Central memory CD8+ T cells induce graft-versus-host disease and mediate graft-versus-leukemia

Abstract

In allogeneic hemopoietic stem cell transplantation, mature donor alphabeta T cells in the allograft promote T cell reconstitution in the recipient and mediate the graft-vs-leukemia (GVL) effect. Unfortunately, donor T cells can attack nonmalignant host tissues and cause graft-vs-host disease (GVHD). It has previously been shown that effector memory T cells not primed to alloantigen do not cause GVHD yet transfer functional T cell memory and mediate GVL. Recently, central memory T cells (T(CM)) have also been reported to not cause GVHD. In contrast, in this study, we demonstrate that purified CD8(+) T(CM) not specifically primed to alloantigens mediate GVHD in the MHC-mismatched C57BL/6 (B6)-->BALB/c and the MHC-matched, multiple minor histocompatibility Ag-mismatched C3H.SW-->B6 strain pairings. CD8(+) T(CM) and naive T cells (T(N)) caused similar histological disease in liver, skin, and bowel. B6 CD8(+) T(CM) and T(N) similarly expanded in BALB/c recipients, and the majority of their progeny produced IFN-gamma upon restimulation. However, in both models, CD8(+) T(CM) induced milder clinical GVHD than did CD8(+) T(N). Nonetheless, CD8(+) T(CM) and T(N) were similarly potent mediators of GVL against a mouse model of chronic-phase chronic myelogenous leukemia. Thus, in contrast to what was previously thought, CD8(+) T(CM) are capable of inducing GVHD and are substantially different from T(EM) but only subtly so from T(N).

Figure 1.. CD8⁺ T_CM induce milder but definite GVHD in the B6→BALB/c strain pairing.

A. Cell sorting. T_N and T_CM CD8 cells were purified and sorted from splenocytes as described in Methods. We gated on CD8⁺ cells (first panel), which were sorted into T_N and T_CM based on the expression of CD62L and CD44. B and C. GVHD in B6→BALB/c model (data combined from 2 experiments). BALB/c mice were lethally irradiated and reconstituted with TCD B6 BM, with no T cells or 1×10⁶ CD8⁺ T_N or T_CM. B. Weight change. P<0.005 at days 5, 8, 12 and 14 comparing recipients of BM versus CD8⁺ T_N or T_CM; P=0.013 comparing BM alone versus T_N on day 20. P<0.01 at day 20, 23, 27, 30, 34, 37 and 40 comparing recipients of CD8⁺ T_N versus CD8⁺ T_CM. C. Liver pathology. Each symbol represents a score from an individual mouse; horizontal lines are mean scores. P=0.0018 comparing BM and CD8⁺ T_N recipients; P=0.0085 comparing BM and CD8⁺ T_CM recipients. P=0.086 comparing CD8⁺ T_N versus CD8⁺ T_CM. D and E. BALB/c mice were irradiated and reconstituted with TCD B6 BM, with no T cells, 1×10⁶ T_N, 1×10⁶ T_CM or 2×10⁴ T_N (T_N control) B6 CD8 cells. D. Weight change. E. Liver pathology scores. P<0.005 for recipients of T_N or T_CM versus T_N control. P value is not significant between BM and T_N control recipients.

Figure 2.. CD8⁺ T_CM induce milder but definite GVHD in C3H.SW→B6 strain pairing.

Data combined from 3 experiments. B6 mice were lethally irradiated and reconstituted with TCD C3H.SW BM, with no T cells, 1.5×10⁶ CD8⁺ T_N or T_CM. A. Survival. P=0.0029 comparing BM and T_N; P=0.2138 comparing BM and T_CM; P=0.054 comparing T_N and T_CM. B. Weight change. P<0.001 at days 26, 30, 34, 37 and 43 comparing recipients of BM only versus CD8⁺ T_N; P<0.05 at days 26 and 43 comparing recipients of T_N versus T_CM. P values not significant at any time-point comparing BM alone versus T_CM. C. Pathology scores. Each symbol represents a score from an individual mouse; horizontal lines are mean scores. P values are noted below each panel.

