Tumour-infiltrating lymphocytes (TILs) are influential components of the tumour microenvironment and implications of their accumulation in breast cancers have been evaluated extensively [
1,
2]. Extent of lymphocytic infiltration has been found to be associated with a variety of prognostic factors [
3,
4], outcomes [
5,
6] and, in some cases, outcomes after specific therapies [
6,
7]. Despite this wealth of published data, key reproducible findings remain elusive, in part due to the range of methodologies used and variety of cohorts studied. In terms of methodologies, TILs were identified in many studies based on morphology using haematoxylin and eosin stained tissue, and indeed this was the recommendation by the International TILs Working Group [
8]. However, this does not allow separate quantification of distinct TIL subsets, such as B and T cells, that may have differential recruitment to tumours and differential associations with prognosis [
9]; the lack of this distinction is a potential cause of some conflicting findings in the literature. Accordingly, there is a growing literature describing specific TIL subtypes, using immunohistochemistry for detection of individual subtype-specific markers [
10,
11], or recently multiplexed simultaneous imaging of subtypes [
12]. A further difficulty is variation in methods for quantification of TILs and their locations. Some authors have quantified in descriptive terms only, such as defining infiltration as mild, moderate or heavy [
13,
14], while others have assessed percentages of tissue area occupied by TILs [
5,
6], or have counted individual cells [
3,
10]. In terms of tissue regions, some have made no distinction between different tumour areas [
3,
6], while others have limited their analyses to single regions, most commonly tumour stroma [
4,
5,
7] for which quantification methods have been mainly standardised [
8], or have quantified in a number of different regions separately [
10,
11], for example in tumour stroma, closely associated with tumour cells, or adjacent to the tumour margin, although analysis of all these regions remains exceedingly rare.
The result of these differing methodologies is that summarising overall conclusions from the field is challenging, although reviews have been published [
15,
16]. Some generalizable themes are: (1) There are typically more TILs in tumours showing markers of poor prognosis; (2) Higher levels of TILs are associated with better responses to neoadjuvant chemotherapy; (3) Different subclasses of TILs, such as cytotoxic T, helper T, or regulatory T cells, have different relationships with outcomes.
Here, we have taken a thorough approach of quantifying the presence, relationships between, and prognostic importance of four different subclasses of TILs (cytotoxic T, helper T, regulatory T, and B cells) in three separate tumoural compartments in a breast cancer cohort that we have characterised previously. In addition, uniquely, we have assessed how these levels correlate with the matched circulating levels of these cells, to infer whether TILs recruitment is tumour-driven independently of systemic levels – and if so for which TIL subtypes and in which tumoural compartments.