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(American Journal of Pathology. 2000;156:1067-1071.)
© 2000 American Society for Investigative Pathology

Regular Articles

Single-Cell PCR Analysis of T Helper Cells in Human Lymph Node Germinal Centers

Axel Roers*, Martin Leo Hansmann{dagger}, Klaus Rajewsky* and Ralf Küppers*

From the Institute for Genetics,*
University of Cologne, Cologne; and the Institute for Pathology,{dagger}
University of Frankfurt, Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
The T helper cell population of human lymph node germinal centers (GCs) was analyzed for clonality and signs of antigen selection. Frozen sections of lymph node biopsies taken from three different individuals were used to micromanipulate single T cells from one particular GC for each of the specimens. T cell receptor (TCR) ß gene rearrangements were amplified from these single cells and directly sequenced. Although only unique rearrangements were amplified from T cells of GC2 and GC3, 11 of 28 potentially functional rearrangements amplified from GC1 originated from four different clones. In all three GCs, TCR gene rearrangements neither showed obvious biases in gene segment usage nor similarities in complementarity determining region 3 amino acid sequence. Thus, it appears that T lymphocytes in human GCs usually represent a diverse population of cells. Sequence analysis of V region genes did not provide evidence that in the human the process of somatic hypermutation acts on the TCRß loci. For one of the GCs (GC3), immunoglobulin heavy chain (IgH) gene rearrangements were amplified and sequenced from single micromanipulated GC B cells. The detection of clonal expansions accounting for more than half of the sampled B cells in addition to ongoing somatic hypermutation of Ig V region genes suggested that GC3 was a fully developed GC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
Germinal centers (GCs) are the sites of maturation of T-cell-dependent antibody responses.1 T helper (TH) cells account for 5 to 10% of all GC cells and are mainly found in the light zone (LZ) of the GC.2 GC T cells represent a specialized subset of CD4+ T cells, which seem to be specific for the immunizing antigen.3 Analysis of the response of mice to nitrophenyl-pigeon cytochrome c (NP-PCC) revealed that although they drive GC B cell development and selection, GC T cells themselves are under selection for antigen recognition.4 By day 14 after immunization with NP-PCC, the GC T cell population is dominated by cells expressing characteristic T cell receptor (TCR) chains composed of particular gene segments and complementarity determining region 3 (CDR3) sequence motifs known to confer recognition of PCC.4 It had earlier been shown that in human lymph nodes (LNs), as in hapten-immunized mice, clonal competition within the B cell population of a particular GC results in dominance of few clones that account for the bulk of the population.5 We have now addressed the question whether TH cells in GCs of human LNs, like the B cells with which they interact, are an oligoclonal population as a result of selection for peptide/MHC recognition. Single GC T cells were micromanipulated from histological sections of human LNs. TCRß gene rearrangements were amplified from these single cells and their sequences compared for signs of antigen selection. Because somatic mutations have been observed in TCR genes of murine GC T cells6 and CD8+ T cells invading GCs in HIV patients,7 the sequences of TCRß genes amplified from single micromanipulated GC T cells were also analyzed for mutations which may have been introduced by the process of somatic hypermutation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Patients

Patients 1, 2, and 3 were female. At the time of tissue sampling they were 25, 30, and 17 years of age, respectively.

Immunostaining and Micromanipulation

Frozen sections of LN tissue were stained with the monoclonal antibody Ki67 (Dako, Glostrup, Denmark) or antibodies against {alpha}ß TCR (ßF1, T Cell Diagnostics, Woburn, MA), CD8 (C8/144B, Dako) or CD20 (L26, Dako) as described.8 Single cells were mobilized and transferred into polymerase chain reaction (PCR) tubes using hydraulic micromanipulators as described.5,8

Amplification of TCR and Ig Gene Rearrangements from Single Cells

TCR Vß gene rearrangements were amplified in a seminested PCR as described.8 Briefly, a first round of amplification was performed using a mix of 25 Vß gene family-specific and seven Jß-specific primers. In a second round of PCR, the same Vß primers were used together with a mix of seven internal Jß-specific primers.

