Immune Function & Modulation Assays
Germinal Center Reaction Assay
Does your compound modulate Tfh–B-cell interactions relevant to germinal-center biology and tertiary lymphoid structures (TLS)?
Our human tonsil-derived assay combines autologous T follicular helper (Tfh) cells and memory B cells to characterize compound effects on B-cell function and Tfh-supported responses. IgG secretion provides a primary functional readout, while complementary immunophenotyping and molecular profiling can characterize changes in B-cell differentiation, phenotype and associated immune programs.
Germinal center reaction assay: format & readouts
- Format
- 96-well plate co-culture
- Model
- Autologous human tonsil-derived T follicular helper cells (CD4+/CXCR5+) and memory B cells (Bmem, CD19+/CD27+)
- Activation
- Staphylococcal enterotoxin B (SEB)
- Functional readout
- Secreted IgG as a measure of antibody secretion
- Response
- Detects increases or decreases in IgG output following compound treatment
- Cellular phenotyping
- Flow cytometry can be incorporated to characterize Tfh and B-cell phenotypes and compound-associated changes in B-cell differentiation or activation, according to the study design
- Reference controls
- SEB; checkpoint inhibitors including nivolumab, atezolizumab and ipilimumab; and the CD40-blocking antibody bleselumab, selected according to the biological question
- Additional profiling
- Extended cytokine profiling and complementary proteomic, transcriptomic or signaling analyses to characterize cellular and molecular changes associated with compound response
Human tonsil tissue provides the cellular foundation of the assay
Immunohistofluorescence of a human tonsil illustrates a germinal center within a lymphoid follicle and the spatial organization of Tfh and B-cell populations. Human tonsils provide a rich source of the autologous Tfh and memory B cells used in our functional co-culture assay.
Germinal-center biology is not only about which immune cells are present, but how they function together. By probing Tfh–B-cell interactions, our assay provides a controlled way to investigate whether a candidate modulates a key component of the humoral immune response. IgG secretion provides a functional endpoint, while complementary cellular and molecular profiling can characterize the biological changes associated with that response and add a functional dimension to TLS-related research.
Imane Nafia, PhD
Chief Scientific Officer, Explicyte
Assay principle
From Tfh–B-cell co-culture to functional and phenotypic readouts
1
Isolate and co-culture
Autologous Tfh cells and memory B cells are isolated from human tonsil and co-cultured with the test compound, alone or in combination with selected immune modulators according to study design.
2
Activate the co-culture
SEB provides an activation stimulus that establishes a functional Tfh–B-cell response against which compound-associated changes can be evaluated.
3
Measure the functional response
Secreted IgG is quantified in the supernatant as a functional measure of antibody secretion, enabling detection of compound-associated increases or decreases in the response.
4
Characterize cellular and molecular changes
Flow cytometry immunophenotyping and complementary cytokine, proteomic or transcriptomic analyses can be incorporated to characterize changes in Tfh and B-cell phenotype, differentiation and associated biological programs.
illustrative Germinal center reaction data
Tfh–B-cell co-culture: example results
Isolation and phenotypic characterization of Tfh and memory B cells
Freshly isolated Tfh and memory B cells were characterized by multiparameter flow cytometry before and after isolation. Tfh cells were identified as CD4+/CXCR5+, while memory B cells were identified as CD19+/CD27+, confirming enrichment of the intended cell populations used in the functional co-culture assay.
Reference immune modulators increase or decrease IgG secretion
Tfh and memory B cells were co-cultured under SEB activation, and secreted IgG was quantified in the supernatant on day 7. The checkpoint inhibitors nivolumab, atezolizumab and ipilimumab increased IgG secretion, whereas the CD40-blocking antibody bleselumab decreased it.
These results illustrate the assay’s ability to detect both increases and decreases in Tfh-supported B-cell responses using reference immune modulators with distinct mechanisms of action.
Concentration-dependent suppression of IgG secretion by kynurenine
In the same SEB-activated Tfh–B-cell co-culture, kynurenine induced a concentration-dependent decrease in IgG secretion, both in the presence and absence of anti-PD-1 treatment with nivolumab.
These results illustrate the assay’s ability to quantify concentration-dependent suppression of the functional response and to evaluate how compound-associated effects behave in a checkpoint-modulated context.
Tfh–B-cell interactions shape the cytokine and B-cell-associated soluble-factor response
Memory B cells isolated from human tonsil were cultured either under IL-21/IL-4/CD40L stimulation, reproducing key signals delivered by Tfh cells (top panel), or in co-culture with autologous Tfh cells under SEB activation (bottom panel).
Supernatants were analyzed using a multiplex approach for IFN-γ, TNF-α, TNF-β, IL-4, IL-6, IL-10, BAFF and APRIL. The two conditions generate distinct soluble-factor profiles, illustrating that direct Tfh–B-cell interactions provide an additional level of regulation beyond defined Tfh-mimetic stimulation.
These data demonstrate the assay’s ability to characterize how Tfh–B-cell cross-talk modulates cytokine and B-cell-associated responses, complementing IgG secretion with a broader functional view of germinal-center-related biology.
Making a difference as a preclinical CRO in oncology
Why work with Explicyte for germinal-center and B-cell functional assays
100+ in vitro campaigns
More than 100 in vitro campaigns over ten years, with a dedicated PhD-level study director overseeing each program from experimental planning through final report discussion.
