. 2022 Apr 15;376(6590):eabi9591.

doi: 10.1126/science.abi9591. Epub 2022 Apr 15.

KIR⁺CD8⁺ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

Jing Li¹, Maxim Zaslavsky², Yapeng Su³, Jing Guo⁴, Michael J Sikora⁵, Vincent van Unen¹, Asbjørn Christophersen^{6

7

8}, Shin-Heng Chiou¹, Liang Chen¹, Jiefu Li⁹, Xuhuai Ji¹⁰, Julie Wilhelmy¹, Alana M McSween¹, Brad A Palanski¹¹, Venkata Vamsee Aditya Mallajosyula¹, Nathan A Bracey¹, Gopal Krishna R Dhondalay¹², Kartik Bhamidipati¹³, Joy Pai¹³, Lucas B Kipp¹⁴, Jeffrey E Dunn¹⁴, Stephen L Hauser¹⁵, Jorge R Oksenberg¹⁵, Ansuman T Satpathy¹⁶, William H Robinson^{17

18}, Cornelia L Dekker¹⁹, Lars M Steinmetz^{5

20

21}, Chaitan Khosla^{11

22

23}, Paul J Utz^{1

18}, Ludvig M Sollid^{6

7

8

24}, Yueh-Hsiu Chien^{1

4}, James R Heath^{3

25}, Nielsen Q Fernandez-Becker²⁶, Kari C Nadeau^{1

12}, Naresha Saligrama¹, Mark M Davis^{1

9

4}

Affiliations

¹ Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
² Program in Computer Science, Stanford University, Stanford, CA, USA.
³ Institute for Systems Biology, Seattle, WA, USA.
⁴ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
⁶ K.G. Jebsen Coeliac Disease Research Centre, University of Oslo, Oslo, Norway.
⁷ Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
⁸ Department of Immunology, University of Oslo, Oslo, Norway.
⁹ The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Human Immune Monitoring Center, Stanford University School of Medicine, Stanford, CA, USA.
¹¹ Department of Chemistry, Stanford University, Stanford, CA, USA.
¹² Sean N. Parker Center for Allergy and Asthma Research, Department of Medicine, Stanford University, Stanford, CA, USA.
¹³ Program in Immunology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁴ Division of Neuroimmunology, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
¹⁵ Department of Neurology and UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA, USA.
¹⁶ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁷ VA Palo Alto Health Care System, Palo Alto, CA, USA.
¹⁸ Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁹ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
²⁰ Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
²¹ European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
²² Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
²³ Stanford ChEM-H, Stanford University, Stanford, CA, USA.
²⁴ Department of Immunology, Oslo University Hospital, Oslo, Norway.
²⁵ Department of Bioengineering, University of Washington, Seattle, WA, USA.
²⁶ Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University, Stanford, CA, USA.

PMID: 35258337
PMCID: PMC8995031
DOI: 10.1126/science.abi9591

KIR⁺CD8⁺ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

Jing Li et al. Science. 2022.

. 2022 Apr 15;376(6590):eabi9591.

doi: 10.1126/science.abi9591. Epub 2022 Apr 15.

Authors

Affiliations

¹ Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
² Program in Computer Science, Stanford University, Stanford, CA, USA.
³ Institute for Systems Biology, Seattle, WA, USA.
⁴ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
⁶ K.G. Jebsen Coeliac Disease Research Centre, University of Oslo, Oslo, Norway.
⁷ Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
⁸ Department of Immunology, University of Oslo, Oslo, Norway.
⁹ The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Human Immune Monitoring Center, Stanford University School of Medicine, Stanford, CA, USA.
¹¹ Department of Chemistry, Stanford University, Stanford, CA, USA.
¹² Sean N. Parker Center for Allergy and Asthma Research, Department of Medicine, Stanford University, Stanford, CA, USA.
¹³ Program in Immunology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁴ Division of Neuroimmunology, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
¹⁵ Department of Neurology and UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA, USA.
¹⁶ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁷ VA Palo Alto Health Care System, Palo Alto, CA, USA.
¹⁸ Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁹ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
²⁰ Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
²¹ European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
²² Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
²³ Stanford ChEM-H, Stanford University, Stanford, CA, USA.
²⁴ Department of Immunology, Oslo University Hospital, Oslo, Norway.
²⁵ Department of Bioengineering, University of Washington, Seattle, WA, USA.
²⁶ Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University, Stanford, CA, USA.

