[Characteristics and courses of COVID-19 in patients from the first through fifth waves of the disease: Focused on a single public health center in Tokyo, Japan].

Objective　Coronavirus disease 2019 (COVID-19) became a global public health threat, and local public health centers in Japan implemented an infectious disease response to support patients. The response was subsequently modified to meet the needs for each of the five waves of infection. The study aim was to analyze the characteristics and courses of the disease in patients with COVID-19 at a single public health center. The study period included the first through fifth waves of the disease.Methods　We utilized a descriptive epidemiological design in this study and data of patients with COVID-19 from one administrative district in Tokyo, Japan. We analyzed age, gender, nationality, symptoms at diagnosis, the route of infection, the recovery environment, and associated morbidity intervals, including case fatality rate, days from symptom onset to diagnosis, days from diagnosis to hospitalization, and recovery time for each of the first through fifth waves.Results　From February 2020 to November 2021, 11,252 patients were diagnosed with COVID-19. Specifically, in the first wave, 151 patients were diagnosed, followed by 803 in the second wave, 2,406 in the third wave, 1,480 in the fourth wave, and 6,412 in the fifth wave. Hospitalization was the primary recovery environment during the first wave, while home recovery became the primary approach from the third wave onward. The case fatality rate was highest during the first wave, likely because of limited testing and treatment options for severe cases. The median time from onset to diagnosis was seven days in the first wave, significantly longer than for the other waves. The median time from diagnosis to hospitalization was one day in the first through fourth waves but three days in the fifth wave. The extension of this interval suggests that hospitalization was delayed in this wave as the number of severe patients increased rapidly, likely because of the novel COVID-19 variant.Conclusion　This study of patients testing positive for COVID-19 provides valuable insights into the characteristics and courses of the pandemic within this district. These findings can inform regarding the development of effective strategies to manage the ongoing COVID-19 pandemic and other future emerging infectious diseases.

Themis2 lowers the threshold for B cell activation during positive selection.

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.

Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function.

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.

Themis, a T cell-specific protein important for late thymocyte development.

During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell-specific protein, Themis, that serves a distinct function during this stage. In Themis(-/-) mice, thymocyte selection was impaired and the number of transitional CD4(+)CD8(int) thymocytes as well as CD4(+) or CD8(+) single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis(-/-) CD4(+)CD8(int) thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.

Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.

Thymus seeding by T-lymphoid progenitor cells is a prerequisite for T-cell development. However, molecules guiding thymus colonization and their roles before and after thymus vascularization are unclear. Here we show that mice doubly deficient for chemokine receptors CCR7 and CCR9 were defective specifically in fetal thymus colonization before, but not after, thymus vascularization. The defective prevascular fetal thymus colonization was followed by selective loss of the first wave of T-cell development generating epidermal Vgamma3(+) gammadelta T cells. Unexpectedly, CCL21, a CCR7 ligand, was expressed not by Foxn1-dependent thymic primordium but by Gcm2-dependent parathyroid primordium, whereas CCL25, a CCR9 ligand, was predominantly expressed by Foxn1-dependent thymic primordium, revealing the role of the adjacent parathyroid in guiding fetal thymus colonization. These results indicate coordination between Gcm2-dependent parathyroid and Foxn1-dependent thymic primordia in establishing CCL21/CCR7- and CCL25/CCR9-mediated chemokine guidance essential for prevascular fetal thymus colonization.

Premature expression of chemokine receptor CCR9 impairs T cell development.

During thymocyte development, CCR9 is expressed on late CD4-CD8- (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.

Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent mu to gamma1 class switch recombination in CD38-activated B cells.

Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to mu to gamma1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce mu to gamma1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces mu to gamma1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce mu to gamma1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce mu to gamma1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR.

A role for CCR9 in T lymphocyte development and migration.

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic alphabeta-T cell development, and thymocyte selection appeared unimpaired in adult CCR9-deficient (CCR9(-/-)) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9(-/-) mice was less efficient at repopulating the thymus of lethally irradiated Rag-1(-/-) mice than bone marrow from littermate CCR9(+/+) mice. CCR9(-/-) mice had increased numbers of peripheral gammadelta-T cells but reduced numbers of gammadeltaTCR(+) and CD8alphabeta(+)alphabetaTCR(+) intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating the development and/or migration of both alphabeta(-) and gammadelta(-) T lymphocytes.

Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69+ thymocytes and gammadeltaTCR+ thymocytes preferentially respond to CCL25.

CCR9 mediates chemotaxis of thymocytes in response to CCL25/thymus-expressed chemokine, and its mRNA is selectively expressed in thymus and small intestine, the two known sites of T lymphopoiesis. To examine the expression of CCR9 during lymphocyte development, we generated polyclonal Ab that recognizes murine CCR9. CCR9 was expressed on the majority of immature CD4+CD8+ (double-positive) thymocytes, but not on immature CD4(-)CD8(-) (double-negative) thymocytes. CCR9 was down-regulated during the transition of double-positive thymocytes to the CD4+ or CD8+ (single-positive) stage, and only a minor subset of CD8+ lymph node T cells expressed CCR9. All CCR9+ thymocyte subsets migrated in response to CCL25; however, CD69+ thymocytes demonstrated enhanced CCL25-induced migration compared with CD69(-) thymocytes. Ab-mediated TCR stimulation also enhanced CCL25 responsiveness, indicating that CCL25-induced thymocyte migration is augmented by TCR signaling. Approximately one-half of all gammadeltaTCR+ thymocytes and peripheral gammadeltaTCR+ T cells expressed CCR9 on their surface, and these cells migrated in response to CCL25. These findings suggest that CCR9 may play an important role in the development and trafficking of both alphabetaTCR+ and gammadeltaTCR+ T cells.