Depletion-Resistant CD4 T Cells Enhance Thymopoiesis During Lymphopenia

Abstract

Lymphoablation is routinely used in transplantation, and its success is defined by the balance of pathogenic versus protective T cells within reconstituted repertoire. While homeostatic proliferation and thymopoiesis may both cause T cell recovery during lymphopenia, the relative contributions of these mechanisms remain unclear. The goal of this study was to investigate the role of the thymus during T cell reconstitution in adult allograft recipients subjected to lymphoablative induction therapy. Compared with euthymic mice, thymectomized heart allograft recipients demonstrated severely impaired CD4 and CD8 T cell recovery and prolonged heart allograft survival after lymphoablation with murine anti-thymocyte globulin (mATG). The injection with agonistic anti-CD40 mAb or thymus transplantation only partially restored T cell reconstitution in mATG-treated thymectomized mice. After mATG depletion, residual CD4 T cells migrated into the thymus and enhanced thymopoiesis. Conversely, depletion of CD4 T cells before lymphoablation inhibited thymopoiesis at the stage of CD4^- CD8^- CD44^hi CD25⁺ immature thymocytes. This is the first demonstration that the thymus and peripheral CD4 T cells cooperate to ensure optimal T cell reconstitution after lymphoablation. Targeting thymopoiesis through manipulating functions of depletion-resistant helper T cells may thus improve therapeutic benefits and minimize the risks of lymphoablation in clinical settings.

Keywords: Immunosuppressant; T cell biology; basic (laboratory) research/science; immunobiology; immunosuppression/immune modulation; immunosuppressive regimens; induction; lymphocyte biology; polyclonal preparations: rabbit anti-thymocyte globulin; thymus/thymic biology.

Figure 1. The thymus is required for rapid reconstitution of T cells following mATG depletion

Thymectomized or euthymic B6 mice were transplanted with BALB/c heart allografts and treated with mATG on day 0 and 4 post-transplant. A. Experimental design. B–C. CD8 and CD4 T cell reconstitution in thymectomized vs euthymic mice treated with mATG. D. Numbers of live cells per 50 ml of peripheral blood before, early (d. 15) and late (d. 60–100) after heart transplantation and mATG treatment. E–F. CD8 and CD4 T cell reconstitution in mATG treated vs untreated thymectomized mice. Data are shown as mean ± SD, n = 4–12 mice/group pooled from three independent experiments. G. Survival of BALB/c cardiac allografts. N = 5–13 mice/group. H. Donor-specific T cell responses measured in the spleen by an IFNγ ELISPOT assay at the time of rejection. Data are calculated as numbers of IFNγ spots per 1 × 10⁶ spleen T cells and shown as mean ± SD, n = 3–6 mice/group. I. Donor-specific antibody serum titers at the time of rejection. Symbols represent serum IgG titers in individual recipients. The titers of third party SJL-reactive alloantibody was <135 for all IgG isotypes in all tested samples. ns – p>0.05, * – p<0.05, ** – p< 0.001, *** – p<0.0001.

Figure 2. CD4 T cell numbers are reduced following thymectomy in naïve non-transplanted mice

Percentages (top) and numbers (bottom) of CD4 and CD8 T cells in the peripheral blood (A), CD4, CD8 and total CD3 T cells in spleen (B) or inguinal lymph nodes (C) of naïve non-transplanted euthymic and thymectomized B6 mice analyzed between 1 and 11 weeks after thymectomy. D. Percentage of CD44^hi cells among CD4 and CD8 T cell populations. Symbols represent data from individual mice analyzed in two independent experiments. ns – p>0.05.

Figure 3. CD4 T cell transfer or treatment with agonistic anti CD40 antibody facilitates T cell reconstitution in thymectomized heart allograft recipients

A. Thymectomized B6 mice were transplanted with BALB/c heart allografts and treated with mATG on days 0 and 4 posttransplant. One group of recipients was injected with 10 × 10⁶ CD4 T cells isolated from naïve B6 mice one day prior to heart transplantation (top). Another group was injected with agonistic anti-CD40 mAb (clone FGK4.5, 100ug i.v.) on days 0 and 1 after heart transplantation (bottom). B–C. CD8 (B) and CD4 (C) T cell recovery in the peripheral blood of recipients injected with CD4 T cells. Data are shown as mean ± SD, n = 10–14 mice/group pooled from three independent experiments. D–E. CD8 (D) and CD4 (E) T cell recovery in the peripheral blood of recipients injected with anti-CD40 mAb. Data are shown as mean ± SD, n = 6–10 mice/group. H. Heart allograft survival. n = 10–11 mice/group. ns – p>0.05, * – p<0.05, ** – p< 0.001, *** – p< 0.0001.