Ig heavy chain gene rearrangements were amplified from samples containing two cells each as previously described.9,10 Forty samples were analyzed using VH FRI family-specific primers,10 and 46 samples were analyzed using VH leader region family-specific primers9 together with a collection of JH segment primers in a seminested PCR using internal JH primers in the second round of amplification.

Sequence Analysis

PCR products were gel-purified and directly sequenced using the Ready Reaction DyeDeoxyTerminator cycle sequencing kit (Perkin Elmer, Foster City, CA) and an ABI 377 automatic sequencer. DNASIS software (Pharmacia, Freiburg, Germany) and the GenBank data library (release 93.0) were used to analyze the V gene sequences. Sequences were deposited in the EMBL database under accession numbers AJ389943-AJ390023 (TCR sequences) and AJ389914-AJ389942 (Ig sequences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Micromanipulation and PCR Analysis of GC T Cells

Biopsies of cervical LNs from three patients were analyzed. All patients had suffered from lymphadenopathy, and LNs were excised to rule out a neoplastic process. The lymph node swelling was clinically manifest several weeks and several months before biopsy in patient 1 and 2, respectively. (This information was not available for patient 3.) Histopathologically, a normal LN architecture with numerous GCs was found. An immune response against an unknown pathogen was considered the most likely explanation for the clinical symptoms.

Frozen sections were stained for the {alpha}ßTCR, CD20, and Ki67 to identify T cells, B cells, and proliferating cells, respectively. For each LN specimen, one GC was selected (GC1, GC2, and GC3 from tissue samples of patients 1, 2, and 3, respectively) that showed clear polarization into a dark zone (DZ) with predominantly proliferating Ki67+ cells and few T cells and a LZ mainly composed of Ki67- cells and numerous T cells.

Sections stained for the {alpha}ßTCR were used to isolate single GC T cells. Most T cells were micromanipulated from the LZ of the GCs. For GC1, a minor fraction (about 10%) and about one-third of the T cells in GC2 and 3 were micromanipulated from the junction of the LZ and the follicular mantle. The cells were analyzed for TCR Vß gene rearrangements on the level of genomic DNA by single cell PCR using mixtures of Vß family-specific and Jß-specific primers.8 {alpha}ßTCR- cells were micromanipulated from the same GC as negative controls. Aliquots of the buffer covering the sections during the micromanipulation procedure were aspirated as additional negative control (buffer samples, Table 1 ). Single T cells from blood of healthy donors were sorted into PCR tubes as described earlier8 and served as positive controls. All control samples were analyzed in parallel with the GC T cells. Of the sorted single T cells, 75% were positive for at least one specific product. The sequences obtained from these cells were all unique and unrelated to those obtained from GC T cells. Of 355 T cells micromanipulated in total from the three GCs, 93 (26%) yielded a specific product. One of 34 tubes containing PCR buffer but no cell (water controls, Table 1 ) yielded a unique Vß gene rearrangement, which likely represents a rare incidence of PCR product contamination. The results of the control amplifications are in accordance with our earlier data, which suggest that the single-cell amplification of TCRß gene rearrangements is reliable with respect to unbiased amplification of the majority of all possible rearrangements and the assignment of sequences to individual cells.8


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  		Table 1. Summary of the Single-Cell Analysis of GC T Cells

 
Expanded GC T Cell Clones Are Detectable in Only One of Three GCs

Twenty-eight potentially functional Vß gene rearrangements were obtained from T cells of GC1 (Table 1) . Among these, the rearrangements from five different cells were identical to each other, indicating that these cells belonged to the same T cell clone (clone 1). Three additional rearrangements were amplified from two different cells each (clones 2–4). Clone 3 also harbored an out-of-frame rearrangement. The remaining 17 potentially functional and seven nonfunctional rearrangements were unique, ie, they were each amplified from one cell only. Members of the four expanded clones in GC1 were in three instances located in proximity to a clonally related cell compatible with recent division of a common progenitor, whereas other clonally related cells were found at locations distant from each other (data not shown).

All 16 and 24 potentially functional rearrangements (and all four and eight non-functional rearrangements) amplified from T cells of GC2 and GC3, respectively, were unique.

