Memory CD4+ T cells do not induce graft-versus-host disease (original) (raw)
Memory CD4 cells do not cause GVHD. To compare the GVHD-inducing potency of naive and memory T cells, we used the B10.D2 (H-2d) → BALB/c (H-2d) model of chronic GVHD. In this model CD4 cells are both required and sufficient for GVHD, whereas, in contrast, CD8 cells alone are incapable of causing GVHD (27, 28). The syndrome induced with purified CD4 cells is indistinguishable from that induced by unfractionated splenocytes. In this model, GVHD is primarily cutaneous, manifested by alopecia, erythema, ulceration, and fibrosis.
Naive and memory phenotype CD4 cells were purified by magnetically depleting B cells and granulocytes from spleens, followed by FACS sorting. Naive CD4 cells were defined as CD62L+CD44–, whereas memory cells were CD62L–CD44+ (Figure 1). BALB/c recipients were irradiated and reconstituted with B10.D2 T cell–depleted BM along with no T cells, 107 unfractionated spleen cells containing 1.2 × 106 CD4 cells, 106 memory CD4 cells, or 106 naive CD4 cells.
Memory CD4+ T cells do not cause GVHD. Naive and memory T cells were purified as described in Methods. After gating on CD4+ T cells (a), cells were sorted into CD62L+CD44– naive and CD62L–CD44+ memory fractions (b). Reanalyses of sorted populations are shown in (c). BALB/c mice were lethally irradiated and reconstituted with 8 × 106 B10.D2 T cell–depleted BM alone (thin dashed line, n = 9) or with 107 B10.D2 total spleen cells (thin solid line, n = 25), 106 naive T cells (thick solid line, n = 20), or 106 memory T cells (thick dashed line, n = 10). Data are combined from two independent experiments. GVHD incidence and mean clinical score are shown in d and e. Statistical comparisons are as follows: (d). P < 0.0001 for GVHD incidence in recipients of memory CD4 versus spleen cells or naive CD4 cells. (e) For clinical score, *P < 0.05 (time points 1–3) and ‡P < 0.01 (time points 4–6) for recipients of naive versus total spleen cells; †P < 0.05. §P < 0.001 (time points 2–10) for recipients of memory versus total spleen cells. P < 0.0001 for recipients of memory versus naive cells at all time points. BM control mice and BM plus memory cell groups did not get GVHD, but the clinical score lines were offset for clarity. Pathology scores from representative mice are shown in (f). Mean scores are indicated by horizontal bars. ††P < 0.005 and P < 0.0004 for recipients of memory versus total (unfractionated) spleen cells and memory versus naive CD4 cells, respectively.
Strikingly, in two independent experiments, memory CD4 cells did not induce clinical or histologic GVHD, while naive CD4 cells caused more severe GVHD than unfractionated splenocytes containing a greater number of CD4 cells (Figure 1, d–f). Between the two experiments, a total of ten mice received memory cells, and none developed GVHD. In contrast, 19 of 20 recipients of naive cells and 23 of 25 spleen cell recipients developed GVHD (P < 0.0001 for both memory versus spleen and memory versus naive). Affected naive CD4 recipients had a mean clinical score of 2.5 compared with 1.5 in spleen cell recipients (P < 0.05 for time points 1–3; P < 0.01 for time points 4–6). Memory cell recipients had a score of 0 (P < 0.05 for time point 1 and P < 0.001 for time points 2–10, memory versus spleen; P < 0.0001 for all time points, memory versus naive). Histologically, memory cell recipients had scores indistinguishable from hosts that received no T cells. In contrast, recipients of spleen cells and naive T cells had mean scores of 5.2 and 5.9, respectively (P < 0.005, memory versus spleen; P < 0.0004, memory versus naive cells). Thus, GVHD in this model is induced only by naive and not memory CD4 cells. The differences in clinical scores between naive and spleen cell recipients were not reflected by histologic analysis of affected skin as the clinical score measures the extent of skin involvement. The nature of the lesions in affected skin was quantitatively and qualitatively similar in the total spleen cell and naive groups.
Memory CD4 cells depleted of regulatory CD4+CD25+ cells do not cause GVHD. Differential GVHD-inducing capacity could be an intrinsic property of naive and memory T cells. Alternatively, the CD44+CD62L– population that contains memory T cells might also contain a regulatory cell that suppresses GVHD that is not present among the naive cell population. Potential candidate regulatory cells include CD4+CD25+ T regulatory cells (Treg), which have been shown to suppress GVHD in MHC-incompatible GVHD models (22–24). We therefore enumerated the percentage of CD4+CD25+ cells present in B10.D2 naive and memory CD4 cells. We found that 5.5% of naive cells and 33% of memory CD4 cells were CD25+ (Figure 2). Thus, memory cells could have been less efficacious at inducing GVHD due to the large number of putative Treg cells present.
