Cancer Immunology, Immunotherapy

, Volume 65, Issue 2, pp 171–180 | Cite as

Interrogation of individual intratumoral B lymphocytes from lung cancer patients for molecular target discovery

  • Michael J. Campa
  • M. Anthony Moody
  • Ruijun Zhang
  • Hua-Xin Liao
  • Elizabeth B. Gottlin
  • Edward F. PatzJr.Email author
Original Article


Intratumoral B lymphocytes are an integral part of the lung tumor microenvironment. Interrogation of the antibodies they express may improve our understanding of the host response to cancer and could be useful in elucidating novel molecular targets. We used two strategies to explore the repertoire of intratumoral B cell antibodies. First, we cloned VH and VL genes from single intratumoral B lymphocytes isolated from one lung tumor, expressed the genes as recombinant mAbs, and used the mAbs to identify the cognate tumor antigens. The Igs derived from intratumoral B cells demonstrated class switching, with a mean VH mutation frequency of 4 %. Although there was no evidence for clonal expansion, these data are consistent with antigen-driven somatic hypermutation. Individual recombinant antibodies were polyreactive, although one clone demonstrated preferential immunoreactivity with tropomyosin 4 (TPM4). We found that higher levels of TPM4 antibodies were more common in cancer patients, but measurement of TPM4 antibody levels was not a sensitive test for detecting cancer. Second, in an effort to focus our recombinant antibody expression efforts on those B cells that displayed evidence of clonal expansion driven by antigen stimulation, we performed deep sequencing of the Ig genes of B cells collected from seven different tumors. Deep sequencing demonstrated somatic hypermutation but no dominant clones. These strategies may be useful for the study of B cell antibody expression, although identification of a dominant clone and unique therapeutic targets may require extensive investigation.


Non-small cell lung cancer Intratumoral B lymphocytes Tumor-infiltrating lymphocytes Target discovery Tropomyosin 4 



Complementarity-determining region

CT antigens

Cancer-testis antigens


Germinal center


Intratumoral lymphocyte


Mass spectrometry


Next-generation sequencing


Non-small cell lung cancer


Polyvinylidene fluoride

ROC analysis

Receiver-operating characteristic analysis


Tropomyosin 4


Variable heavy chain immunoglobulin domain


Variable light chain immunoglobulin domain



We thank Thaddeus C. Gurley and Minyue Wang for expert technical assistance, and gratefully acknowledge the Duke Human Vaccine Institute Flow Cytometry Shared Resource Facility for the isolation of tumor B cells. This work was supported by Grants from the LUNGevity Foundation and the Department of Defense (W81XWH-13-1-0189) to Edward F. Patz, Jr.

Compliance with ethical standards

Conflict of interest

None of the authors of this paper have a conflict of interest.

Supplementary material

262_2015_1787_MOESM1_ESM.pdf (2.2 mb)
Supplementary material 1 (PDF 2216 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Michael J. Campa
    • 1
  • M. Anthony Moody
    • 2
    • 3
  • Ruijun Zhang
    • 3
  • Hua-Xin Liao
    • 3
    • 4
  • Elizabeth B. Gottlin
    • 1
  • Edward F. PatzJr.
    • 1
    • 5
    Email author
  1. 1.Department of RadiologyDuke University Medical CenterDurhamUSA
  2. 2.Department of PediatricsDuke University Medical CenterDurhamUSA
  3. 3.Department of Duke Human Vaccine InstituteDuke University Medical CenterDurhamUSA
  4. 4.Department of MedicineDuke University Medical CenterDurhamUSA
  5. 5.Department of Pharmacology and Cancer BiologyDuke University Medical CenterDurhamUSA

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