ICOS expression is required for maintenance but not the formation of germinal centers in the spleen in response to P. yoelii infection.

Inducible T cell co-stimulator (ICOS) plays a key role in the differentiation and maintenance of follicular helper T (Tfh) cells and thus germinal center (GC) formation. Previously, our lab showed in a Plasmodium chabaudi infection model that Icos -/- mice were significantly impaired in their ability to form GCs despite a persistent infection and thus a continued antigen (Ag) load. Here, we show that resolution of a primary infection with P. yoelii , was delayed in Icos -/- mice. This phenotype was associated with a reduction in the accumulation of Tfh-like and GC Tfh cells and an early deficiency in Ag-specific antibody (Ab) production. However, Icos -/- mice could form GCs, though they were less frequent in number than in wild-type (WT) mice. Nonetheless, the Ag-specific Abs from Icos -/- mice lacked signs of affinity maturation, suggesting functional defects associated with these GCs. Eventually, these GC structures dissipated more rapidly in Icos -/- mice than in WT mice. Moreover, the ability of Icos -/- mice to form these GC structures is not reliant on the high Ag load associated with P. yoelii infections, as GC formation was preserved in Icos -/- mice treated with atovaquone. Finally, mice were unable to form secondary GCs in the absence of ICOS after re-challenge. Overall, these data demonstrate the necessity of ICOS in the maintenance of Tfh cells, the formation and maintenance of sufficient numbers of functioning GCs, and the ability to generate new GC structures after re-infection with P. yoelii .

Recall of B cell memory depends on relative locations of prime and boost

Re-entry of memory B cells to recall germinal centers (GCs) is essential for updating their B-cell antigen receptors (BCRs). Using single B-cell culture and fate-mapping, we have characterized BCR repertoires in recall GCs following boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit to recall GCs progeny of primary GC B cells more efficiently than do distal boosts. Recall GCs following local boosts contain significantly more B cells with elevated levels of Ig mutations and higher avidity BCRs. This local preference is unaffected by blockade of CD40:CD154 interaction that terminate active, primary GC responses. Local boosts with heterologous antigens elicit secondary GCs with B-cell populations enriched for cross-reactivity to the priming and boosting antigens; in contrast, cross-reactive GC B cells are rare following distal boosts. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.

Follicular T cells are clonally and transcriptionally distinct in B cell-driven mouse autoimmune disease

Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their contribution to autoantibody-mediated disease remains unclear. Here we perform single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in a mouse model of autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRαβ repertoires at single cell resolution. A minority of clonotypes are preferentially shared amongst autoimmune follicular T cells and clonotypic expansion is associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches indicates convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures are preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoantibody development.

Human B cell lineages associated with germinal centers following influenza vaccination are measurably evolving

The poor efficacy of seasonal influenza virus vaccines is often attributed to pre-existing immunity interfering with the persistence and maturation of vaccine-induced B cell responses. We previously showed that a subset of vaccine-induced B cell lineages are recruited into germinal centers (GCs) following vaccination, suggesting that affinity maturation of these lineages against vaccine antigens can occur. However, it remains to be determined whether seasonal influenza vaccination stimulates additional evolution of vaccine-specific lineages, and previous work has found no significant increase in somatic hypermutation (SHM) among influenza-binding lineages sampled from the blood following seasonal vaccination in humans. Here, we investigate this issue using a phylogenetic test of measurable immunoglobulin sequence evolution. We first validate this test through simulations and survey measurable evolution across multiple conditions. We find significant heterogeneity in measurable B cell evolution across conditions, with enrichment in primary response conditions such as HIV infection and early childhood development. We then show that measurable evolution following influenza vaccination is highly compartmentalized: while lineages in the blood are rarely measurably evolving following influenza vaccination, lineages containing GC B cells are frequently measurably evolving. Many of these lineages appear to derive from memory B cells. We conclude from these findings that seasonal influenza virus vaccination can stimulate additional evolution of responding B cell lineages, and imply that the poor efficacy of seasonal influenza vaccination is not due to a complete inhibition of vaccine-specific B cell evolution.

Wnt5A Signaling Blocks Progression of Experimental Visceral Leishmaniasis

Visceral Leishmaniasis, caused by L. donovani infection is fatal if left untreated. The intrinsic complexity of visceral leishmaniasis complicated further by the increasing emergence of drug resistant L. donovani strains warrants fresh investigations into host defense schemes that counter infections. Accordingly, using a mouse model of experimental visceral leishmaniasis we explored the utility of host Wnt5A in restraining L. donovani infection, using both antimony sensitive and antimony resistant L. donovani strains. We found that Wnt5A heterozygous (Wnt5A +/-) mice are more susceptible to L. donovani infection than their wild type (Wnt5A +/+) counterparts as depicted by the respective Leishman Donovan Units (LDU) enumerated from the liver and spleen harvested from infected mice. Higher LDU in Wnt5A +/- mice correlated with increased level of plasma gammaglobulin, liver granuloma and disorganization of splenic germinal centers. Progression of infection in mice by both antimony sensitive and antimony resistant strains of L. donovani could be prevented by activation of Wnt5A signaling as evident from the lowered LDU and gammaglobulin level, and intactness of splenic germinal centers through intravenous administration of rWnt5A prior to L. donovani infection. Wnt5A mediated blockade of L. donovani infection correlated with the preservation of splenic macrophages and activated T cells, and a TH1 like cytokine thrust. Taken together our results indicate that depletion of Wnt5A promotes susceptibility to visceral leishmaniasis and revamping Wnt5A signaling in the host is able to curb L. donovani infection irrespective of antimony sensitivity or resistance and mitigate the progression of visceral leishmaniasis.

