Rapid reconstitution of antibody responses following transplantation of purified allogeneic hematopoietic stem cells

Abstract

Allogeneic hematopoietic cell transplantation has broad clinical applications extending from the treatment of malignancies to induction of immunologic tolerance. However, adaptive cellular and humoral immunity frequently remain impaired posttransplantation. Here, recovery of T-dependent and T-independent Ab responses was evaluated in mice transplanted with purified hematopoietic stem cells (HSCs) devoid of the mature immune cells believed to hasten immune recovery. Mixed and full donor chimeras were created by conditioning recipients with sublethal or lethal irradiation, respectively, across different donor/host genetic disparities. By 6 wk posttransplantation, all animals demonstrated robust T-independent Ab responses, and all mixed chimeras and recipients of MHC-matched or haploidentical HSCs with a shared MHC haplotype had T-dependent Ab responses equivalent to those of untransplanted controls. Full chimeras that received fully MHC-disparate HSCs showed delayed T-dependent Ab responses that recovered by 12 wk. This delay occurred despite early reconstitution and proper migration to germinal centers of donor-derived T(follicular helper) (T(FH)) cells. Congenic transplants into T(FH)-deficient CD4(-/-) mice revealed restoration of T-dependent Ab responses by 6 wk, leading us to conclude that MHC disparity caused delay in humoral recovery. These findings, together with our previous studies, show that, contrary to the view that depletion of graft lymphocytes results in poor posttransplant immunity, elimination of immune-suppressing graft-versus-host reactions permits superior immune reconstitution. This study also provides insight into the regeneration of T(FH) cells and humoral immunity after allogeneic HSC transplantation.

FIGURE 1

Ab responses to novel T-dependent Ags 6 wk post-HSCT. Fully MHC-disparate (A, D), haploidentical (B, E), and MHC-matched (C) full and mixed chimeras were challenged with the novel T-dependent Ag TNP-OVA (A–C) or TNP-KLH (D, E) 6 wk after transplant. Serum levels of Ag-specific Ig 9 d after immunization were measured using ELISAs. Results are shown as OD readings with untransplanted controls of donor and host strains. Ab levels were statistically lower than those of untransplanted controls in fully MHC-disparate full chimeras challenged with TNP-OVA (p < 0.0001) and in fully MHC-disparate full and mixed chimeras challenged with TNP-KLH (p < 0.006). For all other conditions, Ab levels were not statistically different from those of untransplanted controls.

FIGURE 2

Donor- and host-derived TNP-OVA specific Abs 6 wk post-HSCT. Allotype-specific Abs were used to detect the origin, donor or host, of IgG_2a/c. Donor-derived IgG_2a/c were completely absent from fully MHC-disparate full and mixed chimeras (A). All IgG_2a/c seen in fully MHC-disparate mixed chimeras were of host origin (B). Haploidentical (C) and MHC-matched (E) full and mixed chimeras showed significant donor-derived responses. Host-derived IgG2_a/c were observed in haploidentical (D) and MHC-matched (F) mixed but not full (D) chimeras.

FIGURE 3

T-independent Ab responses 6 wk post-HSCT. Fully MHC-disparate full and mixed chimeras were challenged with TNP-Ficoll 6 wk after transplantation, and serum levels of TNP-Ficoll specific Ab were measured. Transplanted animals show total TNP-specific Ig levels equivalent to those of untransplanted controls of donor and host strains (A). Allotype analysis showed high levels of donor-derived IgG2_a/c in both full and mixed chimeras (B) and host-derived Ab responses in mixed chimeras (C).

FIGURE 4

Donor- and host-derived T_FH cells. Lymph nodes harvested 9 d after T-dependent Ag challenge with TNP-OVA from fully MHC-disparate and haploidentical chimeras as well as untransplanted controls were analyzed for the percentage of T_FH (CD4⁺CD44⁺CXCR5⁺ICOS⁺) cells using the gating strategy shown (A). The percentage of T_FH cells was then calculated for each group and showed no significant differences between chimeric animals and untransplanted controls (B). The numbers in parentheses show the number of animals used for each condition. At least half of the T_FH cells in fully MHC-disparate animals were of donor origin in full chimeras, and the donor-derived percentage of T_FH cells in mixed chimeras reflected the overall chimerism of the CD4⁺ T cell lineage (C). The numbers in parentheses show the number of animals used for each condition.

FIGURE 5

Donor-derived T_FH localization to germinal centers. Lymph nodes were harvested from fully MHC-disparate full (A) and mixed (B) chimeras, haploidentical full (C) and mixed (D) chimeras, and an untransplanted donor control (E). Sections were stained with peanut agglutinin (in red) to mark germinal centers and Thy1.1 (in green) to mark donor-derived T cells. Small germinal centers formed in all transplant recipients, and donor-derived T cells were present in those germinal centers. Mixed chimeras showed more robust germinal center formation, although haploidentical full chimeras had larger germinal centers than did fully MHC-disparate full chimeras. Original magnification ×20.

FIGURE 6

Recovery of T-dependent Ab responses in fully MHC-disparate full chimeras. Fully MHC-disparate full chimeras were challenged with TNP-OVA (A) or TNP-KLH (B) at 6, 9, 12, and 15 wk after transplant. Nine days after immunization, serum levels of Ag-specific Ig were measured, and each mark represents an individual animal. TNP-OVA specific serum levels remained significantly lower than those of untransplanted controls at 9 wk (p < 0.0005) but reached levels equivalent to those of untransplanted controls by 12 wk post-HSCT.

FIGURE 7

Donor- and host-derived TNP-OVA specific Abs in fully MHC-disparate chimeras at 15 wk posttransplant. Allotype-specific Abs were used to detect the origin, donor or host, of TNP-OVA specific IgG_2a/c. Donor-derived (A) but not host-derived (B) IgG_2a/c is present in fully MHC-disparate full chimeras at 15 wk. Only host-derived Ag-specific Ab is detected in mixed chimeras.

FIGURE 8

Functionality of donor-derived T_FH cells at 6 wk after transplant. Congenic HSCs were transplanted into CD4^−/− hosts to determine if newly arising donor-derived T cells could drive Ab responses against the T-dependent Ag TNP-OVA. Six weeks after congenic transplantation into CD4^−/− or wild-type (WT), recipients and untransplanted controls were immunized. Each mark represents the total Ig level for an individual animal. Ab responses in CD4^−/− hosts transplanted with HSCs were significantly lower than those of untransplanted controls (p = 0.012) but not significantly different from those of wild-type hosts transplanted with congenic HSCs (p = 0.61).