Figure 2.
HLA-DM–mediated modulation of alloreactive T-cell responses to HLA-DP in healthy individuals and transplanted patients. (A-B) Activation of CD4+ T cells from healthy HLA-DP401+ individuals in response to permissive HLA-DP402 (blue dots) or nonpermissive HLA-DP10 (red dots). Shown is the percentage of activated (CD137+) CD4+ T cells after restimulation with HeLa-DP as indicated (see also supplemental Figure 1). Values are reported after subtraction of background activation against HeLa-DP expressing the autologous allotype in the presence of HLA-DM. Statistical comparisons by 1-way ANOVA. ***P < .001, ****P < .0001. (A) CD4+ T cells from healthy individuals (n = 18) were cocultured and restimulated with the same allogeneic HLA-DP/HLA-DM combination as indicated. Mean ± SD percentage autologous background was 7.63 ± 4.65. (B) CD4+ T cells from healthy individuals (n = 6) were cocultured with HLA-DP402 without HLA-DM and restimulated with allogeneic HLA-DP/HLA-DM/HLA-DO combinations as indicated. Mean ± SD percentage autologous background was 16.0 ± 9.6. (C-D) The alloreactive TCRβ repertoire of activated CD4+CD137+ T cells in representative healthy individuals from panel A, characterized by next-generation sequencing. (C) Relative frequency of top-10 or background clonotypes in 3 responders (R1-R3), indicated as colored or gray bars, respectively. (D) Rarefaction curves of TCRβ clonotypes in R1 (R2 and R3 in supplemental Figure 7). Curves are interpolated from 0 to the size of each sample (solid lines) and extrapolated to the size of the largest sample (dashed lines). Numbers in the legend indicate unique clonotypes in each repertoire at the maximum sample size. (E-G) CD4+ T-cell activation and alloreactive TCRβ repertoire in transplanted patients after HCT from UDs mismatched for permissive HLA-DP402 (IZTF-3) or nonpermissive HLA-DP10 (IZTF-28). Full donor CD4+ T cells were isolated from the patients’ peripheral blood at days 363 and 346, respectively (supplemental Table 1), and used for coculture and restimulation with HeLa-DP expressing the mismatched allotype in the presence or absence of HLA-DM. (E) Fluorescence activated cell sorting plots of CD137 activation assays. Specific percentages of alloresponses with or without HLA-DM was 3.87 and 41.91 (IZTF-3) or 41.82 and 47.84 (IZTF-28). (F) Heatmap of cytokine production. Background response to autologous HLA-DP was <100 pg/mL. (G) Alloreactive CD4+ TCRβ clonotypes after in vitro culture (2 rightmost bars) were traced ex vivo in peripheral blood or bone marrow from the patients or their respective UD at the indicated time points. Shown is the cumulative frequency of TCRβ clonotypes found only in the presence (blue) or absence (purple) of HLA-DM, or in both (green), with an expanded clonotype in IZTF-3 in lighter green. Both patients experienced early CMV reactivation as indicated. BM, bone marrow; IL, interleukin; PB, peripheral blood.

HLA-DM–mediated modulation of alloreactive T-cell responses to HLA-DP in healthy individuals and transplanted patients. (A-B) Activation of CD4+ T cells from healthy HLA-DP401+ individuals in response to permissive HLA-DP402 (blue dots) or nonpermissive HLA-DP10 (red dots). Shown is the percentage of activated (CD137+) CD4+ T cells after restimulation with HeLa-DP as indicated (see also supplemental Figure 1). Values are reported after subtraction of background activation against HeLa-DP expressing the autologous allotype in the presence of HLA-DM. Statistical comparisons by 1-way ANOVA. ***P < .001, ****P < .0001. (A) CD4+ T cells from healthy individuals (n = 18) were cocultured and restimulated with the same allogeneic HLA-DP/HLA-DM combination as indicated. Mean ± SD percentage autologous background was 7.63 ± 4.65. (B) CD4+ T cells from healthy individuals (n = 6) were cocultured with HLA-DP402 without HLA-DM and restimulated with allogeneic HLA-DP/HLA-DM/HLA-DO combinations as indicated. Mean ± SD percentage autologous background was 16.0 ± 9.6. (C-D) The alloreactive TCRβ repertoire of activated CD4+CD137+ T cells in representative healthy individuals from panel A, characterized by next-generation sequencing. (C) Relative frequency of top-10 or background clonotypes in 3 responders (R1-R3), indicated as colored or gray bars, respectively. (D) Rarefaction curves of TCRβ clonotypes in R1 (R2 and R3 in supplemental Figure 7). Curves are interpolated from 0 to the size of each sample (solid lines) and extrapolated to the size of the largest sample (dashed lines). Numbers in the legend indicate unique clonotypes in each repertoire at the maximum sample size. (E-G) CD4+ T-cell activation and alloreactive TCRβ repertoire in transplanted patients after HCT from UDs mismatched for permissive HLA-DP402 (IZTF-3) or nonpermissive HLA-DP10 (IZTF-28). Full donor CD4+ T cells were isolated from the patients’ peripheral blood at days 363 and 346, respectively (supplemental Table 1), and used for coculture and restimulation with HeLa-DP expressing the mismatched allotype in the presence or absence of HLA-DM. (E) Fluorescence activated cell sorting plots of CD137 activation assays. Specific percentages of alloresponses with or without HLA-DM was 3.87 and 41.91 (IZTF-3) or 41.82 and 47.84 (IZTF-28). (F) Heatmap of cytokine production. Background response to autologous HLA-DP was <100 pg/mL. (G) Alloreactive CD4+ TCRβ clonotypes after in vitro culture (2 rightmost bars) were traced ex vivo in peripheral blood or bone marrow from the patients or their respective UD at the indicated time points. Shown is the cumulative frequency of TCRβ clonotypes found only in the presence (blue) or absence (purple) of HLA-DM, or in both (green), with an expanded clonotype in IZTF-3 in lighter green. Both patients experienced early CMV reactivation as indicated. BM, bone marrow; IL, interleukin; PB, peripheral blood.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal