In Utero Exposure to Exosomal and B-Cell Alloantigens Lessens Alloreactivity of Recipients' Lymphocytes Rather than Confers Allograft Tolerance

Abstract

According to actively acquired tolerance, antigen exposure before full immune development in fetal or early neonatal life will cause tolerance to this specific antigen. In this study, we aimed to examine whether allogeneic tolerance could be elicited by in utero exposure to surface MHC antigens of allogenic cells or soluble form of MHC exosomes. Gestational day 14 FVB/N fetuses were subjected to intraperitoneal injection of allogeneic major histocompatibility complex (MHC) exosomes or highly enriched B-cells. Postnatally, the recipients were examined for the immune responses to donor alloantigens by lymphocyte proliferative reactions and skin transplantation. In utero exposure to allogeneic MHC exosomes abolished the alloreactivity of recipients' lymphocytes to the alloantigens, but could not confer skin allograft tolerance. In utero transplantation of highly enriched allogeneic B-cells generated low-level B-cell chimerism in the recipients. However, it only extended the survivals of skin allograft by a few days despite the lack of donor-specific alloreactivity of recipients' lymphocyte. Thus, an early in utero contact with exosomal or B-cell alloantigens did not lead to full skin tolerance but rather, at best, only to delayed skin rejection in the presence of microchimerism made by B-cell inocula. These results argued against the theory of actively acquired tolerance, and implicated that in utero exposure to marrow cells in previous studies was a unique model of allo-tolerance induction that involved the establishment of significant hematopoietic chimerism. Taken together with the discovery of in utero sensitization to ovalbumin in our previous studies, the immunological consequences of fetal exposure to foreign antigens might vary according to the type or nature of antigens introduced.

Keywords: B-cells; alloreactivity; exosome; in utero injection; major histocompatibility complex; tolerance induction.

Figure 1

Quantification and verification of major histocompatibility complex (MHC) exosomes. Exosomes were generated from A20 cell line at the concentration of 5 × 10⁵ A20 cells/ml. Following the centrifugation and ultracentrifugation, the final supernatant and pellet were collected, respectively. (A) BCA assay showed that protein concentration of resuspended pellets was proportional to the A20 cell number used for exosome generation. Pearson’s correlation coefficient (0.961) was significant at the 0.01 level with P-value of 0.002. (B) Under the transmission electron microscope, there were bilayer membrane-bound vesicles, sized around 100 nm in the resuspended pellet. (C) Immunoblotting demonstrated the expression of MHC class I (H-2K^d) in the resuspended pellet regardless of 0.22-µm filtration. Controls were samples from the supernatant after the final ultracentrifugation.

Figure 2

FVB/N lymphocyte proliferation in response to H-2^d (A20 cell line) major histocompatibility complex (MHC) exosomes. (A) FVB/N lymphocytes were cultured with various doses of H-2^d MHC exosomes for 5 days. The controls were lymphocyte cultures without adding H-2^d MHC exosomes. The proliferative response (n = 3) was measured by the readout of incorporated tritium as counts per minute (CPM). The proliferative response was evident when there was the exosome concentration of ≥5 μg/ml. (B) Proliferative colonies of FVB/N lymphocytes in response to H-2^d MHC exosomes were observed under an Olympus BX50 microscope.

Figure 3

Immune reactivity to H-2^d alloantigens in FVB/N mice with in utero exposure to H-2^d exosomes. Gestational day 14 FVB/N murine fetuses were subjected to in utero exposure to H-2^d exosomes from A20 cell lines (IU exosome). (A) Postnatally (at 6–8 weeks old without skin transplantation), their lymphocytes did not exhibit proliferative responses in vitro to H-2^d exosomes (P = 0.979, n = 3), whereas the lymphocytes of the controls with in utero saline injection (IU NS) significantly proliferated (P = 0.001, n = 3). Lymphocyte proliferation in response to exosomes also reached statistical significance between IU exosome and IU NS (P = 0.019). (B) FVB/N recipient mice were also subjected to transplantation of BALB/C (H-2^d) allogeneic skin. All the allogeneic skin grafts (n = 18) were rejected within 14 days after transplantation, similar to the graft survivals of IU NS (P = 0.800, n = 7).

Figure 4

Enrichment of splenic B-cells. C57BL/6 splenic B-cells were negatively selected by Pan B Cell Isolation Kit (Miltenyi Biotec). A representative experiment showed that CD3 T-cells and CD19 B-cells were 38.69 and 45.10%, respectively, in splenic lymphocytes before enrichment (left panel). Following negative selection, CD3 T-cells were less than 0.5% and B-cells examined by either anti-CD19 (middle panel) or anti-CD45R (B220) (right panel) were above 90%.

Figure 5

Peripheral chimerism after in utero injection of C57BL/6 (H-2^b) B-cells into FVB/N (H-2^q) fetuses. FVB/N recipients were examined for peripheral chimerism at their age of 4–6 weeks. (A) 5.0–7.5 × 10⁶ B-cells (5.0–7.5, n = 6) generated significantly higher peripheral chimerism (P = 0.006) than 2.5–5.0 × 10⁶ B-cells (2.5–5.0, n = 14). The significance of differences was measured by non-parametric Mann–Whitney U test. The boxplot shows the median as a horizontal line inside the box and the interquartile range (between the 25 and 75th percentiles) as the length of the box. The whiskers (line extending from the top and bottom of the box) represent the minimum and maximum values when they are within 1.5 times the interquartile range from either end of the box. A score greater than 1.5 times the interquartile range is out of the boxplot and is considered as outliers (circle), and that greater than three times the interquartile range is extreme outliers (asterisk). (B) A representative recipient had the low-level chimerism of 0.47% (H-2K^b+) in the circulation. The engrafted donor cells were shown to be CD45R-positive, indicating donor B-cell origin.

Figure 6

Alloreactivity of FVB/N mice after in utero injection of C57BL/6 B-cells. (A) Following donor skin transplantation, FVB/N recipients had better survivals of C57BL/6 skin grafts than the controls with in utero saline injection (P = 0.029 for 2.5–5.0 and P = 0.025 for 5.0–7.5). However, it made no difference in graft survivals between two B-cell doses of 2.5–5.0 × 10⁶ and 5.0–7.5 × 10⁶ used (P = 0.273). (B) Mixed lymphocyte reactions in response to FVB/N, C57BL/6, and BALB/C antigens were examined. Lymphocytes from FVB/N recipients with in utero B-cell injection (IU B-cells, n = 5) were responsive to third-party BALB/C stimulators (P < 0.001), but not to donor-specific C57BL/6 stimulators (P = 0.919). However, the mice with in utero saline injection (IU NS, n = 3) were both responsive to C57BL/6 (P = 0.004) and BALB/C alloantigens (P = 0.005). Lymphocytic proliferative responses to donor-specific C57BL/6 stimulators also reached significant difference between IU B-cells and IU NS (P = 0.005). Nil: no stimulator added.