Rearrangements that potentially encode a functional TCRß V region may have been subject to selection by antigen. Therefore, the potentially functional rearrangements obtained from each GC were compared with each other and with a collection of 60 potentially functional Vß gene rearrangements that were amplified from blood of four healthy donors8 in an attempt to find signs of antigen selection. However, the usage of gene segments from the various Vß gene families and of Jß gene segments as well as the distribution of CDR3 lengths in TCRß gene rearrangements from GC T cells did not show obvious biases in comparison to the control collection8 (clonally related sequences from GC1 were counted only once; data not shown).

The Vß gene rearrangements were also compared with respect to deduced CDR3 amino acid sequences. Sequences were aligned according to the conserved CASS amino acid motif encoded by Vß and the conserved Jß-encoded FG motif. In GC1, the TCRß gene rearrangement of clone 4 showed some similarity to a TCR V region sequence, which was obtained from one cell only. The rearrangements used Vß gene segments from closely related Vß families (BV5 and BV23), used the same Jß gene segment, and had an identical CDR3 length. The two amino acids encoded by the hypervariable VDJ junction, however, were dissimilar (CASSTRSYEQYFG (clone 4) and CASSLDSYEQYFG).

TCRß Gene Rearrangements of Human GC T Helper Cells Are Not Subject to Somatic Hypermutation

Because products of single target amplification were directly sequenced without prior subcloning, the sequences are essentially devoid of errors introduced by the thermostable polymerases. Therefore, the sequences amplified from GC T cells allow investigation at a high level of resolution of whether somatic mutations are introduced into TCRß V region genes of these cells in the course of the GC reaction. An unambiguously readable Vß gene segment sequence (1.7 x 104 bp) from GC T cells contained only 1 bp that could not be matched with published human Vß gene segment sequences and may thus represent either a rare mutation event or a polymorphism.

B Cells of GC3 Are Characterized by Clonal Expansions and Ongoing Hypermutation of Ig V Region Genes

The composition of the GC T cell population is likely dependent on the maturity of the GC reaction. All three GCs analyzed showed the clear polarization into DZ and LZ that usually develops several days after the onset of the GC reaction. Clonal expansion of GC B cells and the load of mutations introduced into Ig V region genes are additional indicators of GC maturity. Therefore, for one of the GCs, GC3, single B cells were micromanipulated from Ki67-stained sections and analyzed for rearrangements of the IgH locus by single cell PCR. Single Ki67+ and Ki67- cells were isolated from DZ and LZ. Because B cells account for about 90% of all GC cells, most of the cells isolated in this manner are B cells.5 A total of 172 cells yielded 29 VH gene rearrangements. As positive control for the VH gene PCR, single sorted B cells were used. Twenty-eight VH gene rearrangements (2 VH1, 15 VH3, 9 VH4, and 2 VH5) were amplified from 45 B cells, demonstrating that VH region genes of various families were efficiently amplified.

Several reasons might account for the relatively low efficiency of the VH gene PCR analysis of GC cells. First, about 10% of the micromanipulated cells may have been non-B cells, since the sections were not stained with a B cell-specific antibody (see above). Second, somatic mutations in a VH region gene can prevent amplification. If such mutations are present in a VH region gene of a large B cell clone, many cells will remain negative in the PCR analysis. Third, because centroblasts are large cells, often a fraction of the nucleus will not be present in the section used for single cell isolation (the thickness of the section was in the same range as the diameter of small lymphocytes, ie, about 10 µm).

Among 18 potentially functional rearrangements amplified from GC B cells of GC3, 8 belonged to one of three clones (Table 2) . In addition, 8 of 11 non-productive rearrangements could be assigned to two clones (Table 2) . Thus, besides several unique sequences, three to five expanded B cell clones were detected in GC3. Twenty-eight of the 29 VH region genes were somatically mutated, with an average mutation frequency of 3%. Intraclonal diversity of V gene rearrangements was observed in each of the five groups of clonally related sequences. Clonally related sequences differed from each other by 4 to 24 sequence differences (single nucleotide exchanges, deletions, and/or duplications; not shown), demonstrating ongoing somatic hypermutation during clonal expansion of the respective GC B cells.