PMID: 35258337
PMCID: PMC8995031
DOI: 10.1126/science.abi9591

Abstract

In this work, we find that CD8⁺ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49⁺CD8⁺ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8⁺ T cells efficiently eliminated pathogenic gliadin-specific CD4⁺ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR⁺CD8⁺ T cells, but not CD4⁺ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49⁺CD8⁺ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8⁺ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

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Figures

None — **The proposed role of CD8⁺ regulatory T cells in peripheral tolerance.**
KIR⁺CD8⁺ T cells are the equivalent of mouse Ly49⁺CD8⁺ T cells in humans, with similar regulatory functions. During an infection, this kind of CD8⁺ regulatory T cell is induced and suppresses those CD4⁺ T cells with a strong reactivity to self, which may cause autoimmunity without interfering with the immune responses against pathogens.

**Fig. 1.. Increased KIR⁺CD8⁺ T cells in patients with autoimmune diseases.**
(A) Representative contour plots and a summary histogram showing the frequency of KIR⁺CD8⁺ T cells in the peripheral blood of HCs (N = 16) and patients with SLE (N = 22), MS (N = 10), or CeD (N = 21) analyzed by flow cytometry. KIR⁺ cells were detected by PE-conjugated antibodies against KIR2DL1 (clone no. 143211), KIR2DL2/L3 (Dx27), KIR2DL5 (UP-R1), KIR3DL1 (Dx9), and KIR3DL2 (clone no. 539304). *P < 0.05; **P < 0.01; Kruskal-Wallis test followed by multiple comparisons test. (B) Representative contour plots and a summary histogram showing the frequency of KIR⁺CD8⁺ T cells in the duodenum of normal controls (N = 4), CeD in remission (N = 5), and active CeD (N = 5) analyzed by flow cytometry. *P < 0.05; ***P < 0.001; Kruskal-Wallis test followed by multiple comparisons test. (C) Expression of *KIR* transcripts (*KIR3DL1*, *KIR2DL3*, and *KIR2DL2*) in CD8⁺ T cells from healthy kidneys (control, N = 6) versus SLE nephritis kidneys (N = 20). (D) Expression of *KIR* transcripts (*KIR3DL1*, *KIR2DL3*, and *KIR2DL2*) in synovial CD8⁺ T cells and expression of *FOXP3* in synovial CD4⁺ T cells from RA (N = 18) and OA (N = 3).

**Fig. 2.. Elimination of gliadin-specific CD4⁺ T cells by KIR⁺CD8⁺ T cells.**
(A) Representative contour plots showing tetramer-bound CD4⁺ T cells before and after enrichment and summary of the number of gliadin-specific CD4⁺ T cells per 1 × 10⁶ CD4⁺ T cells on day 6. Experiments were repeated using PBMCs from 11 CeD patients. **P < 0.01; ***P < 0.001; Friedman test followed by multiple comparisons test. Unstim., unstimulated. (B) Representative contour plots and a summary graph displaying annexin V binding of gliadin-specific CD4⁺ T cells from the culture harvested on day 3 (N = 6). *P < 0.05; **P < 0.01; Friedman test followed by multiple comparisons test. (C) Frequency of gliadin-specific CD4⁺ T cells from the cell cultures in the presence or absence of KIR^– or KIR⁺CD8⁺ T cells or with KIR⁺CD8⁺ T cells separated by a 4-μm insert in a transwell plate (N = 6). *P < 0.05; **P < 0.01; Friedman test followed by multiple comparisons test. (D) Frequency of gliadin-specific CD4⁺ T cells from the PBMC cultures in the presence or absence of KIR^– or KIR⁺CD8⁺ T cells with or without preactivation (N = 8). **P < 0.01; ***P < 0.001; Friedman test followed by multiple comparisons test. (E) Frequency of gliadin-specific CD4⁺ T cells from the PBMC cultures in the presence or absence of preactivated KIR^– or KIR⁺CD8⁺ T cells with isotype control, anti–HLA-ABC, or anti–HLA-E blockade (N = 9). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Friedman test followed by multiple comparisons test.