Figure 4. Thymus transplantation enhances T cell reconstitution following mATG depletion in thymectomized mice

A. Thymectomized B6 mice received BALB/c heart transplants and were treated with mATG on d. 0 and 4 post-transplant. During the transplantation surgery, either a whole RAG1^−/− or half of a CD45.1 WT thymus was placed under the left kidney capsule. B–C. CD8 (B) and CD4 (C) T cell recovery in mATG treated thymectomized mice transplanted with one half of WT thymus. Data are shown as mean ± SD, n = 6–13 mice/group pooled from three independent experiments. D–E. CD8 (D) and CD4 (E) T cell recovery in mATG treated thymectomized mice transplanted with a whole RAG1^−/− thymus. Data are shown as mean ± SD, n = 5–11 mice/group. F) Heart allograft survival. n = 6–11 mice/group. ns – p>0.05, *=p<0.05, ** – p< 0.001, *** – p< 0.0001.

Figure 5. Peripheral CD4 T cells migrate into the thymus in lymphopenic mice

A. 13–19 × 10⁶ CD4 T cells isolated from naïve B6.CD45.1⁺ mice were intravenously injected into thymectomized B6.CD45.2⁺ mice. On the day of surgery, these mice were transplanted with BALB/c heart allografts and a thymus lobe from B6.CD45.1/2 F1 mice. All recipients were treated with mATG on days 0 and 4, and thymic and peripheral T cells were evaluated on day 15 after transplantation. All data are shown as mean ± SD, n = 8–12 mice/group pooled from three independent experiments. B. Representative dot plots of CD45.1/CD45.2 staining of cells isolated from the transplanted thymi. C. Percentages and numbers of transferred CD4 T cells within transplanted thymi. D. Numbers of CD24^lo (left) and Qa2^hiCD24^lo (right) cells among CD45.2⁺ host-derived single positive CD4 thymocytes.

Figure 6. Peripheral CD4 T cells enhance thymopoiesis in lymphopenic mice

Recipients depicted in Figure 5 were analyzed at 15 days after transplantation. A. Comparison of the thymus size and total numbers of cells in the thymus. B, D. Host-derived (B) or thymus donor-derived (D) cells presented as percentage of total thymocytes or absolute numbers. C, E. Numbers of host-derived (C) and thymus donor-derived (D) cells among double negative CD4⁻CD8⁻ (DN), double positive CD4⁺CD8⁺ (DP) and single positive (SP) CD4⁺ or CD8⁺ thymic subsets. On the right, absolute numbers of CD44^hi CD25⁻ (DN1), CD44^hi CD25⁺ (DN2), CD44^low CD25⁺ (DN3) and CD44^low CD25⁻ (DN4) cells within host-derived and donor-derived DN populations. F. Total numbers of cells in spleens. G. Anti-donor T cells responses in the spleen were measured by IFNγ ELISPOT assay in response to donor antigen stimulation and presented as numbers of IFNγ spots per 1 × 10⁶ spleen T cells. Symbols represent data from individual mice. n=10–13 pooled from three independent experiments. ns – p>0.05.

Figure 7. Peripheral CD4 T cells enhance thymopoiesis following mATG depletion by interfering in the process at the DN2 stage

B6 mice were transplanted with BALB/c heart allografts and treated with mATG on days 0 and 4 post-transplant. Groups of recipients were additionally treated with depleting anti-CD4 mAbs (clones YTS191 and GK1.5, 200 μg of each i.p. on d. -3, -2, -1 relative to transplantation). A. Absolute numbers of thymic cell subsets on day 6 (top) or day 15 (bottom) after transplantation. Please note that the fluorochrome-labeled anti-CD4 mAb used for flow cytometry analyses (clone RM4-5) is different from YTS191 and GK1.5 mAbs used for CD4 T cell depletion. Symbols represent data from individual mice. B. Numbers of DN1 (CD44^hiCD25^low), DN2 (CD44^hiCD25^hi), DN3 (CD44^loCD25^hi) and DN4 (CD44^loCD25^lo) thymic cells on day 15 after transplantation. Data are shown as mean ± SD, n = 4–11 mice/group pooled from two independent experiments. ns – p>0.05. P values are calculated between mATG treated and aCD4 + mATG treated groups.