Unraveling graft-versus-host disease and graft-versus-leukemia responses using TCR Vβ spectratype analysis in a murine bone marrow transplantation model

Abstract

The optimum use of allogeneic blood and marrow transplantation (BMT) as a curative therapy for hematological malignancies lies in the successful separation of mature donor T cells that are host reactive and induce graft-versus-host disease (GVHD) from those that are tumor reactive and mediate graft-versus-leukemia (GVL) effects. To study whether this separation was possible in an MHC-matched murine BMT model (B10.BR→CBA) with a CBA-derived myeloid leukemia line, MMC6, we used TCR Vβ CDR3-size spectratype analysis to first show that the Vβ13 family was highly skewed in the B10.BR anti-MMC6 CD8(+) T cell response but not in the alloresponse against recipient cells alone. Transplantation of CD8(+)Vβ13(+) T cells at the dose equivalent of their constituency in 1 × 10(7) CD8(+) T cells, a dose that had been shown to mediate lethal GVHD in recipient mice, induced a slight GVL response with no concomitant GVHD. Increasing doses of CD8(+)Vβ13(+) T cells led to more significant GVL responses but also increased GVHD symptoms and associated mortality. Subsequent spectratype analysis of GVHD target tissues revealed involvement of gut-infiltrating CD8(+)Vβ13(+) T cells accounting for the observed in vivo effects. When BMT recipients were given MMC6-presensitized CD8(+)Vβ13(+) T cells, they displayed a significant GVL response with minimal GVHD. Spectratype analysis of tumor-presensitized, gut-infiltrating CD8(+)Vβ13(+) T cells showed preferential usage of tumor-reactive CDR3-size lengths, and these cells expressed increased effector memory phenotype (CD44(+)CD62L(-/lo)). Thus, Vβ spectratyping can identify T cells involved in antihost and antitumor reactivity and tumor presensitization can aid in the separation of GVHD and GVL responses.

Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1. B10.BR CD8+ T cells induce both lethal GVHD and GVL effects in MMC6-challenged CBA recipients

CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 2×106 B10.BR ATBM cells with or without 1×107 CD8+ T cells. The next day, mice were challenged i.p. with 5×103 MMC6 tumor cells. Results were combined from 4 separate experiments, each with 3-5 mice per group. Statistical significance between survival curves was determined using the non-parametric Wilcoxon test. ATBM + MMC6 versus ATBM + CD8+ T cells + MMC6, p < 0.01; ATBM + CD8+ T cells versus ATBM + CD8+ T cells + MMC6, p > 0.2.

FIGURE 2. Representative spectratype histograms in the B10.BR anti-CBA and anti-MMC6 CD8+Vβ13+ T cell responses in lymphoid tissue

Spectratype analyses were performed on CD8+ T cell populations as described in Materials and Methods. (A) CD8+ splenic T cells harvested from naïve B10.BR mice acted as the control group. (B) For alloreactive responses (anti-CBA), CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 1-2×107 B10.BR CD8+ T cells. Splenocytes were harvested at 10-13 d post-BMT. (C) For the anti-tumor response (anti-MMC6), B10.BR mice were presensitized (i.p.) with 1×107 MMC6 tumor cells (40 Gy), followed by a second injection of 1×106 MMC6 cells (40 Gy) in the footpad 17-21 d later. After 1 wk, draining lymph nodes were harvested and CD8+ T cell isolated. Skewing was defined as a peak area greater than the mean of the control plus 5×SD. CDR3 lengths are shown under each peak. Skewed peaks are marked with an arrow.

FIGURE 3. GVHD and GVL potential of B10.BR CD8+Vβ13+ donor T cells in MMC6-challenged CBA recipients

A and B, CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 2×106 ATBM cells alone or along with either unseparated CD8+ T cells (1×107) or CD8+Vβ13+ T cells (1x, 8.34×105; 2x, 1.67×106; or 3x, 2.5×106) from B10.BR donor mice. A, Statistical significance between survival curves was determined using the non-parametric Wilcoxon test. ATBM + CD8+ T cells versus ATBM + CD8+Vβ13+ T cells (1x), p < 0.02; ATBM + CD8+ T cells versus ATBM + CD8+Vβ13+ T cells (3x), p = 0.9. B, All groups except ATBM alone were challenged with MMC6 tumor cells (5×103) on d 1 post-BMT. Experiments were repeated twice with 3-5 mice per group. ATBM + MMC6 versus ATBM + MMC6 + CD8+Vβ13+ T cells (1x), p < 0.01; ATBM + MMC6 versus ATBM + MMC6 + CD8+Vβ13+ T cells (2x), p < 0.01; ATBM + MMC6 versus ATBM + MMC6 + CD8+Vβ13+ T cells (3x), p < 0.01; ATBM + MMC6 + CD8+Vβ13+ T cells (1x) versus ATBM + MMC6 + CD8+Vβ13+ T cells (2x), p = 0.59; ATBM + MMC6 + CD8+Vβ13+ T cells (1x) versus ATBM + MMC6 + CD8+Vβ13+ T cells (3x), p = 0.05; ATBM + MMC6 + CD8+Vβ13+ T cells (2x) versus ATBM + MMC6 + CD8+Vβ13+ T cells (3x), p < 0.02.