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  		Table 2. Summary of VH Gene Analysis of B Cells Isolated from GC3

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
In the present study, clonal composition and TCRß repertoire of the T helper cell population in human LN GCs were analyzed. For each of three patients investigated, T cells were micromanipulated from one particular GC, and TCRß gene rearrangements were amplified from these single cells. This technique8 allows comprehensive, quantitative analysis of the TCRß repertoire expressed by small T cell populations.

In all three GCs analyzed, the GC reaction had reached a degree of maturity characterized by clear polarization into DZ and LZ as revealed by immunohistochemical staining for a proliferation marker. Furthermore, sequence analysis of IgH V region genes from B cells of GC3 demonstrated the presence of expanded B cell clones. Eight of 18 potentially functional and 8 of 11 nonfunctional IgH gene rearrangements amplified from B cells could be assigned to one of few clones. The finding of these expanded B cell clones, which were all characterized by somatically mutated Ig genes showing considerable intraclonal diversity, suggests that in GC3 the GC reaction must have commenced several days before the biopsy.

The TCRß V region sequences from GC T cells were compared for clonal identity, Vß and Jß gene segment usage and CDR3 amino acid sequence. In GC1, 11 of 28 potentially functional rearrangements originated from four different clones while the remaining 17 rearrangements were unique. In GC2 and GC3, only unique TCRß gene rearrangements were obtained. Thus, the clonal composition of the B and T cell populations of GC3 differed significantly from each other, since about half of the IgH V region sequences originated from three to five expanded B cell clones, showing that the GC T cell population can be much more diverse than the GC B cells with which they are interacting. It is possible that the immune response was less mature in GC2 and GC3 as compared to GC1. At late stages of the GC response, effects of T cell selection, including expansion of clones with optimal specificities, may be more evident. This interpretation is not supported by the fact that the duration of the lymphadenopathy before biopsy was longer in patient 2 (GC2) as compared to patient 1 (GC1). However, this time interval may not be a reliable measure of the duration of a GC reaction, as the reaction may go on for long periods of time without clinically manifest lymph node swelling. On the other hand, lymphadenopathy may be sustained by different subsequent immune responses. An alternative explanation for the difference observed between GC1 and the GCs 2 and 3 is that GC1 was involved in a response against an antigen that selects a more restricted repertoire of T cell specificities.

Except for the clonal expansions detected in GC1, comparison of TCR V region sequences provided little evidence that the micromanipulated T cells were sampled from a population of cells selected by one or few antigenic peptides. The gene rearrangements did not exhibit apparent biases in V and J gene segment usage or CDR3 length in any of the three GCs analyzed. Similarities in CDR3 amino acid sequence, which might reveal antigen selection, were rare. Taken together, it appears that the human lymph node GC T cell population is usually not dominated by a few expanded clones. One may speculate that the selection of GC T cells allows survival of a broad spectrum of different clones. The diversity of the GC T cells may also reflect a high number of T cell epitopes linked to one or few B cell epitopes that are recognized by the oligoclonal population of GC B cells.

There is evidence that the process of somatic hypermutation, which introduces point mutations into Ig V region genes of GC B cells, is not entirely specific for cells of the B cell lineage. Somatically mutated TCR{alpha}, but not TCRß V region, genes were described in murine GC T cells,6 and ongoing mutation of a TCR{alpha} gene rearrangement was observed in vitro in a murine T cell hybridoma.11 Recently, somatically mutated TCRß V region genes were detected in CD8 T cells invading GCs of HIV patients,7 implying that the human TCRß locus may be accessible for hypermutation.

Among 68 potentially functional as well as 21 nonproductive TCRß gene rearrangements amplified in total from GC T cells of three individuals in the present study, only a single sequence contained one mismatch with published TCR Vß gene segment sequences. This mismatch may be due to a so far undescribed polymorphism of the gene segment. We conclude that TCRß V region genes of human GC T helper cells are in general not subject to somatic hypermutation. Mutation of TCRß V region genes in CD8 T cells recovered from GCs of HIV+ individuals may therefore be a phenomenon unique to HIV infection.

