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In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of 5 healthy donors and 13 patients with COVID-19, including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in patients with COVID-19 showed a strong interferon-α response and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4 + effector-GNLY (granulysin), CD8 + effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during disease progression. Severe COVID-19 is characterized—among other things—by a hyperinflammatory state. Wang and colleagues describe the single-cell transcriptional landscape of moderate, severe and convalescent cases of patients with COVID-19.

Long-lived tissue-resident memory T cells (T RM cells) confer fast, robust protection after pathogen rechallenge. Gebhardt and colleagues show that skin T RM cells arise from KLRG1 – cells that differentiate in situ in response to IL-15 and TGF-β. Tissue-resident memory T cells (T RM cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103 + CD8 + T RM cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-β (TGF-β) was required for the formation of these long-lived memory cells. Notably, differentiation into T RM cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.

The authors use tiled CRISPR activation for functional enhancer discovery across two autoimmunity risk loci, CD69 and IL2RA , and identify elements with features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours a fine-mapped autoimmunity risk variant. CRISPRa mapping of enhancer functions Enhancers are gene regulatory elements that shape cell-type-specific transcriptional programs and responses to specific extracellular cues. Mapping enhancer function is challenging because of our limited understanding of the cellular context in which each enhancer contributes to gene regulation. Here, Alexander Marson and colleagues use a tiled CRISPR activation (CRISPRa) approach for functional enhancer discovery across two autoimmunity risk loci: CD69 and IL2RA. They identify several elements with features of stimulus-responsive enhancers, including an IL2RA enhancer that contains an autoimmunity risk variant. This approach should be useful for discovering functional enhancers without prior knowledge of their specific biological context. The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues 1 , 2 , 3 . Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption 4 , 5 , 6 , but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) 7 to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA . We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T H 17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Jameson and colleagues show that the establishment of resident memory CD8 + T cells in nonlymphoid tissues requires transcriptional downregulation of the trafficking molecule S1P 1 , mediated by induced loss of the transcription factor KLF2. Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T RM cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T RM cells is unknown. We found that CD8 + T RM cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P 1 , a receptor for sphingosine 1-phosphate). Forced expression of S1P 1 prevented the establishment of T RM cells. Cytokines that induced a T RM cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P 1 provides a switch that dictates whether CD8 + T cells commit to recirculating or tissue-resident memory populations.

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

B cell depletion via anti-CD20 antibodies is a highly effective treatment for multiple sclerosis (MS). However, little is known about the maturation/activation stage of the returning B cell population after treatment cessation and the wider effects on other immune cells. In the present study, 15 relapsing-remitting MS patients receiving 1,000 mg of rituximab were included. B, T, and myeloid cells were analyzed before anti-CD20 administration and in different time intervals thereafter over a period of 24 mo. In comparison to the phenotype before anti-CD20 treatment, the reappearing B cell pool revealed a less mature and more activated phenotype: 1) reappearing B cells were significantly enriched in transitional (before: 10.1 ± 1.9%, after: 58.8 ± 5.2%) and mature naive phenotypes (before: 45.5 ± 3.1%, after: 25.1 ± 3.5%); 2) the frequency of memory B cells was reduced (before: 36.7 ± 3.1%, after: 8.9 ± 1.7%); and 3) reappearing B cells showed an enhanced expression of activation markers CD25 (before: 2.1 ± 0.4%, after: 9.3 ± 2.1%) and CD69 (before: 5.9 ± 1.0%, after: 21.4 ± 3.0%), and expressed significantly higher levels of costimulatory CD40 and CD86. T cells showed 1) a persistent increase in naive (CD4⁺: before: 11.8 ± 1.3%, after: 18.4 ± 3.4%; CD8⁺: before: 12.5 ± 1.4%, after: 16.5 ± 2.3%) and 2) a decrease in terminally differentiated subsets (CD4⁺: before: 47.3 ± 3.2%, after: 34.4 ± 3.7%; CD8⁺: before: 53.7 ± 2.1%, after: 49.1 ± 2.7%).

B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers. Antigen escape represents a potential drawback of chimeric antigen receptor T cell (CAR-T) therapy targeting a single tumor-associated antigen. To reduce the risk of antigen escape, here the authors report the design and characterization of a BAFF ligand CAR-T that can recognize three different receptors (BAFF-R, BCMA and TACI), demonstrating in vitro and in vivo cytotoxicity against multiple B cell cancer models.

The costimulatory molecule ICOS is important for the development of both interleukin 17–producing and follicular T helper cells. Kuchroo and colleagues find that ICOS induces the transcription factor c-Maf, which regulates the population expansion of both helper cell types. The inducible costimulatory molecule ICOS has been suggested to be important in the development of interleukin 17 (IL-17)-producing helper T cells (T H -17 cells) and of follicular helper T cells (T FH cells). Here we show that ICOS-deficient mice had no defect in T H -17 differentiation but had fewer T H -17 cells after IL-23 stimulation and fewer T FH cells. We also show that T FH cells produced IL-17 and that T FH cells in ICOS-deficient mice were defective in IL-17 production. Both T H -17 and T FH cells had higher expression of the transcription factor c-Maf. Genetic loss of c-Maf resulted in a defect in IL-21 production and fewer T H -17 and T FH cells. Thus our data suggest that ICOS-induced c-Maf regulates IL-21 production that in turn regulates the expansion of T H -17 and T FH cells.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8 + T cells. For unbiased assessment of cellular immunopathogenesis, here we perform single-cell (sc) transcriptome, surface proteome, and T cell receptor (TCR) sequencing on unaffected skin, affected skin, and blister fluid from 15 SJS/TEN patients. From 109,888 cells, we identify 15 scRNA-defined subsets. Keratinocytes express markers indicating HLA class I-restricted antigen presentation and appear to trigger the proliferation of and killing by cytotoxic CD8 + tissue-resident T cells that express granulysin, granzyme B, perforin, LAG3 , CD27 , and LINC01871 , and signal through the PKM , MIF , TGFβ , and JAK-STAT pathways. In affected tissue, cytotoxic CD8 + T cells express private expanded and unexpanded TCRαβ that are absent or unexpanded in unaffected skin, and mixed populations of macrophages and fibroblasts express pro-inflammatory markers or those favoring repair. This data identifies putative cytotoxic TCRs and therapeutic targets. Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening severe cutaneous drug reaction mediated by CD8 + T cells. Here the authors characterise the immune response in skin samples at the site of tissue damage from patients affected with SJS/TEN and compare this to healthy skin or blister fluid and find populations of CD8 + T cell clonotypes expressing cytotoxic mediator molecules.

Memory T-helper (Th) lymphocytes are crucial for the maintenance of acquired immunity to eliminate infectious pathogens. We have previously demonstrated that most memory Th lymphocytes reside and rest on stromal niches of the bone marrow (BM). Little is known, however, regarding the molecular basis for the generation and maintenance of BM memory Th lymphocytes. Here we show that CD69-deficient effector CD4 T lymphocytes fail to relocate into and persist in the BM and therefore to differentiate into memory cells. Consequently, CD69-deficient CD4 T cells fail to facilitate the production of high-affinity antibodies and the generation of BM long-lived plasma cells in the late phase of immune responses. Thus, CD69 is critical for the generation and maintenance of professional memory Th lymphocytes, which can efficiently help humoral immunity in the late phase. The deficit of immunological memory in CD69-deficient mice also highlights the essential role of BM for the establishment of Th memory.