Immune cell repertoires in breast cancer patients after adjuvant chemotherapy

Abstract

Adjuvant chemotherapy in breast cancer patients causes immune cell depletion at an age when the regenerative capacity is compromised. Successful regeneration requires the recovery of both quantity and quality of immune cell subsets. Although immune cell numbers rebound within a year after treatment, it is unclear whether overall compositional diversity is recovered. We investigated the regeneration of immune cell complexity by comparing peripheral blood mononuclear cells from breast cancer patients ranging from 1-5 years after chemotherapy with those of age-matched healthy controls using mass cytometry and T cell receptor sequencing. These data reveal universal changes in patients' CD4+ T cells that persisted for years and consisted of expansion of Th17-like CD4 memory populations with incomplete recovery of CD4+ naive T cells. Conversely, CD8+ T cells fully recovered within a year. Mechanisms of T cell regeneration, however, were unbiased, as CD4+ and CD8+ T cell receptor diversity remained high. Likewise, terminal differentiated effector memory cells were not expanded, indicating that regeneration was not driven by recognition of latent viruses. These data suggest that, while CD8+ T cell immunity is successfully regenerated, the CD4 compartment may be irreversibly affected. Moreover, the bias of CD4 memory toward inflammatory effector cells may impact responses to vaccination and infection.

Keywords: Cell Biology; Cellular immune response.

Figure 1. Effect of adjuvant chemotherapy on circulating immune cell subsets in breast cancer patients.

(A) Frequencies of immune cell populations in peripheral blood from healthy controls (n = 15) and chemotherapy treated breast cancer patients (n = 15), as determined by hand-gating of data from 33 parameter mass cytometry. P values were determined using Mann-Whitney U tests with Hochberg multiple comparisons. (B) Scaffold plots of live, single cells from 1 healthy control (left) and 1 patient (right) using 200 cluster input. Red indicates that a cluster is statistically significantly higher in patients, and blue is statistically lower in patients, while gray is no difference, based on Q values calculated in Statistical Scaffold analysis comparing all 15 patients to 15 age-matched healthy controls. Black nodes are landmark nodes, which indicate where a specific cell population lies in relationship to the cluster. The size of the cluster is relative to its proportion within total cells within a sample, where larger clusters contain a higher number of cells. Distance is relative to how similar clusters are to landmark nodes.

Figure 2. Major alterations in the CD4⁺ T cell compartment with minor CD8⁺ T cell changes in patients postchemotherapy.

(A) Representative scaffold plots for CD4⁺ T cells from 15 patients and 15 healthy controls using 100 clusters. Cluster size is proportional to cell frequencies in that donor. (B) Representative scaffold plots for CD8⁺ T cells from 15 patients and 15 healthy controls using 50 clusters. Cluster size is proportional to cell frequencies in that donor. (C) The percentage of differential clusters between patients and controls in CD4⁺ and CD8⁺ T cell scaffold analyses. (D) The subset composition of differential clusters in CD4⁺ and CD8⁺ T cells, based on the highest predicted nearest landmark node.

Figure 3. Reduced naive CD4⁺ T cells and expanded CD95⁺ naive CD8⁺ T cells in patients.

(A) Representative flow plots of CD4⁺ and CD8⁺ naive (CCR7⁺CD45RA⁺) T cell frequencies in patients and controls. (B) Number of T cell excision circles (TRECs) present in CD4⁺ (upper) and CD8⁺ (lower) naive (CCR7⁺CD45RA⁺) T cells in patients (P, n = 7) and controls (C, n = 10). (C) Frequencies of CD95⁺ and CD95^– CD4⁺ and CD8⁺ naive (CCR7⁺CD45RA⁺) T cell frequencies within total CD4 and CD8 compartments of patients (n = 15) and controls (n = 15). (D) Absolute cell numbers of CD95⁺ and CD95^– CD4⁺ and CD8⁺ naive (CCR7⁺CD45RA⁺) T cells in patients (n = 15) and controls (n = 5). (E and F) Heatmap of median surface marker expression intensity on CD95^– CD4⁺ (E) and CD8⁺ (F) naive (CCR7⁺CD45RA⁺) T cells from patients (n = 10) and controls (n = 10) using unsupervised hierarchical clustering. P values were determined by Mann-Whitney U tests with Hochberg multiple comparisons.

Figure 4. Expansion of Th17-like central memory populations in the CD4⁺ T cell compartment in patients.

