B cells and tertiary lymphoid structures promote immunotherapy response

TLDR: B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders and insights are provided into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Abstract: Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

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Submitted on 29 Jun 2020
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B cells and tertiary lymphoid structures promote
immunotherapy response
Beth A Helmink, Sangeetha M Reddy, Jianjun Gao, Shaojun Zhang, Rafet
Basar, Rohit Thakur, Keren Yizhak, Moshe Sade-Feldman, Jorge Blando,
Guangchun Han, et al.
To cite this version:
Beth A Helmink, Sangeetha M Reddy, Jianjun Gao, Shaojun Zhang, Rafet Basar, et al.. B cells and
tertiary lymphoid structures promote immunotherapy response. Nature, Nature Publishing Group,
2020, 577 (7791), pp.549-555. �10.1038/s41586-019-1922-8�. �hal-02456277�

B Cells and Tertiary Lymphoid Structures Promote Immunotherapy Response
Beth A. Helmink
1
*, MD PhD; Sangeetha M. Reddy
2
*, MD MSci; Jianjun Gao
3
, MD PhD*; Shaojun
Zhang
4
*, PhD.; Rafet Basar
5
, MD PhD; Rohit Thakur
1
, PhD; Keren Yizhak
6
, PhD; Moshe Sade-
Feldman
6,7
, PhD; Jorge Blando
8
, DVM; Guangchun Han
4
; Vancheswaran Gopalakrishnan
1
, PhD;
Yuanxin Xi
10
, PhD; Hao Zhao
8
, PhD; Wenbin Liu
8
; Valerie LeBleu
9
, PhD; Fernanda G.
Kugeratsk
9
, PhD; Hussein A. Tawbi
11
, MD PhD; Rodabe N. Amaria
11
, MD; Sapna Patel
11
, MD;
Michael A. Davies
11
, MD PhD; Patrick Hwu
11
, MD; Jeffrey E. Lee
1
, MD; Jeffrey E. Gershenwald
1
,
MD; Anthony Lucci
1
, MD; Reetakshi Arora
4
, PhD; Scott Woodman
11
, MD PhD; Emily Z. Keung
1
,
MD; Pierre-Olivier Gaudreau
1
, MD; Alexandre Reuben
12
, PhD; Christine N. Spencer
13
, PhD; Alex
P. C ogd i l l
1
,
MEng; Elizabeth M. Burton
1
, MBA; Lauren E. Haydu
1
, PhD; Alexander J. Lazar
4,14,15
,
MD PhD; Roberta Zapassodi
16
, PhD; Courtney W. Hudgens
14
, BS; Deborah A. Ledesma
14
, PhD;
SuFey Ong
17
, PhD; Michael Bailey
17
, PhD; Sarah Warren, PhD; Disha Rao
17
, MS; Oscar
Krijgsman
18
, PhD; Elisa A. Rozeman
18
, MD; Daniel Peeper
18
, PhD; Christian U. Blank
18
, MD
PhD; Ton N. Schumacher
18
, PhD; Lisa H. Butterfield
19
, PhD; Raghu Kalluri
9
, MD PhD; James
Allison
8
, PhD; Florent Petitprez, PhD
20,21,22
; Wolf Herman Fridman, MD PhD
20,21
; Catherine
Sautes-Fridman, PhD
20,21
; Nir Hacohen
6,8
, PhD; MD PhD; Katayoun Rezvani
5,≠
, MD PhD;
Michael T. Tetzlaff
14,15,≠
, Padmanee Sharma
3,8,≠
, MD PhD; Linghua Wang
4,≠
, PhD; Jennifer A.
Wargo
1,4,≠
, MD MMSc.
1: Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center
2: Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center
3: Department of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center
4: Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center
5: Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD
Anderson Cancer Center
6: Department of Medicine, Massachusetts General Hospital Cancer Center
7: Broad Institute of the Massachusetts Institute of Technology

8: Department of Immunology, The University of Texas MD Anderson Cancer Center
9: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center
10: Department of Bioinformatics and Computational Biology, The University of Texas MD
Anderson Cancer Center
11: Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer
Center
12: Department of Thoracic / Head and Neck Medical Oncology, The University of Texas MD
Anderson Cancer Center
13: Parker Institute for Cancer Immunotherapy
14: Department of Pathology, The University of Texas MD Anderson Cancer Center
15: Department of Translational and Molecular Pathology, The University of Texas MD Anderson
Cancer Center
16: Immunology Program, Sloan Ketterin Institute, Memorial Sloan Kettering Cancer Center.
17: Nanostring Technologies, Seattle, WA
18: Division of Molecular Oncology and Immunology, The Netherlands Cancer Institute
19: Departments of Medicine, Surgery, Immunology and Clinical and Translational Science,
University of Pittsburgh
20: INSERM, UMR_S 1138, Cordeliers Research Center, Team Cancer, immune control and
escape, Paris, France
21:
University Paris Descartes Paris 5, Sorbonne Paris Cite, UMR_S 1138, Centre de
Recherche des Cordeliers, Paris, France
22: Programme Cartes d'Identité des Tumeurs, Ligue Nationale Contre le Cancer, Paris, France
*Contributed equally
≠
Shared senior authorship
Corresponding Author:
Jennifer A. Wargo, MD, MMSc
1515 Holcombe Blvd, Unit 1484