Figure 3.. CD8⁺ T_N and T_CM infiltrate small bowel in the C3H.SW→B6 strain pairing.

Frozen sections of small bowel from recipients of only TCD BM and T_N or T_CM recipients with confirmed histologic GVHD (by blinded scoring) were stained with anti-CD8 (Alexa647; rendered in green). DAPI-stained nuclei are rendered in blue. Shown in A are representative sections from two recipients of T_N, T_CM or only TCD BM. In T_N and T_CM recipients, CD8 cells percolate through the bowel and invade crypts, characteristic of GVHD. B, quantitation of the number of CD8 cells. CD8 cells were counted in 3–4 40× fields from two mice from each group. Each circle represents the number of CD8 cells in an individual field; horizontal lines are means. P=0.003 comparing BM alone versus T_N or T_CM; P=0.8 comparing T_N and T_CM.

Figure 4.. CD8⁺ T_CM mediate GVL against mCP-CML.

B6 mice were irradiated and reconstituted with TCD C3H.SW BM, B6 mCP-CML with no T cells, 3×10⁵ CD8⁺ T_N, 3×10⁵ CD8⁺ T_CM, or 7.5×10³ CD8⁺ T_N (T_N control) C3H.SW CD8 cells. A. Survival data. P<0.001 for recipients of CD8⁺ T_N or CD8⁺ T_CM versus only BM. B. Representative serial flow cytometry of peripheral blood. Each column is from an individual mouse; two representative mice are shown for T_N and T_CM recipients. C. Numbers of NGFR⁺ cells in the peripheral blood of transplanted mice at different time points. Each symbol represents an individual animal; solid lines are mean values.

Figure 5.. Both T_N and T_CM expand in syngeneic and allogeneic recipients and produce IFN-γ.

BALB/c (CD45.2) and B6 (CD45.2) mice were irradiated and reconstituted with TCD B6 CD45.2 BM and 10⁶ CD8⁺CD45.1+ T_N or T_CM. Seven days post BMT, spleen and LN cells were stimulated with PMA and ionomycin and then stained for CD45.1, CD8, IFN-γ and TNF-α. Positive gates for IFN-γ were based on isotype staining (not shown). A and B, representative flow cytometry of CD8⁺CD45.1⁺ splenocytes and LN cells, respectively. The percentage of IFN-γ+ and IFN-γ very bright cells are shown in the upper left and upper right corners, respectively. Total numbers of CD45.1⁺CD8⁺, CD45.1⁺CD8⁺IFN-γ⁺ or IFN-γ-bright and the percent of CD8⁺CD45.1⁺ cells that are IFN-γ⁺ is shown for spleen (C) and LN (D). Each circle is the value from an individual mouse; horizontal lines are mean values. E, IFN-γ expression after PMA/ionomycin treatment of freshly isolated T_CM (right panel) and T_N (left panel).

Figure 6.. Allogeneic T_CM and T_N induce serum IFN-γ.

Serum was harvested from B6→B6 and B6→BALB/c recipients of B6 T_N or T_CM on day +7. Cytokine concentrations were determined by Bio Plex beads. IFN-γ but not IL-2 or TNF-α were elevated in allogeneic T_CM or T_N recipients. Each symbol is a measurement from an individual mouse; horizontal lines are means.

Figure 7.. Allogeneic BALB/c B cells are preferentially killed in vivo in recipients of B6 T_CM or T_N 7 days post transplant.

Representative flow cytometry of splenocytes (SPL) and lymph node cells (LN). Plots are gated on B220⁺ cells. Note that greater than 94% of CFSE⁺B220⁺ cells are H-2K^b+ and therefore B6 in recipients of T_N or T_CM.