**Fig. 3.. Increased KIR⁺CD8⁺ T cells in infectious diseases.**
(A) Representative contour plots and summary scatter plots showing the percentage of KIR⁺ cells in CD8⁺ T cells from the blood of 17 HCs and 53 COVID-19 patients with varying disease severity (mild, N = 23; moderate, N = 17; severe, N = 13). ****P < 0.0001; Mann-Whitney test (left). **P < 0.01; ***P < 0.001; ****P < 0.0001; Kruskal-Wallis test followed by multiple comparisons test (right). (B) Frequency of KIR⁺CD8⁺ T cells, CD4⁺ T_regs (CD25^hiCD127^lo), and KIR⁺ NK cells in the blood of COVID-19 patients with or without vasculitis. **P < 0.01; Mann-Whitney test. (C) Frequency of CD8⁺ T cells expressing *KIR* transcripts (*KIR3DL1*, *KIR3DL2*, *KIR2DL3*, or *KIR2DL1*) in the bronchoalveolar lavage fluid of HCs (N = 4) versus COVID-19 patients (N = 9) (left; **P < 0.01; Mann-Whitney test) and HCs versus COVID-19 patients with moderate (N = 3) or severe (N = 6) disease (right; *P < 0.05; Kruskal-Wallis test followed by multiple comparisons test). (D) Frequency of KIR⁺CD8⁺ T cells and CD4⁺ T_regs (CD25^hiCD127^low) in the blood of HCs (N = 17) versus influenza-infected patients (N = 7). ***P < 0.01; Mann-Whitney test.

**Fig. 4.. scRNA-seq analysis of KIR⁺CD8⁺ T cells in the blood.**
(A and B) scRNA-seq analysis of total CD8⁺ T cells from the blood of HCs (N = 10), MS patients (N = 6), and COVID-19 patients (N = 25) by 10X Genomics. (A) UMAP plot of the eight subpopulations identified by unsupervised clustering. (B) UMAP plots showing the distribution of KIR⁺CD8⁺ T cells (expressing *KIR3DL1*, *KIR3DL2*, *KIR2DL1*, or *KIR2DL3* transcripts) and KIR^–CD8⁺ T cells from HCs, MS patients, and COVID-19 patients. (C to F) KIR⁺CD8⁺ T cells in the blood of HCs (N = 10) and patients with MS (N = 2), SLE (N = 6), and CeD (N = 5) were sorted for scRNA-seq using the Smart-seq2 protocol and analyzed using the R package “Seurat.” (C) UMAP plots showing KIR⁺CD8⁺ T cells segregated into six clusters (top) and the distribution of expanded (≥2 cells expressing same TCR) and unexpanded (cells expressing unique TCRs) cells (bottom). (D) UMAP plots of KIR⁺CD8⁺ T cells from MS, SLE, and CeD patients and HCs are shown, with expanded and unexpanded cells annotated with different colors (expanded, red; unexpanded, blue; other diseases, gray). (E) Cluster compositions of expanded KIR⁺CD8⁺ T cells from each individual. (F) Heatmap showing expression of the top 10 genes differentially expressed in each cluster, with the categories of each group of genes annotated on the left.

**Fig. 5.. Analysis of TCR sequences of KIR⁺CD8⁺ T cells.**
(A and B) Summary histograms showing Shannon-Wiener indices (A) and Chao estimates (B) of TCRs of KIR^– versus KIR⁺CD8⁺ T cells from 26 subjects, including 11 healthy donors, two MS, five SLE, three CeD, and five T1D patients as evaluated by VDJtools. ****P < 0.0001; Wilcoxon matched-pairs signed-rank test.

