Beta2 integrins separate graft-versus-host disease and graft-versus-leukemia effects

Abstract

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.

Figure 1

CD18 affects GVHD development. Lethally irradiated BALB/c mice were given intravenous injections of 5 × 10⁶ TCD BM cells from B6 donors alone or with T cells from WT or CD18^−/− B6 donors. (A) Survival of recipients given transplants of TCD BM cells alone (n = 4) or with 1 × 10⁶ WT (n = 8) or CD18^−/− (n = 8) total T cells. (B) Mean changes in percentage of body weights for the recipients as in panel A. (C) Survival of recipients given transplants of TCD BM cells alone (n = 4) or 2 × 10⁶ WT (n = 7) or CD18^−/− (n = 7) T cells after removal of CD25⁺ cells. (D) Mean changes in percentage of body weights for the recipients as in panel B. P values indicate the differences between recipients of WT versus CD18^−/− cells.

Figure 2

Naive T cells from CD18^−/− donors have diminished ability to induce GVHD. Spleen and lymph nodes were harvested from WT or CD18^−/− donors, and CD25⁻CD62L⁺ naive T cells were purified with magnetic beads as described in “Methods.” CD25 and CD62L expression were shown on WT or CD18^−/− CD4⁺ and CD8⁺ T cells before (A) and after (B) purification. Numbers in the panels indicate the percentages of cells in the gate as shown among total splenocytes (left panels), CD4⁺ cells (middle panels), or CD8⁺ cells (right panels). Lethally irradiated BALB/c mice were given intravenous injections of 5 × 10⁶ TCD-BM cells alone or with naive T cells from WT or CD18^−/− B6 donors. (C) Survival of recipients given TCD BM cells alone (n = 12) or with WT or CD18^−/− naive T cells at 0.5 × 10⁶ (0.5 M) or 2 × 10⁶ (2 M) per mouse. The recipients with donor T cells at 2 M were 12 mice per group, and there were 6 mice per group at 0.5 M. (D) Mean changes in percentage of body weights for the recipients as in panel C.

Figure 3

CD18^−/− T cells have intact activation and expansion in response to alloantigen in vivo. WT or CD18^−/− T cells (CD25⁻CD62L⁺) were labeled with CFSE and then injected intravenously into lethally irradiated BALB/c mice at 5 × 10⁶ cells/mouse. Cell division and expansion were determined 4 days after cell transfer. (A) Mean of percentage donor (H2^b+) CD4 (top panels) and CD8 T cells (bottom panels) are shown in spleens of recipients given WT (left panels) or CD18^−/− (middle panels) T cells. CFSE profiles are shown on gated CD4 and CD8 cells (right panels) from WT (black lines) or CD18^−/− (gray lines) donors. (B) Mean of absolute numbers of CD4 or CD8 cells per spleen in recipients that were given WT (■) or CD18^−/− (▩) donor T cells as shown in panel A. Brackets show SEM from 3 mice each group, and data represent 1 of 2 replicate experiments. (C) WT or CD18^−/− T cells (CD25⁻CD62L⁺) were injected intravenously into lethally irradiated BALB/c mice at 2 × 10⁶ cells/mouse, and cell expansion was determined 19 days after cell transfer. Mean of absolute numbers of CD4 or CD8 cells per spleen was shown in recipients given WT (■) or CD18^−/− (▩) donor T cells. Brackets indicate standard errors of the mean from 3 to 4 mice each group, and data represent 1 of 4 replicate experiments.

Figure 4

CD18^−/− T cells produce comparable levels of cytokines as WT cells in allogeneic recipients. Indicated cytokines were measured in recipient serum on day 4 (A) or day 19 (B) after BMT as described in “Methods.” The data are presented as means ± SD of 5 to 6 mice per group, and represent 1 of 3 replicate experiments.

Figure 5

Recipients of CD18^−/− donor T cells have significant less pathologic damage in intestine and liver. Lethally irradiated BALB/c mice were given intravenous injections of 5 × 10⁶ TCD BM cells alone or with naive T cells from WT or CD18^−/− B6 donors. On day 19, GVHD target organs, including liver, small intestine, colon, skin, and lung, were harvested for evaluation of histopathology as described in “Methods.” (A) Combined results of pathology scores for 5 to 6 mice in each group. Error bars indicate the standard errors of the mean in the group. (B) Representative micrographs from recipient livers and intestines of BM alone, WT, and CD18^−/− groups with original magnification ×600. Images were captured with an Olympus BX 40 microscope (Olympus, Melville, NY) equipped with a 10×/0.40 numerical aperture objective lens. Image acquisition was performed with a JVC GC-Qx 5HDU digital camera (JVC, Wayne, NJ). (C) Donor T-cell infiltration was determined by flow cytometry in liver and intestine. Left panels show the percentage of H2^b+ in total mononuclear cells isolated, and right panels show the percentage of CD4⁺ and CD8⁺ cells in gated H2^b+ cells. The data show average percentage of each cell population among 3 to 4 mice per group, and represent 1 of 3 replicate experiments.

Figure 6

GVL activity is partially preserved in CD18^−/− T cells. Lethally irradiated BALB/c mice underwent BMT with 5 × 10⁶ TCD BM cells alone or plus 2 × 10⁶ naive T cells from WT or CD18^−/− B6 donors. Recipients were given 2 × 10³ A20 tumor cells with luciferase transgene as a separate intravenous injection at the same time of transplantation. Recipient survival (A) and body weight changes (B) are shown. Numbers of recipients given transplants of BM alone or plus WT or CD18^−/− T cells were 7, 11, and 12, respectively, and data are pooled from 2 replicate experiments. (C) Recipients were tracked for in vivo luminescence 10 minutes after intraperitoneal injection of firefly luciferin; data represent 1 of 2 replicate experiments. (D) BMT was set as in panels A and B, and BALB/c recipients of WT or CD18^−/− T cells were killed 2 weeks after transplantation. Splenocytes of each recipient were assayed directly for cytotoxicity without in vitro restimulation. The activity of cytolytic effectors was measured in a 4- to 5-hours cytotoxic assay against A20 or Yac-1 at an E/T ratio of 100:1. The data represent the means (± 1 SD) of percentage of specific killing from 3 to 4 replicate mice each group, and the percentage of killing is normalized based on the number of total T cells in the spleen. The assay was run in triplicate with less than 5% SE, and data represent 1 of 2 replicate experiments. (E) Splenocytes from WT or CD18^−/− B6 donors were stimulated with IL-2 for 3 days and used as effector cells to kill Yac-1 (NK-sensitive targets) or A20 cells an E/T ratio of 100:1. The data represent the means (± 1 SD) of percentage of specific killing from 2 replicate mice each group, and triplicate wells were set in vitro with less than 5% SE.