD CD4 T cells is needed to achieve full reversal of isletitis by mixed chimerism induction from a protective donor strain, but it is not required for induction of specific tolerance to donor alloantigens. Thus, the requirements for tolerizing alloreactive and autoreactive NOD CD4 cells are distinct.

Figure 1. Mixed allogeneic chimerism in NOD mice receiving incomplete CD4-depleting conditioning regimen

A) Six to eight-week-old pre-diabetic female NOD mice received conditioning with anti-CD8 α.1 (Days −1,0,1,6,7,8), anti-Thy1.2 (Days −6, −1), anti-CD154 (Day 0) mAbs, and 4 Gy TBI (Day 0) followed by transplantation with 30 × 10⁶ allogeneic C57BL/6 bone marrow cells. The mean levels of donor chimerism in NOD mouse blood were measured by FACS at various time-points post-BMT (n=9). B) CD4⁺ T cell recovery in B6-->NOD (diabetic) mixed allogeneic chimeras prepared with non-CD4-depleting, non-myeloablative conditioning. The mean levels of donor (white bars) and recipient CD4⁺ T cells (black bars) in blood of pre-diabetic B6→NOD chimeras, as measured by FACS at various time points post-BMT.

Figure 2. Peripheral T cells of NOD mice resist depletion by conditioning regimen

The level of conditioning regimen-induced T cell depletion in spleens and lymph nodes of conditioned NOD and BALB/c mice were analyzed on Day 4. Pre-diabetic NOD mice (n=4) were treated with combinations of anti-CD4 (Days −6 and −1), anti-CD8α.1 (Days −6, −5, −4, −1, 0 and 1), anti-Thy1.2 (Days −6, and −1) and anti-CD154 mAb (Day 0). Age and sex-matched BALB/c mice (n=4) received anti-CD4 (Days −6 and −1), anti-CD8α.2 (Days −6 and −1), anti-Thy1.2 (Days −6, and −1) and anti-CD154 mAb (MR1). NOD mice received 4 Gy TBI (Day 0) and Balb/c mice received 3 Gy TBI (Day 0) without donor BMT. The mean percentages (left panel) and the mean total numbers (right panel) of CD4⁺ and CD8⁺ T cells in NOD (black bars) and BALB/c (white bars) spleens and lymph nodes were measured by FACS at Day 4. P values compare NOD and BALB/c mice.

Figure 3. Donor-specific skin graft tolerance in NOD mice receiving non-myeloablative conditioning, followed by B6 bone marrow cells

NOD mice received non-myeloablative conditioning followed by transplantation of 30×10⁶ allogeneic C57BL/6 (B6) bone marrow cells (BMC). Durable mixed chimerism was established in all recipients. Donor C57BL/6 and third party Balb/c skin was grafted 14 weeks post-BMT. Donor C57BL/6 skin grafts were accepted through the entire observation period (left panel). Third-party skin grafts (Balb/c) were rejected within 18 days after skin grafting (n=5 per group) (right panel).

Figure 4. Conditioning with or without BMT delays the development of diabetes in NOD mice

Six to eight week old pre-diabetic female NOD mice received no treatment (untreated, n=20) or conditioning Regimens A–H (conditioning regimen n=55). Additional animals received similar conditioning followed by transplantation of 30 × 10⁶ allogeneic C57BL/6 bone marrow cells (n=12). Diabetes was assessed by bi-weekly measurement of blood glucose using Dextrostix and a glucometer. Animals were considered to be diabetic after three sequential glucose measurements above 200mg/dl (hyperglycemia).

Figure 5. B6 mixed chimerism induced in pre-diabetic NOD mice with partially T cell depleting conditioning reverses autoimmunity in both islets and salivary glands. Conditioning alone does not

A, B) Pancreata and salivary glands of conditioned pre-diabetic NOD mice receiving CD4 partially-depleting conditioning regimens with or without allogeneic BMT, as well as non-treated control mice, were analyzed for the presence of inflammatory infiltrates at 31 weeks after conditioning. C, D) Pancreata and salivary glands of conditioned pre-diabetic NOD mice receiving non-CD4-depleting conditioning regimen with or without allogeneic BMT, as well as non-treated control mice, were analyzed for the presence of inflammatory infiltrates at 38 weeks after conditioning.

Figure 6. Progression of autoimmunity in NOD mice conditioned with Regimen F, with or without B6 BMT

Six-week-old NOD mice were conditioned with Regimen F, with or without BMT from B6 donors. At intervals of 5 weeks (5,10,15,20 weeks), 8–10 mice per group were sacrificed and analyzed for isletitis and sialadenitis. An additional cohort of mice received conditioning at 10 weeks of age and was sacrificed for histological analysis 30 weeks later. A shows the level (left) and incidence (right) of isletitis measured using the scale described in (22) and B shows the level (left) and incidence (right) of sialadenitis measured using the following scale: 0, no inflammation; 1, focal inflammation or lymphoid aggregates; 2, mild sialadenitis; 3, moderate sialadenitis; 4, severe sialadenitis.

Figure 7. Donor cells suppress autoimmunity in mixed chimeras prepared with Regimen F

Six-week-old NOD mice were conditioned with Regimen F, with or without BMT from B6 donors. Six weeks later the animals were euthanized and T cells were purified from splenocytes. Two million purified splenic T cells from naive NOD mice or conditioned control (CC NOD) mice were adoptively transferred to NOD-scid recipients. Two million NOD T cells from mixed chimeras were transferred to additional groups with (Mixed Chimerism (MC) NOD donor cell-depleted) or without (in 8 million total T cells with approximately 75% donor-type T cell chimerism at the time of sacrifice) (MC NOD) depletion of donor cells. Almost all transferred T cells from conditioned NOD mice were CD4+ and CD8−.