FIGURE 4. Representative spectratype histograms in the B10.BR anti-CBA and anti-MMC6 CD8+Vβ13+ T cell responses in intestinal tissue

(A) For alloreactive responses (anti-CBA), CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 1-2×107 B10.BR CD8+ T cells. Intestinal tissue was harvested at 10-13 d post-BMT. (B) For the anti-tumor response in the intestine, CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 2×107 MMC6 presensitized CD8+ T cells and tissue was harvested at 10-13 d post-BMT. Tissues were processed and spectratype analyses were performed. CD8+ splenic T cells harvested from naïve B10.BR mice acted as the control group (shown in Fig. 2A). Skewing was defined as a peak area greater than the mean of the control plus 5×SD. CDR3 lengths are shown under each peak. Skewed peaks are marked with an arrow.

FIGURE 5. Tumor-presensitized donor B10.BR CD8+Vβ13+ T cells elicit a significant GVL effect with minimal GVHD

CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with ATBM cells (2×106) alone or along with either CD8+ T cells (1×107) or CD8+Vβ13+ T cells (1x, 8.34×105 or 3x, 2.5×106) from MMC6 presensitized or naïve donor B10.BR mice. Transplanted mice were challenged with MMC6 tumor cells (5×103) on d 1 post-BMT. Experiments were repeated twice, each with 4-5 mice per group. Statistical significance between survival curves was determined using the non-parametric Wilcoxon test. ATBM + MMC6 versus ATBM + MMC6 + CD8+Vβ13+ T cells (1x presensitized), p <0.01; ATBM + CD8+ T cells + MMC6 versus ATBM + MMC6 + CD8+Vβ13+ T cells (1x presensitized), p < 0.05; ATBM + MMC6 + CD8+Vβ13+ T cells (1x), versus ATBM + MMC6 + CD8+Vβ13+ T cells (1x presensitized), p = 0.03.

FIGURE 6. CBA recipients of tumor-presensitized B10.BR CD8+Vβ13+ T cells exhibit minimal histological evidence of GVHD

CBA mice were exposed to lethal irradiation (11 Gy, split dose) and transplanted with 2×106 ATBM cells alone or in combination with either CD8+ T cells (1×107) or CD8+Vβ13+ T cells (1x, 8.34×105 or 3x, 2.5×106) from MMC6 presensitized or naïve B10.BR donor mice, as indicated. A, The mean ± SE % initial body weight of surviving mice in each group was derived relative to the mean weight of the group on d 0. n = # mice surviving at termination of experiment/ # mice at initiation of experiment. B, Comparison of the dermis and epidermis of the ear between mice transplanted with ATBM cells alone, CD8+ T cells, or CD8+Vβ13+ T cells (1x presensitized). H&E reveals normal and regular thickness of the epithelium (double arrows) of the ATBM [a] and CD8+Vβ13+ T cells (1x presensitized) specimens [c], while there is severe irregularity of the epidermal-dermal border (single arrows) in the CD8+ T cell sample [b] with evident hyperplasia of the epidermis. Likewise, the collagen layer in the dermis (double arrow) is regular and well arranged in the ATBM and CD8+Vβ13+ T cell sample [a&c] and has low cellularity [d&f]. In contrasts, the dermis in the CD8+ T cell sample exhibits increased thickness and vast cellularity (double arrow) [e]. C, comparison of the epithelium of the small intestine between mice transplanted with ATBM cells alone, CD8+ T cells, or CD8+Vβ13+ T cells (1x presensitized). H&E reveals the integrity of the epithelium in the ATBM specimen [a] while there is loss of the architecture of the villi and crypts in the CD8+ T cell sample (arrows) [b] compared to the more defined epithelial structure observed in the CD8+Vβ13+ T cell (1x presensitized) sample (arrows) [c].

FIGURE 7. Memory (CD44+) CD8+Vβ13+ T cells from the spleens and lymph nodes of tumor pre-sensitized B10.BR mice exhibit decreased CD62L expression as compared to naïve mice

B10.BR mice were injected i.p. with 1×107 MMC6 cells (40Gy). Three weeks later, cell suspensions from spleens and lymph nodes were stained for flow cytometric analysis. Cells were first gated on CD8+ Vβ13+ cells (left panel) followed by CD44 expression (middle panel). The percent expression of CD62L on naïve cells is shown in the top right panel compared to the MMC6 presensitized cells in the bottom right panel. One representative experiment is shown.

FIGURE 8. Tumor-presensitized memory (CD44+) CD8+Vβ13+ T cells from the mesenteric lymph nodes of B10.BR mice upregulate the expression of α4β7 integrin

B10.BR mice were injected i.p. with 1×107 MMC6 cells (40 Gy). Mesenteric lymph nodes were harvested 17-21 d later for flow cytometric analysis. Cells were first gated on CD8+Vβ13+ cells (left panel) followed by expression of CD44 (middle panels). The percent expression of α4β7 on naïve cells is shown in the top right panel compared to the MMC6-presensitized cells in the bottom right panel. One representative experiment is shown.