CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.


Identification and quantification of TLTs
In the present study, we defined TLTs as organized lymphocyte clusters exhibiting the signs of proliferation, as previously described (1). Identification and diagnosis of TLTs in the renal cortex was based on the unique perivascular localization of mononuclear cell aggregates by light microscopy and the presence of lymphocyte aggregates with Ki67 signals inside by immunofluorescence. Determination of TLT stages in the renal cortex was examined with immunofluorescence of (i) CD3e and CD20 and (ii) Ki67 and CD21 in two serial sections for each mouse and human, and was assessed by an experienced renal pathologist. TLTs containing FDC were defined as advanced-stage TLTs. The number of TLTs was shown as the number per one section.

Quantification of TLT size
The renal TLT sizes were examined in the PAS-stained sections of injured kidneys as described previously (2). TLT size was defined as the total cumulative size of the TLTs in the renal cortex of the sample. Pictures that included TLTs were taken with the same size and resolution, and TLT size was measured by an experienced renal pathologist using Adobe Photoshop software.

Renal histochemistry
Mouse kidneys were harvested, cut along the short axis at the maximum area of the whole kidney, fixed in Carnoy's solution, embedded in paraffin, sectioned (2.0 µm thickness), and then stained with periodic acid-Schiff (PAS) or Masson's trichrome (MTC).

Renal immunofluorescence
For immunofluorescence studies of mouse kidneys, kidneys were fixed in 4% paraformaldehyde (PFA), incubated in 20% sucrose for 6 h, and then incubated in 30% sucrose in PBS at 4°C overnight. Optimal cutting temperature compound (OCT)-embedded (Sakura Finetek) kidneys were cryosectioned into 6.0 µm sections and mounted on Superfrost slides (Matsunami Glass).
All staining samples were visualized using the appropriate secondary antibodies (1/300), counterstained with DAPI, and analyzed using a confocal microscope (FV1000D; Olympus).

Renal immunohistochemistry
Human kidneys were fixed in formalin, embedded in paraffin, and sectioned (3.0 µm thickness).
For immunohistochemistry, paraffin-embedded tissue sections were rehydrated, and endogenous peroxidase was blocked using 3% H2O2. Antigen retrieval was performed with trypsin antigenretrieval solution (catalog ab970; Abcam), and then tissue sections were stained with anti-CD153 primary antibody (catalog PAB26949; Abnova). Antibody labeling was performed with a DAB reagent (Vector Laboratories). Sections were counterstained with hematoxylin.

RNA in situ hybridization (RNA-ISH) of Tnfsf8 and Tnfrsf8
Detection of mouse Tnfsf8 and Tnfrsf8 RNA was performed on cryosections described above and

Soluble CD30 concentration in plasma
The levels of sCD30 were measured in murine plasma specimens with an enzyme-linked immunosorbent assay (ELISA) kit (catalog DY852; R&D Systems) according to the manufacturer's instructions.

Bone marrow transplantation experiments
Bone marrow transplantation experiments were performed as previously described (3). Briefly, bone marrow cells obtained from donor mice were washed with cold PBS and injected intravenously (3.0 x 10 6 cells) into 4 Gy-irradiated 10-month-old male Rag2 knockout mice. After 6 weeks, the mice were subjected to unilateral renal IRI.

Quantitative analysis for renal fibrosis and tubular injury
Fibrosis score was quantified as aSMA-positive area (pixel) in the renal interstitial space as described previously (1). Briefly, six images of each kidney section at the cortical and corticomedullary field, except for the TLT area, were taken. All images were obtained with the same laser power and gain intensity using a confocal microscope (FV1000D; Olympus). aSMApositive areas, except for vascular smooth muscle cells, were automatically calculated by Adobe Photoshop software. Tubular injury scores were examined in PAS-stained sections of aged kidneys 28 days after the initiation of adenine feeding. Tubular injury scores were graded by an experienced renal pathologist blinded to the sample information using a semi-quantitative scale.

Real-time RT-PCR analysis
RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR) were performed as described previously (2). The primer sequences are listed in Table S1. Expression levels were normalized to those of Gapdh and expressed relative to the levels in aged wild-type kidneys on day 0 (IRI) ( Figure 1D), in aged CD153 -/and CD30 -/mouse kidneys subject to IRI on day 45 ( Figure 6B and Figure 7B, respectively), and in aged CD153 -/mouse kidneys subject to adenine nephropathy on day 28 ( Figure 7I).

