Th2 Cell Therapy of Established Acute Graft-Versus-Host Disease Requires IL-4 and IL-10 and Is Abrogated by IL-2 or Host-Type Antigen-Presenting Cells

Delayed administration of Th2 cells improves balance of GVHD and GVT effects. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow alone (“BMT”); marrow and host-type TS/A tumor cells (“TSA”); or marrow, tumor cells, and additional donor T cells (“GVHD”). Other cohorts received marrow, tumor cells, donor T cells, and additional donor CD4⁺ Th2 cells generated ex vivo in high-dose rapamycin (10 μM; administered on day 14 post-BMT); Th2 cells were generated from either wild-type donors (“Th2”) or IL-4-deficient donors (“Th2 4^−/−”) or IL-10-deficient donors (“Th2 10^−/−”). On day 19 post-BMT, lungs from treatment cohorts were removed to evaluate tumor burden; liver, stomach, small intestine, large intestine, cecum, and skin were harvested to assess GVHD. (A) For GVHD, each organ was scored on a scale of 0 to 4, with cumulative GVHD severity score shown (maximum value = 24). (B) For tumor burden, a semiquantitative determination of tumor cell infiltration was scored on a scale of 0 to 4; n = 7 subjects were evaluated in each cohort.

Donor Th2 cells inhibit donor CD8⁺ T cell expansion after allogeneic BMT. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow alone (“BMT”); marrow and TS/A tumor cells (“TSA”); or marrow, tumor cells, and additional donor T cells (“GVHD”). Other cohorts received marrow, tumor cells, donor T cells, and additional donor CD4⁺ Th2 cells generated from either wild-type donors (“Th2”) or IL-4 deficient donors (“Th2 4^−/−”) or IL-10 deficient donors (“Th2 10^−/−”). On day 19 post-BMT, spleen cells were isolated and the absolute number of donor CD8⁺ T cells (A) and CD4⁺ T cells (B) were calculated from flow cytometry data (cells per spleen, ×10⁶). Data shown are mean ± SEM (n = 7 per cohort).

In vivo IL-2 administration abrogates Th2 cell therapy: histology analysis. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow alone (“BMT”); marrow and TS/A tumor cells (“TSA”); marrow, tumor cells, and additional donor T cells (“GVHD”); or marrow, tumor cells, donor T cells, and IL-2 therapy (“GVHD+IL-2”; 50,000 I.U., twice a day from day 14 to day 18 post-BMT). Other cohorts received marrow, tumor cells, donor T cells, and donor Th2 cells on day 14 post-BMT either alone (“Th2”) or with IL-2 infusion (“Th2+IL-2”). On day 19 post-BMT, lungs from treatment cohorts were removed to evaluate tumor burden; liver, stomach, small intestine, large intestine, cecum, and skin were harvested to assess GVHD. (A) For GVHD, each organ was scored on a scale of 0 to 4, with cumulative GVHD score shown (maximum value = 24). (B) For tumor burden, a semiquantitative determination of tumor cell infiltration was scored on a scale of 0 to 4; n = 5 subjects were evaluated per cohort.

Th2 cell inhibition of CD8⁺ T cell expansion is abrogated by IL-2. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow alone (“BMT”); marrow and TS/A tumor cells (“TSA”); marrow, tumor cells, and additional donor T cells (“GVHD”); or marrow, tumor cells, donor T cells, and IL-2 therapy (“GVHD+IL-2”; 50,000 I.U., twice a day from day 14 to day 18 post-BMT). Other cohorts received marrow, tumor cells, donor T cells, and donor Th2 cells on day 14 post-BMT either alone (“Th2”) or with IL-2 infusion (“Th2+IL-2”). On day 19 post-BMT, spleen cells were isolated and the absolute number of donor CD8⁺ T cells (A) and CD4⁺ T cells (B) emanating from the initial allograft, and the absolute number of CD90.1+Th2 cells [C] were calculated from flow cytometry data (cells per spleen, ×10⁶). Data shown are mean ± SEM (n = 5 per cohort).

Host-type APC infusion abrogates Th2 cell anti-GVHD effect: histology. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow (“BMT”); marrow and TS/A tumor cells (“TSA”); marrow, tumor cells, and donor T cells (“GVHD”); marrow, tumor cells, donor T cells, and donor-type APC (“GVHD + Donor DC”); or marrow, tumor cells, donor T cells and host-type APC (“GVHD + Host DC”). Other cohorts received marrow, tumor cells, donor T cells, and donor Th2 cells either alone (“Th2”; day 14 post-BMT), with donor-type APC (“Th2 + Donor DC”), or with host-type APC (“Th2 + Host DC”). On day 19 post-BMT, lungs from treatment cohorts were removed to evaluate tumor burden; liver, stomach, small intestine, large intestine, cecum, and skin were harvested to assess GVHD. (A) For GVHD, each organ was scored (scale = 0 to 4), with cumulative GVHD score shown (maximum value = 24). (B) For tumor burden, a semiquantitative determination of tumor cell infiltration was scored (scale = 0 to 4); n = 5 subjects were evaluated per cohort.

Host-type APC transfer abrogates Th2 cell therapy: donor T cell tracking. B6-into-BALB/c BMT was performed (850 cGy host irradiation). Cohorts received donor marrow alone (“BMT”); marrow and TS/A tumor cells (“TSA”); marrow, tumor cells, and additional donor T cells (“GVHD”); marrow, tumor cells, donor T cells, and donor-type APC (“GVHD + Donor DC”); or marrow, tumor cells, donor T cells and host-type APC (“GVHD + Host DC”). Other cohorts received marrow, tumor cells, donor T cells, and donor Th2 cells either alone (“Th2”; day 14 post-BMT), with donor-type APC (“Th2 + Donor DC”), or with host-type APC (“Th2 + Host DC”). On day 19 post-BMT, spleen cells were isolated and the absolute number of donor CD8⁺ T cells (A) and CD4⁺ T cells (B) emanating from the initial allograft, and the absolute number of CD90.1+Th2 cells [C] were calculated from flow cytometry data (cells per spleen, ×10⁶). Data shown are mean ± SEM (n = 5 per cohort).