**Fig. 6.. Exacerbated autoimmunity in *Klra6*^creDTA mice after viral infection.**
(A) Frequency of Ly49⁺CD8⁺ T cells in the blood of DTA mice (N = 8) and *Klra6*^creDTA mice (N = 5) 0, 5, 8, and 12 days after LCMV-Armstrong infection. **P < 0.01; ***P < 0.001; repeated measures (RM) two-way analysis of variance (ANOVA) followed by multiple comparisons test. p.i., postinfection. (B) Representative contour plots and summarized scatter plots displaying frequency and absolute number of PD1⁺CXCR5⁺CD4⁺ T cells (T_FH) and CD38^–CD95⁺ GC B cells in the spleen of DTA mice (N = 8) and *Klra6*^creDTA mice (N = 5) 30 days after LCMV-Armstrong infection. *P < 0.05; **P < 0.01; Mann-Whitney test. Tfh, T follicular helper cell. (C) Representative kidney pathology of day-30 LCMV-infected DTA mice and *Klra6*^creDTA mice assessed by PAS staining (630X; scale bars, 20 μm). (D) IgG deposition in glomeruli of the kidneys of DTA mice (N = 8) and *Klra6*^creDTA mice (N = 5) 30 days after LCMV infection accessed by immunofluorescence staining (400X; scale bars, 20 μm) and quantified by ImageJ. *P < 0.05; Mann-Whitney test. DAPI, 4′,6-diamidino-2-phenylindole. (E) Frequency of Ly49⁺CD8⁺ T cells in the blood of DTA mice (N = 6) and *Klra6*^creDTA mice (N = 5) after influenza A-PR8 infection. *P < 0.05; **P < 0.01; ****P < 0.0001; RM two-way ANOVA followed by multiple comparisons test. (F) Microscopy of representative H&E staining of lung sections from DTA mice and *Klra6*^creDTA mice 60 days after influenza infection (top, 20X; scale bars, 2 mm) (bottom, 630X; scale bars, 50 μm). Representative data from two independent experiments are shown. The means ± SEMs are indicated.

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Update of

Human KIR ⁺ CD8 ⁺ T cells target pathogenic T cells in Celiac disease and are active in autoimmune diseases and COVID-19.
Li J, Zaslavsky M, Su Y, Sikora MJ, van Unen V, Christophersen A, Chiou SH, Chen L, Li J, Ji X, Wilhelmy J, McSween AM, Palanski BA, Aditya Mallajosyula VV, Dhondalay GKR, Bhamidipati K, Pai J, Kipp LB, Dunn JE, Hauser SL, Oksenberg JR, Satpathy AT, Robinson WH, Steinmetz LM, Khosla C, Utz PJ, Sollid LM, Heath JR, Fernandez-Becker NQ, Nadeau KC, Saligrama N, Davis MM. Li J, et al. bioRxiv [Preprint]. 2021 Dec 25:2021.12.23.473930. doi: 10.1101/2021.12.23.473930. bioRxiv. 2021. Update in: Science. 2022 Apr 15;376(6590):eabi9591. doi: 10.1126/science.abi9591. PMID: 34981055 Free PMC article. Updated. Preprint.

Comment in

Regulatory CD8⁺ T cells suppress disease.
Levescot A, Cerf-Bensussan N. Levescot A, et al. Science. 2022 Apr 15;376(6590):243-244. doi: 10.1126/science.abp8243. Epub 2022 Apr 14. Science. 2022. PMID: 35420956
CD8⁺ Tregs kill pathogenic cells to avert autoimmunity.
Hu D, Murugaiyan G. Hu D, et al. Trends Immunol. 2022 Jun;43(6):415-416. doi: 10.1016/j.it.2022.04.006. Epub 2022 May 5. Trends Immunol. 2022. PMID: 35527183
KIRs mark killers suppressing autoimmunity.
Koh JY, Shin EC. Koh JY, et al. Immunity. 2022 May 10;55(5):735-737. doi: 10.1016/j.immuni.2022.04.014. Immunity. 2022. PMID: 35545025

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KIR⁺CD8⁺ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

Affiliations

KIR⁺CD8⁺ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

Authors

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Abstract

Figures

Update of

Comment in

References

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