TCR and BCR repertoire analysis
CD4 + T cell populations and B cell populations were isolated from single-cell suspensions of the spleens and kidneys of two 12-month-old male C57BL/6J mice 45 days after unilateral IRI inductions using FACS Aria II (BD Bioscience). Total RNA was extracted from CD4 + T cells and B cells using a RNeasy Mini Kit (Qiagen) following the manufacturer's recommended protocol. Next-generation sequencing was performed with unbiased T cell receptor (TCR)/ B cell receptor (BCR) repertoire analysis technology (Repertoire Genesis Inc.). Unbiased adaptorligation PCR was performed as described previously (4,5). In brief, total RNA was converted to cDNA with Superscript III reverse transcriptase (Thermo Fisher Scientific). Subsequently, double-strand cDNA (ds-cDNA) was synthesized, and an adaptor was ligated to the 5' end of the ds-cDNA and then cut with Sph I restriction enzyme. For TCR analysis, PCR was performed using P20EA adaptor primer and TCRa-chain constant region-specific (mCA1) or TCRab-chain constant region-specific (mCB1) primers. The second PCR was performed using P20EA and either mCA2 or mCB2 with the same PCR conditions. For BCR analysis, P20EA and IgM constant region-specific (mCM1) or IgG constant region-specific (mCG1) primers were used for the first PCR and then mCM2 or mCG2 primer was used for the second PCR. After Tag PCR amplification, index sequences were added by amplification with a Nextera XT index kit v2 setA (Illumina). Sequencing was performed with the Illumina Miseq paired-end platform (2 × 300 bp).
Data processing, assignment, and data aggregation were performed with repertoire analysis software. TCR and BCR sequences were assigned with the International ImMunoGeneTics (IMGT) reference sequences (http://www.imgt.org). Nucleotide sequences of complementaritydetermining region 3 (CDR3) ranged from conserved cysteine at position 104 (Cys104) of the IMGT nomenclature to conserved phenylalanine at position 118 (Phe118) and the following glycine (Gly119) was translated into amino acid sequences, and the identical V (TCR chain V (TRV) or immunoglobulin heavy chain V (IGHV)), J (TRJ or IGH), and deduced amino acid sequence of the CDR3 were defined as a unique sequence read (USR). The copy number of USRs was automatically counted by RG software. TCR and BCR diversity analysis was performed according to the method described previously (6, 7). The Morisita-Horn similarity index was calculated by 'vegan' package version 2.5-6, and the Venn diagram was depicted by VennDiagram package version 1.6.20 with R version 4.0.2. TCR and BCR sequences are listed in Data file S1.

Reanalysis of published human RNA-seq data
Normalized RNA-seq data available in the National Center for Biotechnology Information's Gene Expression Omnibus (accession number GSE110999) were used and further analyzed in R.
Detailed methods of data normalization were described in previously published articles (8).

Reanalysis of published human scRNA-seq data
scRNA-seq data of joint synovial tissues from patients with RA (9), which is a data from the Accelerating Medicines Partnership project, were preprocessed by Cincinnati Children's Hospital Medical Center's ToppCell project, and a normalized data matrix in logTPM was obtained. Pooled samples from 18 rheumatoid arthritis patients (3 leukocyte poor RA: mean age 64.2 years old and 15 leukocyte-rich RA: mean age was 57.3 years old) and 3 osteoarthritis patients (mean age 71years old) were used in analysis (9).

Analysis of human kidney specimens
All human specimens were procured and analyzed after obtaining informed consent and with the approval of the ethics committee at RWTH University of Aachen Hospitals (Aachen, Germany) and Kyoto University Hospital (Kyoto, Japan). Kidneys from patients with chronic pyelonephritis who underwent nephrectomy at RWTH University of Aachen Hospitals were analyzed (1).

Supplemental Figure 1. Histological analysis of wild-type aged kidneys subject to ischemic reperfusion injury (IRI).
Representative images of aged kidneys 4, 24, 60 days after 45min IRI by PAS staining. Yellow arrow heads indicate the localization of tertiary lymphoid tissues (TLTs). Scale bars: 300 µm. Representative gating strategy of SAT cells from aged injured kidneys 45 days after IRI induction.

Supplemental
Lymphocytes were gated according to their FSC-A/SSC-A profile to exclude debris, and then doublets were also excluded by the FSC-W/FSC-H profile. Subsequently, single cells were gated on live cells and then gated on CD45 + cells. To define SAT cells, CD45 + cells were gated on CD3e + CD44 high , followed by CD4 + populations, and then PD1 + CD153 + T cells were plotted.