In several autoimmune diseases like rheumatoid arthritis, Sjögren’s syndrome, Hashimoto’s disease, and myasthenia gravis, GC-like structures develop in extranodal sites.12 Little is known about T cell differentiation processes taking place in these ectopic GCs in comparison to normal GCs. Moreover, GC T cells are found in association with malignant B cells in follicular lymphoma.13 It will be interesting to investigate whether the T cell populations found in these diseases have a composition distinct from GC T cells in normal GCs. For example, it may well be that protracted interaction of T cells with B cells in autoimmune diseases and B cell lymphomas results in the selective outgrowth of few T cell clones.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
Evidence that T cells in human GCs usually are a diverse population of cells was recently also provided by Golby et al (Eur J Immunol 1999, 29:3729–3736).


   Acknowledgements
 
We thank Andrea Klöckner, Michaela Fahrig, and Julia Jesdinsky for expert technical help.


   Footnotes
 
Address reprint requests to Dr. Axel Roers, Institute for Genetics, Weyertal 121, 50931 Cologne, Germany. E-mail: axel.roers{at}uni-koeln.de

Supported by Deutsche Forschungsgemeinschaft through SFBs 502 and 243, and Deutsche Krebshilfe, Mildred Scheel Stiftung.

Accepted for publication November 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

  1. Rajewsky K: Clonal selection and learning in the antibody system. Nature 1996, 381:751-758[Medline]
  2. Stein H, Bonk A, Tolksdorf G, Lennert K, Rodt H, Gerdes J: Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin’s lymphomas. J Histochem Cytochem 1980, 28:746-760[Abstract]
  3. Fuller KA, Kanagawa O, Nahm MH: T cells within germinal centers are specific for the immunizing antigen. J Immunol 1993, 151:4505-4512[Abstract]
  4. Zheng B, Han S, Zhu Q, Goldsby R, Kelsoe G: Alternative pathways for the selection of antigen-specific peripheral T cells. Nature 1996, 384:263-266[Medline]
  5. Küppers R, Zhao M, Hansmann ML, Rajewsky K: Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J 1993, 12:4955-4967[Medline]
  6. Zheng B, Xue W, Kelsoe G: Locus-specific somatic hypermutation in germinal centre T cells. Nature 1994, 372:556-559[Medline]
  7. Cheynier R, Henrichwark S, Wain-Hobson S: Somatic hypermutation of the T cell receptor V ß gene in microdissected splenic white pulps from HIV-1-positive patients. Eur J Immunol 1998, 28:1604-1610[Medline]
  8. Roers A, Montesinos-Rongen M, Hansmann ML, Rajewsky K, Kuppers R: Amplification of TCRß gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin’s disease are polyclonal. Eur J Immunol 1998, 28:2424-2431[Medline]
  9. Braeuninger A, Kuppers R, Strickler JG, Wacker HH, Rajewsky K, Hansmann ML: Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells. (published erratum appears in Proc Natl Acad Sci USA 1997, 94: 14211). Proc Natl Acad Sci USA 1997, 94:9337-9342[Abstract/Free Full Text]
  10. Kanzler H, Küppers R, Hansmann ML, Rajewsky K: Hodgkin and Reed-Sternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med 1996, 184:1495-1505[Abstract/Free Full Text]
  11. Marshall B, Schulz R, Zhou M, Mellor A: Alternative splicing and hypermutation of a nonproductively rearranged TCR alpha-chain in a T cell hybridoma. J Immunol 1999, 162:871-877[Abstract/Free Full Text]
  12. Berek C, Kim HJ: B-cell activation and development within chronically inflamed synovium in rheumatoid and reactive arthritis. Semin Immunol 1997, 9:261-268[Medline]
  13. Poppema S, Kaleta J, Hugh J, Visser L: Neoplastic changes involving follicles: morphological, immunophenotypic and genetic diversity of lymphoproliferations derived from germinal center and mantle zone. Immunol Rev 1992, 126:163-178[Medline]



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