CD25 expression on naive and memory CD4 subsets. B10.D2 spleen cells enriched for CD4 cells as described in Methods were stained with mAb’s against CD4, CD25, CD62L, and CD44. We found that 12.4% of CD4 cells were CD25+ (a). We gated on CD62L+CD44– naive and CD62L–CD44+ memory CD4 cells (b) and analyzed their expression of CD25 (c and d). Note that 33.1% of cells with a memory phenotype express CD25 versus 5.1% of cells with a naive phenotype.
To evaluate this possibility, we performed GVHD experiments with naive and memory CD4+ T cells that were depleted of CD4+CD25+ cells (see Methods). In two independent experiments (Figures 3 and 4), 106 memory CD4 cells depleted of CD4+CD25+ cells did not cause GVHD, whereas an equal number (Figure 3) or only 250,000 naive CD4 cells (Figure 4) caused severe GVHD.
FACS-sorted memory CD25– T cells do not cause GVHD. Donor B10.D2 spleen cells were enriched for CD4+ T cells using BioMag separation, then stained with mAb’s for CD4, CD25, CD62L, and CD44. After gating on CD4+CD25– cells (a), T cells were sorted on the basis of CD62L and CD44 expression (b). Reanalyses of sorted populations are shown in (c) (CD44 versus CD62L) and (d) (CD4 versus CD25). BALB/c mice were lethally irradiated and reconstituted with 8 × 106 B10.D2 T cell–depleted BM alone (thin dashed line, n = 1) or with 2 × 106 B10.D2 unfractionated CD4+ T cells (thin solid line, n = 4), 106 purified naive CD4+CD25– T cells (thick solid line, n = 9), or 106 memory CD4+CD25– T cells (thick dashed line, n = 3). Incidence of GVHD is shown in (e). P < 0.0082 and P < 0.0005 comparing GVHD incidence in recipients of CD25– memory CD4 versus unfractionated and CD25– naive CD4 cells, respectively. Average clinical disease score for affected mice (f). *P < 0.05 (time points 1–3) and for recipients of CD25– naive cells versus unfractionated CD4 cells. P < 0.02 for all comparisons between recipients of CD25– memory and naive cells. Pathology scoring from representative mice (g). ††P < 0.0034 and P < 0.017 for recipients of memory versus total and naive CD4+ T cells, respectively. **P < 0.016 for recipients of naive versus total CD4+ T cells.
AutoMACS- and FACS-sorted CD25-depleted memory T cells do not cause GVHD. Donor B10.D2 spleen cells enriched for CD4+ T cells using BioMag beads were stained with biotinylated anti-CD62L and anti-CD25 mAb’s, followed by staining with SA-beads. Cells were separated into CD25–CD62L– (negative [neg] fraction) and CD25+CD62L+ (positive [pos] fraction) cells using an AutoMACS. Phenotype of presort CD4+ T cells is shown in (a). Phenotype of CD25–CD62L– negative fraction (memory cells) is shown in (b). CD25+CD62L+ cells (positive fraction) were sorted on a FACStar cell sorter to purify CD25– (c) and CD62L+CD44– cells (d). Reanalysis of the sorted population is not available. BALB/c mice were lethally irradiated and reconstituted with 8 × 106 B10.D2 T cell–depleted BM alone (thin dashed line, n = 5) or with 1.5 × 106 unfractionated B10.D2 CD4+ T cells (thin solid line, n = 10), 2.5 × 105 CD4+CD25– naive T cells (thick solid line, n = 5), or 106 CD4+CD25– memory T cells (thick dashed line, n = 4). Incidence of GVHD (e). P < 0.0002 and P < 0.003 for difference between recipients of CD25– memory and total CD4 and CD25– naive CD4 cells, respectively. Average clinical disease score for mice affected with GVHD (f). *P < 0.02 (all time points) for CD25– memory versus total CD4. P < 0.01 on days 19–43 after transplant for recipients of CD25– memory versus naive CD4 cells. Pathology scoring from representative mice (g). #P < 0.007 and P < 0.014 for recipients of CD25– memory versus total and CD25– naive CD4 cells, respectively.
In the first experiment, naive and memory CD25– cells were isolated by FACS sorting (Figure 3, a–d). Irradiated BALB/c hosts received 8 × 106 B10.D2 T cell–depleted BM with no T cells, 2 × 106 unfractionated CD4 cells, and 106 naive or 106 memory CD4 cells. As in our experiments with CD25-replete naive and memory cells, CD25– memory CD4 cells did not cause GVHD, whereas naive CD25– cells caused more severe GVHD than did an equal number of unfractionated CD4 cells (Figure 3, e–g). Differences in GVHD incidence between recipients of memory versus naive (P < 0.0005) and memory versus total CD4 cells (P < 0.008) were highly significant.