(A) Frequencies of central memory (CCR7⁺CD45RA^–) CD4⁺ T cells within the CD4⁺ T cell compartment of patients (n = 15) and controls (n = 15). (B) Representative flow plots of CXCR3 and CCR6 on central memory CD4⁺ T cells from patients and controls, indicating the associated functional subset definition; Th1, Th17, Th1/17, and CXCR3^–CCR6^– (DN) cells. (C) Frequencies of functional subsets in the central memory CD4⁺ T cell compartment of patients (n = 15) and controls (n = 15). (D) Absolute numbers of Th1, Th17, Th1/17, and DN central memory CD4⁺ T cells in patients (n = 15) and controls (n = 5). (E) Frequencies and absolute number of CD161⁺ central memory CD4⁺ T cells in patients and controls. (F) Frequencies of functional subsets (Tc1, Tc17, Tc1/17, and DN) in the central memory CD8⁺ T cell compartment of patients (n = 15) and controls (n = 15). (G) Frequencies and absolute number of CD161⁺ and CD161⁺⁺ central memory CD8⁺ T cells in patients and controls. P values were determined by Mann-Whitney U tests with Hochberg multiple comparisons.

Figure 5. TCR repertoire diversity in naive and memory T cell compartments after chemotherapy.

TRB sequencing was performed on 7 chemotherapy-treated patients and 7 age-matched healthy controls. (A and B) TCR richness of naive (CCR7⁺CD45RA⁺) (A) and memory (B) CD4⁺ and CD8⁺ T cells in patients and controls. This parameter estimates the number of unique TRB sequences in a repertoire. (C and D) Clonality index of naive (CCR7⁺CD45RA⁺) (C) and memory (D) CD4⁺ and CD8⁺ T cells in patients and controls. This parameter estimates the amount of clonal expansion in a TCR repertoire. Both TCR richness and clonality index are based on TCR-β V (BV), BD, and BJ segment usage and CDR3 amino acid sequence. P values were determined by Mann-Whitney U test. (E and F) Representative Circos plots showing the overlap between the top 300 CDR3 amino acid sequences between naive and CD4⁺ Tem (E) and CD8⁺ T cells (F). Overlaps between naive-naive are green, memory-memory are purple, and naive-memory are orange. N1–N5 indicate replicates of naive and M1–M5 indicate replicates of memory cells from the same donor. (G–I) Quantification of TRB overlap between CDR3 nucleotide sequences between the naive and memory compartment (pMN) (G), in the naive compartment (pNN) (H), and in the memory compartment (pMM) (I) of patients and controls. P values were determined by Mann-Whitney U test. (J) Spearman’s correlation between pNN and pMN in the CD8⁺ T cell compartment from patients (triangles) and controls (circles).

Figure 6. Loss of B cell memory and intermediate monocytes with expansion of naive B cells in patients after chemotherapy.

(A) Frequencies of B cell subsets in patients (n = 15) and healthy controls (n = 15). P values determined by Mann-Whitney U tests with Hochberg multiple comparisons. (B) Frequencies of age-associated B cells (ABC) in patients (n = 15) and healthy controls (n = 15). P values determined by Mann-Whitney U test. (C) Gating strategy for monocyte subsets: classical (CD14⁺⁺CD16^–), intermediate (CD14⁺⁺CD16⁺), and nonclassical (CD14^+/loCD16⁺). (D) Frequencies of monocyte subsets in chemotherapy-treated patients (n = 15) and healthy controls (n = 15). P values determined by Mann-Whitney U tests with Hochberg multiple comparisons.

Figure 7. Persistent alterations in the CD4⁺ T cell compartment of patients after chemotherapy.

(A) Representative scaffold analysis and the percent of differential clusters in B cells between patients < 3 (early) and 3–5 years (late) after chemotherapy compared with healthy controls. (B) Frequencies of B cell subsets in controls, early patients, and late patients determined by hand-gating. (C) Representative scaffold analysis and the percent of differential clusters in CD4⁺ T cell compartment cells between patients < 3 (early) and 3–5 years (late) after chemotherapy compared with healthy controls. Cluster size is proportional to cell frequencies in the individual patient. (D) Frequencies of CD4⁺ T cell subsets in controls, early patients, and late patients determined by hand-gating. P values were determined by 1-way ANOVA with Tukey’s multiple comparison test. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.