Depth in TLS and B cell biology
Co-authors of peer-reviewed publications on the role of TLS in immunotherapy response, we have also developed ISO-certified pathology services for TLS scoring in IO trials.
Reference-anchored functional assessment
Reference immune modulators can be incorporated to contextualize compound-associated increases or decreases in functional and cellular readouts and support interpretation of response magnitude and direction.
Extend beyond the primary functional readout
Flow cytometry immunophenotyping, cytokine profiling, proteomics and transcriptomics can be incorporated to characterize cellular and molecular changes associated with compound response.
Paul Marteau, PharmD (study director), Imane Nafia, PhD (CSO), Loïc Cerf, MSc (COO), Alban Bessede, PhD (founder, CEO), Jean-Philippe Guégan, PhD (CTO)
Contact our team
Discuss your germinal center program
Tell us the question you need the germinal center reaction assay to answer — which compounds, which controls, and the readouts beyond IgG you want on the same co-culture. A PhD-level study director will scope it with you.
Frequently asked questions
Frequently asked questions about germinal center reaction and Tfh–B-cell assays
What does the germinal center reaction assay measure?
The assay models a key functional interaction between human tonsil-derived T follicular helper (Tfh) cells and memory B cells relevant to germinal-center biology. It can be used to evaluate how a test compound modulates Tfh-supported B-cell responses through functional readouts such as antibody secretion, together with cellular phenotyping and complementary cytokine or molecular profiling.
Which cells are used in the Tfh–B-cell co-culture?
The assay uses autologous T follicular helper cells and memory B cells isolated from human tonsil. Tfh cells are characterized as CD4+/CXCR5+, while memory B cells are characterized as CD19+/CD27+. Human tonsil provides a rich source of these interacting immune-cell populations for functional co-culture studies.
Which functional and phenotypic readouts are available?
Secreted IgG provides a primary functional measure of antibody secretion. Flow cytometry immunophenotyping can be incorporated to characterize Tfh and B-cell phenotypes and compound-associated changes in B-cell activation or differentiation. Extended cytokine profiling and complementary proteomic, transcriptomic or signaling analyses can also be integrated according to the study design.
Can the assay assess B-cell differentiation or phenotypic changes?
Yes. Flow cytometry can be incorporated to characterize changes in B-cell phenotype, activation and differentiation alongside the functional response. The markers and cell populations assessed are selected according to the biological question and the expected effect of the test compound.
What can cytokine profiling add to the assay?
Multiplex cytokine profiling can provide an additional functional layer beyond IgG secretion. Depending on the study design, analytes can include IFN-γ, TNF-α, TNF-β, IL-4, IL-6, IL-10, BAFF and APRIL. Profiling B-cell cultures and Tfh–B-cell co-cultures under defined experimental conditions can help characterize cytokine and soluble-factor responses associated with B-cell activation and Tfh–B-cell interactions.
How is antibody secretion measured?
Secreted IgG is quantified in the co-culture supernatant and used as a functional readout of the Tfh-supported B-cell response. Changes in IgG secretion can reveal compound-associated enhancement or suppression of antibody production and are interpreted alongside other cellular or molecular readouts when included.
Which reference controls can be included?
SEB is used to establish an activated Tfh–B-cell co-culture response. Reference immune modulators can include checkpoint inhibitors such as nivolumab, atezolizumab and ipilimumab, as well as the CD40-blocking antibody bleselumab. Appropriate controls are selected according to the study design and biological question.
Can the assay detect both enhancement and suppression?
Yes. In representative data, checkpoint inhibitors increase IgG secretion, whereas CD40 blockade with bleselumab decreases it. Kynurenine also produces a concentration-dependent reduction in IgG secretion. These responses illustrate the assay’s ability to detect both enhancement and suppression and to quantify graded compound effects.
How do you distinguish functional suppression from cytotoxicity?
Appropriate viability controls can be incorporated to determine whether reduced IgG secretion or changes in other functional readouts reflect modulation of the Tfh–B-cell response rather than loss of viable cells. Cellular phenotyping can provide additional context when needed.
How is this assay related to tertiary lymphoid structure (TLS) biology?
Tfh–B-cell interactions are a key component of germinal-center biology and are also relevant to organized immune responses within mature tertiary lymphoid structures. This in vitro assay provides a controlled system for investigating one functional component of TLS-associated biology, but it does not reproduce the full cellular and spatial complexity of a tertiary lymphoid structure.
How is the germinal center reaction assay different from TLS detection and scoring?
The germinal center reaction assay is an in vitro functional Tfh–B-cell co-culture used to investigate compound-associated changes in defined cellular responses. TLS detection and scoring is a separate tissue-based approach that identifies and characterizes tertiary lymphoid structures in patient samples, including their organization and maturity. The two approaches are complementary: one provides functional information, while the other characterizes TLS biology directly in tissue.
Can compounds be tested in combination?
Yes. Test compounds can be evaluated alone or in combination with selected immune modulators or other relevant agents according to the study design. This allows compound-associated effects to be assessed in different immunological contexts, including checkpoint-modulated conditions.
What additional analyses can be integrated?
Studies can be extended with flow cytometry immunophenotyping, broader cytokine profiling, proteomics, transcriptomics and selected signaling analyses. These complementary readouts can help characterize cellular and molecular changes associated with compound response beyond the primary IgG functional endpoint.
