# Vaccination of calves with Bacille Calmette Guerin increased the frequency but did not affect aggregation or clustering of natural killer cells in draining lymph nodes

## TL;DR

Vaccinating calves with BCG increases the number of natural killer cells in lymph nodes but does not change how they cluster or group together.

## Contribution

The study reveals that BCG vaccination increases NK cell frequency in bovine lymph nodes without altering their spatial organization.

## Key findings

- BCG vaccination increased the frequency of NK cells in draining lymph nodes.
- There were no significant changes in NK cell aggregation or clustering after vaccination.
- Re-vaccination had minimal effect on NK cell numbers or distribution.

## Abstract

Natural killer (NK) cells are central to innate immune responses but they also influence adaptive immunity. Evidence suggests that NK cells are involved in protective immune responses induced by the Bacille Calmette Guerin (BCG) vaccine. In cattle, vaccination with BCG provides significant protection against infection with Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). Bovine NK cells were previously shown to traffic from BCG vaccination sites in afferent lymph, and to be activated reciprocally through interactions with dendritic cells (DC) to drive high-level interferon gamma secretion. To further define roles for bovine NK cells in the induction of BCG vaccine-mediated immunity, we examined alterations in their frequency, location, and aggregation in lymph nodes (LN) draining immunization sites.

Calves were either not vaccinated, vaccinated with BCG once, or were re-vaccinated. The frequency and localization of NK cells in draining LN was examined by immunohistochemistry and immunofluorescence, and statistical analyses of imaging outputs were performed.

While increased numbers of NK cells were found in BCG-draining LN, there were no significant alterations in location, nor the clustering or aggregation of NK cells. Re-vaccination with BCG had little impact on NK cell numbers or location.

BCG vaccination induced changes in NK cell frequency in bovine LN. Further studies of NK cell function and co-localization with subsets of DC and T cells will be important to define the roles of these cells in the induction of protective immunity in bTB.

Graphical AbstractImpact of Bacille Calmette Guerin (BCG) vaccination on natural killer (NK) cell frequency in bovine lymph nodes (LN). In non-vaccinated calves, NK cells are scattered throughout the LN. Following vaccination with BCG, the frequency of NK cells increased, but their location within the LN was not altered. We hypothesize that increased numbers of NK cells increase the potential for cellular interactions that are central to adaptive (protective) immunity. Created in BioRender. Hope, J. (2025) https://BioRender.com/pg277n3Alt Text. Graphical representation of natural killer cells in calf lymph nodes, changes in cell number and potential cell interactions following vaccination with the Bacille Calmette Guerin vaccine.

Impact of Bacille Calmette Guerin (BCG) vaccination on natural killer (NK) cell frequency in bovine lymph nodes (LN). In non-vaccinated calves, NK cells are scattered throughout the LN. Following vaccination with BCG, the frequency of NK cells increased, but their location within the LN was not altered. We hypothesize that increased numbers of NK cells increase the potential for cellular interactions that are central to adaptive (protective) immunity. Created in BioRender. Hope, J. (2025) https://BioRender.com/pg277n3