Adult C57BL/6 and B2m-deficient mice (B6.129P2-B2m^tm1-Unc/J) were purchased from Jackson Laboratories. All mice were bred and housed under barrier conditions in the Division of Veterinary Resources of the University of Miami Miller School of Medicine. Mice were regularly screened for specific common pathogens. Adult mice (6–10 weeks of age) and neonatal mice (7 days of age) were used in experiments. All mice were handled in compliance with the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine, Miami, FL, USA.

Wild-type high-virulence Y. enterocolitica A127/90 serotype 0:8 biotype IB was originally provided by G. R. Cornelis (Universität Basel, Basel, Switzerland). For infection, bacterial frozen stocks (17) were washed twice with Hank’s Balanced Salt Solution (HBSS, Gibco, Grand Island, NY, USA), diluted to the desired concentration, and inoculated with the indicated doses. Adults were inoculated orogastrically with a 22-gauge, round-tipped feeding needle (Fine Science Tools, Foster City, CA, USA) attached to a 1-ml syringe (Becton Dickinson, Franklin Lakes, NJ, USA). Neonates were inoculated orogastrically with PE-10 tubing (polyethylene tubing with an outside diameter of 0.61 mm) (Clay Adams, Sparks, MD, USA) attached to a 30-gauge needle and Hamilton syringe (20). The actual administered dose was determined by plating serial dilutions of the suspensions on Luria Broth plates and incubating for 48 h at 27°C.

Individual MLN from neonates and adults were harvested and placed in cold HBSS containing 1% calf serum (Gibco), 10 mM HEPES (Gibco), and 4 mM sodium azide. Cell suspensions were prepared by mincing tissues with scissors and pressing cells through wire mesh with 74 µm pore size (Compass Wire, Westville, NJ, USA). Cells were incubated in mouse Fc Block (CD16/CD32; BD Pharmingen, San Diego, CA, USA) for 5 min on ice, followed by a 30-min incubation with fluorochrome-conjugated antibodies specific for CD4, CD8, Ki67, or TCRαβ (BD Pharmingen). For intracellular cytokine staining, cells were activated with 50 ng/ml PMA and 0.5 µM ionomycin in the presence of 5 µg/ml brefeldin A, fixed and permeabilized, and stained with fluorochrome-conjugated anti-IFNγ (BD Pharmingen). Samples were run on a Becton Dickinson LSR II flow cytometer and analyzed with FlowJo flow cytometry analysis software.

To measure Y. enterocolitica titers, tissues were weighed and homogenized in HBSS using a Seward Biomaster 80 Stomacher (Brinkman, Westbury, NY, USA) for 4 min at high speed. For small intestine mucosa-associated Y. enterocolitica titers, the small intestine contents were flushed with HBSS prior to homogenization. Individual MLN were homogenized in 400 μl (neonates) or 500 μl (adults) of HBSS using a VWR disposable pellet mixer with cordless motor (VWR International). Y. enterocolitica titers were enumerated by plating dilutions of homogenates on Difco Yersinia Selective Agar Base plates (Becton Dickinson, Sparks, MD, USA).

All experiments were performed at least two times. Statistical tests were performed using GraphPad Prism software, as follows: unpaired t test for the Ki67/CD4/CD8/IFNγ staining experiments; Mann–Whitney test for the bacterial colonization experiments; Log-rank (Mantel–Cox) test for the survival experiments. The significant threshold was P ≤ 0.05.

In the course of characterizing immune responses to Y. enterocolitica infection, we compared MLN cells from uninfected neonates and adults for expression of the proliferation antigen Ki67. Approximately 10% of both CD4⁺ and CD8⁺ cells in adult MLN expressed the Ki67 antigen. A much greater proportion of both T cell types were Ki67⁺ in neonatal MLN (Figures 1A,B). This is consistent with previous studies in which neonatal T cells in mouse spleen and cord blood were found to be spontaneously proliferating at a higher rate than in adults (21, 22). However, the frequency of proliferating cells in the MLN, especially among the CD8⁺ population, is markedly higher than in blood or spleen.

