Normal human lymph node T follicular helper cells and germinal center B cells accessed via fine needle aspirations

Abstract

Germinal centers (GC) are critically important for maturation of the antibody response and generation of memory B cells, processes that form the basis for long-term protection from pathogens. GCs only occur in lymphoid tissue, such as lymph nodes, and are not present in blood. Therefore, GC B cells and GC T follicular helper (T_FH) cells are not well-studied in humans under normal healthy conditions, due to the limited availability of healthy lymph node samples. We used a minimally invasive, routine clinical procedure, lymph node fine needle aspirations (LN FNAs), to obtain LN cells from healthy human subjects. This study of 73 LNs demonstrates that human LN FNAs are a safe and feasible technique for immunological research, and suggests benchmarks for human GC biology under noninflammatory conditions. The findings indicate that assessment of the GC response via LN FNAs will have application to the study of human vaccination, allergy, and autoimmune disease.

Fig. 1.. Ultrasound guided LN FNAs in healthy human donors is well-tolerated.

A) Ultrasound image of LN. Images not captured for all LN FNA attempts. B) Graph of LN sizes, measured via ultrasound. C) Visualization score of LNs images by ultrasound. D) Graph of donor reported discomfort during the LN aspiration procedure. Red bars indicate geometric means

Fig. 2.. Human LN FNA cell recovery.

A) Cell recovery from human LN FNAs. N = 50. Note: Some samples were analyzed directly by flow cytometry and included in later analyses, but no initial count data was obtained. Red bars indicate geometric means.

Fig. 3.. Live T and B cells recovered from healthy human LN FNA samples with minimal blood cells.

A) Representative flow plot of lymphocyte and granulocyte populations. B) Graph of granulocyte percentage of total acquired events. Gray shaded area represents the frequency range of granulocytes found in blood from normal healthy subjects (ref17). C) Representative flow plot of live, CD45+ lymphocytes. D) Quantification of live CD45⁺ lymphocytes. N = 73. E) Representative flow plot of CD4⁺ T cell and B cell gating. F) Quantification of CD4⁺ T cell percentages of live lymphocytes. N = 73. G) Quantification of CD19⁺ B cell percentages of live lymphocytes. N = 73. H) Quantification of CD19⁺ B cell percentages of live lymphocytes. Successful samples, N = 34. Red bars indicate means in B, F, G, H. The Read bar indicates the median in D.

Fig. 4.. Variables affecting LN FNA sampling.

A) Cell yield of live lymphocytes versus donor age. Average of successful Ax LN samples. N = 12 donors. B) Cell yield of live lymphocytes versus donor BMI. Average of successful Ax LN samples. N = 12 donors. C) Cell yield as a function of sampling site and physician. N = 73. D) Cell yield as a function of with or without aspiration. N = 19. Two-tailed Wilcoxon matched-pairs signed rank test. Red bars indicate geometric means.

Fig. 5.. Quantification of human LN GC-TFH cells and GC B cells by LN FNA.

A) Representative flow plot of GC B cell gating post immunization in a draining LN. Gated on CD19⁺ B cells. B) Representative flow plot of GC-T_FH cell gating post immunization in a draining LN. Gated on CD4⁺ T cells. C) Quantification of GC-T_FH % of successful LN samples. Green point, subject reported to have received an immunization one month prior to the LN FNA procedure. N = 34. Frequency in total samples are shown in Fig. S4. D) Quantification of GC-T_FH % of successful LN samples. Green point, subject reported to have received an immunization one month prior to the LN FNA procedure. N = 34. Frequency in total samples are shown in Fig. S4. E) Quantification of GC B cell frequency of successful Ax LN attempts as a function of physician. N = 15. F) Quantification of GC-T_FH cell frequency of successful Ax LN attempts as a function of physician. N = 15. Red bars indicate means.