Houston, TX 77030
jwargo@mdanderson.org

Intro paragraph:
Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy, and efforts
to better understand therapeutic responses are ongoing. To date, predictive biomarkers
1-10
and
strategies to augment clinical response have largely focused on the T-cell compartment.
However other immune subsets (including B-cells and tertiary lymphoid structures, TLS) may
also contribute to anti-tumor immunity
11-15
, though these are not well-studied in ICB
16
. We
conducted a neoadjuvant ICB trial in melanoma patients and demonstrated that B-cell signatures
were enriched in tumors of responders (R) versus non-responders (NR) via targeted expression
profiling
17
.To build on this, we performed bulk RNA sequencing on these tumor specimens and
demonstrated that markers associated with B-cell development and function were the most
differentially expressed genes in R versus NR. Findings were corroborated using a
computational method to estimate immune and stromal composition of tumor samples (MCP
counter
18
), and were corroborated in another melanoma cohort and in a cohort of renal cell
carcinoma (RCC) patients on ICB. Histologic evaluation was performed in these cohorts,
highlighting localization of B-cells within TLS. Potential functional contributions of B-cells were
assessed via bulk and single-cell RNAseq analysis, demonstrating clonal expansion of B-cells in
responders to ICB and unique transcriptional states associated with response. Mass cytometry
(CyTOF) was performed in tumor and blood samples from our cohort demonstrating an
enrichment of switched memory B-cells and decreased naïve B-cells in tumors of R versus NR
to ICB, suggesting that these intra-tumoral B-cells may actively contribute to the anti-tumor
response following ICB. Together, these data provide novel insight into the potential role of B-
cells and TLS in the response to ICB with implications for the development of biomarkers and
potentially therapeutic targets. Further studies to fully elucidate their role are critically needed
and are currently underway.

Frequently asked questions

Q1. What is the role of B-cells in the immune response to ICB?

B-cells can also secrete an array of cytokines, including TNF-α, IL-2, IL-6 and IFNγ, through which they activate and recruit other immune effector cells, including T-cells. 

Q2. What have the authors contributed in "B cells and tertiary lymphoid structures promote immunotherapy response" ?

HAL this paper is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. 

Q3. What is the role of memory B-cells in the TLS?

Memory B-cells may be acting as antigen-presenting cells, driving the expansion of both memory and naive tumor-associated T-cell responses. 

Q4. What was the process used to evaluate the quality of RNA-seq reads?

RNA-seq FASTQ files were first processed through FastQC (v0.11.5)39, a quality control tool to evaluate the quality of sequencing reads at both the base and read levels. 

Q5. How many l of BSA/PBS was added to the mixture?

100µl of 0.1% BSA/PBS was added to beads + exosomes mixture for a final volume of 145 µl (15 µl of exosomes + 30 µl of Dynabeads® + 100 µl of 0.1% BSA/PBS). 

Q6. How many days did the authors exclude patients with Stage IV disease?

to exclude patients with recurrent Stage III disease, the authors excluded all patients for whom the number of days from the diagnosis of the primary to the accession date was > 90 days. 

Q7. What is the role of B-cells in the TLS?

these B-cells are likely acting in concert with other key immune constituents of the TLS by altering T-cell activation and function as well as through other mechanisms. 

Q8. What is the role of T lymphocytes in the treatment of cancer?

It is clear that cytotoxic T lymphocytes play a dominant role in response to ICB and other forms of immunotherapy; however there is a growing appreciation of other components of the tumor microenvironment that may influence therapeutic response – including myeloid cells and other immune cell subsets11. 

Q9. What was the process of generating the RNA-seq BAM files?

After that, RNA-SeQC (v1.1.8)41 was run on the aligned BAM files to generate a series of RNA-seq related quality control metrics including read counts, coverage, and correlation. 

Q10. How was the UV light used to release the oligos?

ROIs was selectively illuminated with UV light to release the indexing oligos by coupling UV LED light with a double digital mirror device (DDMD) module. 

Q11. How did the authors determine the functional contributions of B-cells?

Potential functional contributions of B-cells were assessed via bulk and single-cell RNAseq analysis, demonstrating clonal expansion of B-cells in responders to ICB and unique transcriptional states associated with response. 

Q12. Who provided technical support on multiplex immunofluorescence?

Thank you to Oscar Contrares for technical support onthe multiplex immunofluorescence and for Miles Andrews for technical support on RNAsequencing library preparation. 

Q13. What was the significance of the B-cell signatures in the melanoma patients?

In these analyses, the authors again observed enrichment of a B-cell signature in R versus NR at baseline and early on-treatment (p=0.036 and 0.038, respectively). 

Q14. What are the main pathways upregulated in Rs as compared to NRs?

Pathways upregulated in Rs as compared to NRs include those consistent with increased immune activity including CXCR4 signaling, cytokine receptor interaction and chemokine signaling pathways (Extended Data Fig. 15a and Extended Data Table 10). 

Q15. What is the role of B-cells in anti-tumor immunity?

In these studies, the authors identified significantly increased clonal counts for both immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgL) (p=0.001 and p=0.004, respectively) and increased BCR diversity in Rs as compared to NRs (p=0.002 and p=0.0008) suggesting an active role for B-cells in anti-tumor immunity (Fig. 3a, Extended DataFig. 12-13). 

Q16. What was the FPKM expression matrix used to produce?

The R package software MCP-counter18 was applied to the normalized log2-transformed FPKM expression matrix to produce the absolute abundance scores for 8 major immune cell types (CD3+ 

Q17. What is the significance of the B-cell signatures in the analysis of the tumor?

B-cell signatures alone were predictive of response in univariable analyses (OR 2.6, p=0.02 for their trial, and OR 2.9, p=0.03 for combined melanoma cohorts), but not in multivariable analyses when considering other components of the immune cell infiltrate, suggesting that B-cells are likely acting in concert with other immune subsets and not acting in isolation; however these analyses were limited due to the low sample size (Extended Data Table 4 and 5).