The substantial endogenous proliferation of CD8⁺ cells in uninfected neonates indicated that this population may pre-exist in a state poised for rapid responsiveness to invasive intestinal pathogens. Indeed, within 24 h of infection with Y. enterocolitica, there was a significant increase in the proportion of neonatal CD8⁺ cells while percentages of neonatal CD4⁺ and adult CD4⁺ and CD8⁺ did not increase (Figure 1C). The absolute numbers showed a similar pattern, although neonatal CD4⁺ cells also increased by fivefold while CD8⁺ cell numbers increased eightfold. Neither population increased in absolute numbers in adults.

These results indicated that the neonatal CD8⁺ population was able to expand rapidly in response to Y. enterocolitica infection. The next question was whether this expansion was accompanied by effector function. A prominent effector function of CD8⁺ cells is the production of IFNγ. Our previous studies had shown that IFNγ is required for survival of neonates to Y. enterocolitica infection (18) and that IFNγ production by CD4⁺ cells was an important component of protection. However, IFNγ-producing CD4⁺ cells most likely act in the later stages of primary infection, during the adaptive phase of the response, since death in CD4-deficient neonates is delayed until ≥15 days post infection. In striking contrast, IFNγ-deficient neonates die rapidly, within 7 days of infection. Together, these results suggest that IFNγ may provide important protection both early and late post infection. Thus, we proposed that early activated CD8⁺ cells may be a rapid source of IFNγ production. We first examined early IFNγ production among total MLN cells and found an increase in total cytokine-producing cells in neonates, but not adults (Figure 2A). Indeed, a significant increase in IFNγ-production was selectively observed in the neonatal CD8⁺ population (Figure 2B). Thus, Y. enterocolitica infection of neonates is characterized by the rapid mobilization of CD8⁺ MLN populations with IFNγ-producing effector function.

Our results thus far indicated that neonates infected with Y. enterocolitca mount early CD8⁺ responses. To test whether CD8⁺ cells were essential for protection against infection, survival of wt and B2m^−/− neonates to a threshold lethal dose of Y. enterocolitica was compared. As shown in Figure 3, B2m^−/− neonates were more susceptible to lethal infection and, notably, most deaths occurred prior to 14 days post infection—i.e., well before the time that CD4^−/− neonates begin to succumb (18). In these experiments, we used female and male neonates indiscriminately but subset analyses showed that there were no differences in survival of male or female neonates in either wt or B2m^−/− neonates. Therefore, CD8⁺ cells appear to play an important role in the survival of neonates, regardless of gender.

Yersinia enterocolitica replicates in the small intestines and routinely invades to the MLN but only systemically disseminates if the dose of infection is very high in wt animals or if animals are immunocompromised (23). Significant systemic dissemination most likely leads to death via septic shock. To investigate the potential importance of CD8⁺ cells in confining Y. enterocolitica to the intestinal tissues, we compared early colonization of intestinal and systemic organs in infected wt and B2m^−/− neonates. 48 h post infection, colonization levels of the small intestines and MLN were similar in wt and B2m^−/− neonates (Figures 4A,B). However, at this relatively early time point, both the liver and spleen showed massive bacterial infiltration only in the B2m^−/− neonates (Figures 4C,D). These results indicate that the early activation of CD8⁺ cells in the neonatal MLN may be critical for preventing the systemic spread of Y. enterocolitica.

In adult mice, CD8⁺ cells appear to be essential for protection against secondary infection with the closely related bacterium Yersinia pseudotuberculosis (24). To test whether this was also the case in neonates, B2m^−/− neonates were infected with a sublethal dose (1 × 10⁴ CFU) of Y. enterocolitica. Uninfected littermates were kept as control mice. Eight weeks later, all mice were challenged with 1 × 10⁵ CFU of Y. enterocolitica, and survival was monitored. At this challenge dose, approximately 50% of the control, previously uninfected, mice survived (Figure 5). However, 100% of the B2m^−/− mice previously infected as neonates survived the infection. Therefore, CD8⁺ cells are not required for either the development of immunological memory in neonates or for its manifestation in response to secondary challenge.