Murine neonates develop vigorous in vivo cytotoxic and Th1/Th2 responses upon exposure to low doses of NIMA-like alloantigens

Abstract

Early life exposure to noninherited maternal antigens (NIMAs) may occur via transplacental transfer and/or breast milk. There are indications that early life exposure to NIMAs may lead to lifelong tolerance. However, there is mounting evidence that exposure to NIMAs may also lead to immunologic priming. Understanding how these different responses arise could be critical in transplantation with donor cells expressing NIMAs. We recently reported that murine neonates that received a transplant of low doses of NIMA-like alloantigens develop vigorous memory cytotoxic responses, as assessed by in vitro assays. Here, we demonstrate that robust allospecific cytotoxicity is also manifest in vivo. Importantly, at low doses, NIMA-expressing cells induced the development of in vivo cytotoxicity during the neonatal period. NIMA-exposed neonates also developed vigorous primary and memory allospecific Th1/Th2 responses that exceeded the responses of adults. Overall, we conclude that exposure to low doses of NIMA-like alloantigens induces robust in vivo cytotoxic and Th1/Th2 responses in neonates. These findings suggest that early exposure to low levels of NIMA may lead to long-term immunologic priming of all arms of T-cell adaptive immunity, rather than tolerance.

Figure 1

GFP⁺ donor cells are detectable at NIMA-like levels 24 hours and 48 hours after injection into neonates. BALB/c neonates were injected intravenously with GFP⁺ adult spleen cells. Twenty-four or 48 hours later, spleens from individual mice were harvested for DNA, which was used in a GFP-specific real-time PCR assay. For quantitation, a standard curve consisting of DNA from a 0.001% to 1% GFP⁺ cell mixture was generated in parallel. The limit of sensitivity of this assay was 0.001% (dashed line). Individual animals from 3 independent experiments are shown.

Figure 2

In vivo exposure to NIMA-like allogeneic cells induces striking phenotypic changes on neonatal spleen cells. Neonatal and adult BALB/c (H-2^d) mice were injected with C57BL/6 (H-2^b) adult spleen cells, as described in “Preparation and injection of donor cells.” Seven days later, spleen cells were harvested and analyzed by flow cytometry. Spleens were pooled from 2 to 3 mice per group. (A) Host-specific class I MHC expression. The depicted histogram is representative of 12 independent experiments. (B) Host-specific class II MHC expression. The histogram shown is representative of 8 independent experiments. (C) Expression of class II MHC on F4/80⁺ spleen cells. The histograms are representative of 6 independent experiments.

Figure 3

The development of robust in vivo memory cytotoxic and Th1/Th2 responses upon exposure of neonates to NIMA-like alloantigens. (A) Neonates (n = 22) and adults (n = 17) were injected with allogeneic spleen cells, as described for Figure 2. Five to 15 weeks later, the percentage of allospecific cytotoxicity in individual spleens was determined as described in “In vivo cytotoxicity assays.” A minimum of 1 × 10⁶ total cells were examined for each sample. Data from individual mice from 3 independent experiments are depicted (variations in responses were observed within each experiment). The average percentage of allospecific cytotoxicity was not significantly different (2-tailed t test) between neonates and adults. (B) Neonates were injected with allogeneic spleen cells (SP), bone marrow (BM), or Lin⁻ BM cells, as described in “Preparation and injection of donor cells.” Five to 6 weeks later, the percentage of allospecific cytotoxicity in individual spleens was determined. Data are pooled from 2 independent experiments; n = 9 for SP, n = 10 for BM, and n = 11 for Lin⁻ BM. The average percentage of allospecific cytotoxicity was not significantly different (2-tailed t test) between groups. (C-H) Neonates and adults were injected with allogeneic cells, as described for Figure 2. Memory CD4⁺ cytokine responses were then assayed by ELISA (C-E) and the frequency of cytokine-producing memory cells was determined by ELISPOT (F-H), as described in “Culture conditions for cytokine ELISA and ELISPOT.” The in vitro responses of age-matched, naive controls, determined in parallel, were as follows: for neonates: IFNγ, 155 (± 44) ng/mL and 1728 (± 826) secretors/10⁶ cells; IL-4, 1.0 (± 0.7) ng/mL and 2113 (± 636) secretors/10⁶ cells; for adults: IFNγ, 187 (± 41) ng/mL and 2146 (± 2112) secretors/10⁶ cells; IL-4, 0.28 (± 0.1) ng/mL and 2221 (± 1056) secretors/10⁶ cells. Error bars represent the mean (± SD) of data from 4 independent experiments. Statistical significance was determined using a 2-tailed t test; **P < .005