In the second experiment, naive and memory CD25– cells were isolated by MACS depletion to obtain memory cells, followed by FACS sorting to obtain naive cells (see Methods and Figure 4, a–d). Again, memory CD4 cells caused no clinical GVHD, whereas only 250,000 naive CD25– memory CD4 cells induced GVHD similar to that induced by 1.5 × 106 unfractionated CD4 cells (Figure 4, e–g). As in the first experiment, differences in GVHD incidence between recipients of memory cells versus naive cells (P < 0.0027) and memory versus total CD4 cells (P < 0.0002) were highly significant. Thus, between the two experiments, none of the seven mice that received CD25-depleted memory cells developed GVHD, whereas 14 of 14 recipients of CD25-depleted naive cells got GVHD.
The clinical differences we observed were also borne out histologically. Mice were sacrificed on day 42, and skin histology was scored as described in Methods (Figure 3g and Figure 4g). Memory CD25– T cell recipients had no evidence of GVHD, whereas recipients of total unfractionated CD4 cells (including CD4+CD25+ cells) and naive CD25– CD4 cells developed severe histologic GVHD. Representative histology is shown in Figure 5.
Representative histology. Representative skin histology from BALB/c recipients of B10.D2 T cell–depleted BM alone (a), with memory cells (b), unfractionated CD4 cells (c), or naive CD4 cells (d). Note thickening of keratinocyte layer, interface dermatitis, and ulcerations (c and d) not present in a and b.
Engrafted memory CD4 cells respond to antigen in vivo. For donor memory T cell infusions to be effective in immune reconstituting alloSCT recipients, they must engraft and be able to respond to antigen. We could not ask this in the experiments described above because memory CD4 cell recipients also had donor BM-derived T cells. To evaluate memory donor CD4 cell engraftment and function, we therefore used ATX BALB/c mice as recipients. Because donor BM-derived cells cannot differentiate into T cells due to the absence of a thymus, the only donor-derived T cells in these mice are derived from those infused at the time of transplant. Donor- and host-derived cells can be distinguished by expression of the recipient-specific Ly9.1 isoform.
To mimic the situation of a recipient responding to an antigen against which the donor has already been exposed, we used B10.D2 mice that were immunized 21 days prior to transplant with CGG as CD4+ T cell and BM donors. ATX BALB/c mice were irradiated and reconstituted with T cell–depleted B10.D2 BM, with (a) no CD4 cells, (b) unfractionated CD4 cells, or (c) CD25– memory CD4+ T cells. To test functional in vivo memory of donor T cells, recipients and unmanipulated ATX mice were immunized on day 37 after transplant with CGG or the irrelevant control antigen PCC. Fourteen days later, mice were sacrificed and draining lymph node cells were harvested. Residual recipient host cells were depleted (less than 1% contaminating host cells), and the remaining cells were restimulated in vitro with CGG. Proliferation was measured by [3H]-thymidine incorporation.
As in previous experiments, memory T cell recipients did not develop clinical GVHD (Figure 6a), whereas most recipients of unfractionated CD4 cells did. Donor memory T cells engrafted, with 75% of LN CD4 cells being Ly9.1– and therefore derived from the mature donor T cells given at the time of transplant. These memory cells made strong proliferative recall responses to CGG, even greater than those made by recipients of unfractionated CD4 cells. This is particularly striking because the fraction of CD4+ T cells in the memory group was only one-third of that in the unfractionated CD4 cell group (not shown). Memory recipients immunized with PCC in vivo failed to respond to CGG in vitro, which confirms that proliferation depended on in vivo priming. Importantly, CGG-immunized recipients of T cell-depleted BM had poor responses, demonstrating that residual host CD4 cells could not have made a significant contribution. Thus, donor memory CD4+ T cells both engrafted and responded vigorously to antigen.
Donor memory cells engraft and respond to antigenic challenge. B10.D2 mice were immunized intraperitoneally with CGG in CFA and used 3 weeks later as CD4+ cell and BM donors. ATX BALB/c mice were irradiated and reconstituted with 8 × 106 T cell–depleted BM cells with no CD4 cells (thin dashed line, n = 9), 1.5 × 106 unfractionated CD4 cells (thin solid line, n = 17), or 106 CD4+CD25– memory cells (thick line, n = 14). Incidence of GVHD (a). P < 0.001 for GVHD incidence in recipients of CD25– memory cells versus total CD4 cells. Transplanted memory cells respond to CGG (b). Thirty-seven days after the transplant, recipients and unmanipulated ATX BALB/c mice were immunized with CGG or PCC in CFA. Two weeks later, draining LN cells were collected, depleted of residual recipient cells, and rechallenged with 50 μg CGG in vitro in a standard proliferation assay. Cells were pooled from all animals (n = 3–7) of an experimental group: untransplanted ATX control, BM alone, BM plus unfractionated CD4 cells, BM plus CD25–CD4+ memory cells. Background counts (no antigen) were subtracted from plotted data. P = 0.0002 for proliferation to CGG for BM plus memory cells versus BM plus unfractionated CD4 cells. P < 0.0001 for BM plus memory cells versus BM alone. Error bars indicate standard deviation of samples run in triplicate.