Figure 4

Donor cells persist in the neonate for at least 6 days after injection. (A) Neonates (top panel) and adults (bottom panel) were injected with allogeneic GFP⁺ cells, as described in Figure 2. Two to 9 days later, spleen cells were harvested for DNA isolation. DNA (0.1 μg) from individual animals was used in a quantitative GFP-specific real-time PCR assay to test for the presence of donor GFP⁺ cells, as described in Figure 1. The limit of sensitivity (dotted line) for this assay was 0.001%. Data are pooled from 2 to 3 independent experiments. (B) Cytokine-secreting memory CD4⁺ spleen cells were sorted, and the frequency of N-region addition in the CDR3 region of the TCR was determined as described in “Determination of N-region addition in the CDR3 region of the TCR.” Data represent 19 clones from IFNγ-secreting cells and 24 clones from IL-4–secreting cells.

Figure 5

In vivo cytotoxic responses to NIMA-like alloantigens develop during the neonatal period. (A) Neonates were injected with allogeneic adult spleen cells or PBLs. Seven days later, the percentage of allospecific cytotoxicity in the spleen (SP) and lymph nodes (LNs) was determined, as described for Figure 3. Data are pooled from 2 to 4 independent experiments. (B) (BALB/c × FVB) F1 neonates were injected with semiallogeneic adult (C57BL/6 × FVB) F1 spleen cells, as described for Figure 2. Seven days later, neonates were injected with a 1:1 mixture of Far Red–labeled (C57BL/6 × FVB) F1 and (BALB/c × FVB) F1 cells, as described in “In vivo cytotoxicity assays.” The percentage of allospecific cytotoxicity in the spleen (SP) and lymph nodes (LNs) was determined as described for Figure 3. Data are pooled from 3 independent experiments; n = 31 neonates.

Figure 6

Neonates develop vigorous Th1 and Th2 primary responses to NIMA-like alloantigens. Neonates and adults were injected with allogeneic cells, as described for Figure 2. Seven days later, purified CD4⁺ cells from injected neonates or adults were stimulated with allogeneic APCs and assayed for cytokine production by ELISA after 48 hours (A-C) or frequency of cytokine-producing cells by ELISPOT after 72 hours (D-F) as described in “Culture conditions for cytokine ELISA and ELISPOT.” The in vitro responses of age-matched, naive controls, determined in parallel were as follows: for neonates: IFNγ, 109 (± 145) ng/mL and 1598 (± 534) secretors/10⁶ cells; IL-4, 0.17 (± 0.03) ng/mL and 3798 (± 1412) secretors/10⁶ cells; for adults: IFNγ, 9.4 (± 5.6) ng/mL and 1760 (± 339) secretors/10⁶ cells; IL-4, 0.024 (± 0.024) ng/mL and 2054 (± 91) secretors/10⁶ cells. Error bars represent the mean (± SD) of data from 2 independent experiments. Statistical significance was determined using a 2-tailed t test; **P = .002

Figure 7

IFNγ production by donor cells contributes to the development of neonatal allospecific cytotoxic responses. Adult C57BL/6 (A,B), CD40L^−/− spleen cells (A), or IFNγ^−/− spleen cells (B) were transplanted into BALB/c neonates, as described for Figure 2. Seven days later, the percentage of allospecific cytotoxicity in the spleen (SP) and lymph nodes (LNs) was determined, as described for Figure 3. Data are pooled from 2 to 4 independent experiments. **P = .02; *P = .04, compared with wild-type donor cells by t test.