Safety and Short-Term Efficacy of CART Cells in Treatment of Diffuse Large B-Cell Lymphoma with Follicular Transformation

Background：Tissue transformation to Diffuse large B-cell lymphoma(DLBCL) occurs in some patients with follicular cell lymphoma(FL), and these patients have a poor prognosis, and some patients do not respond well to chemotherapy, relapse or disease progression. In recent years, many clinical trials have found that CART cells are have a relatively good effect on relapsed and refractory DLBCL, but what about the effect on DLBCL transformed by FL? Methods：A total of 13 patients with diffuse large B-cell lymphoma transformed from follicular cell lymphoma who received CART cell therapy in our hospital from January 2020 to January 2021 were observed. All patients had stage III/IV disease (7 patients with stage III and 6 patients with stage IV). Among them, 10 were germinal centers and 3 were non-germinal centers. There were 8 males and 5 females, with a median age of 44 years (33-70 years old), who had undergone more than 2 chemotherapy cycles before admission to our hospital, and the median chemotherapy cycle was 10 cycles (7-19 years old). Among them, 3 had previously undergone other clinical drug trials, 1 had undergone CD20 CART cell immunotherapy, and 1 had undergone radiotherapy. At admission, ECOG was 0-3, and all patients had measurable lesions with a median size of 5cm (1.4cm-12.0cm). After preconditioning, CART cells were reinjected, 12 cases were reinjected with murine anti-CD19-CART, and 1 case was reinjected with humanized anti-CD19-CART. Median volume 3.07×10^6/kg (0.46×10^6/kg -5.43×10^6/kg). The cytokine release syndrome(CRS) and the therapeutic effect of 3 months/6 months after reinfusion were observed, and the endpoint was disease progression or death. Results：The main treatment related adverse reaction was cytokine release syndrome. Among them, there were 12 I CRS, 1 II CRS, and no treatment-related deaths. The short-term efficacy of 3 months after treatment: 4/13CR（30.8%）, 7/13PR（53.8%）, 2/13 SD/PD(15.4%) and ORR was 11/13 (84.6%). The efficacy of 6 months after treatment: 10/13CR（76.9%）, 0/13PR（0%）and ORR was 10/13 (76.9%). One patient with 3-months CR developed disease at 11 months and died at 12 months, and one patient with 3- months PR developed disease at 5 months and died at 8 months. The remaining 6patients with 3- PR have now achieved CR。the median follow-up time was 10 months (6-19months). Of the 2 patients who did not respond to treatment have achieved PR after subsequent treatment. Conclusions: It can be seen from this study that CART cells have obvious efficacy and good safety in the treatment of DLBCL transformed by FL. It should be noted that 7 patients had PR status at 3 months after treatment, 6 patients had CR at 6 months after treatment, and 1 patient had disease progression. Efficacy evaluation of patients with DLBCL with potential transformation from FL after CART cell therapy should be appropriately delayed, except that 6 months may be the best time to evaluate the efficacy. At present, the observation time is short, PFS and OS have not been observed, and long-term observation with a larger sample is under way. Disclosures No relevant conflicts of interest to declare.

Mature tertiary lymphoid structures in lung adenocarcinoma are associated with better progression free survival

Introduction/Objective The presence of inducible lymphoid structures known as tertiary lymphoid structures in the tumor microenvironment has been shown to correlate with positive clinical outcome. However, the maturation states of lymphoid aggregates in lung adenocarcinoma are not completely understood. Methods/Case Report Seventy tumor samples from 69 patients diagnosed with lung adenocarcinoma (Stages I to III) between 2013 and 2015 were included in the study. The presence and maturation states of the lymphoid structures within the tumors were evaluated by conventional and 26 samples were further analyzed by multiplexed immunohistochemistry of formalin fixed paraffin embedded tissues and then quantified. Mature lymphoid follicles containing germinal centers were identified by the presence of CD21+ and BCL-6+ cells in an organized configuration within tight clusters of T and B cells. Results (if a Case Study enter NA) Samples with fully mature lymphoid structures (germinal centers) had larger tumors and higher disease stage. The number of mature lymphoid structures correlated with the total number of lymphoid aggregates present in the tumor microenvironment. Additionally, tumor samples with ≥10 mature lymphoid structures had more primary follicles. While there was no difference in overall survival, progression free survival was significantly longer in patients who had ≥10 mature lymphoid structures in comparison with patients who had <10 mature structures. Conclusion In conclusion, a spectrum of lymphoid aggregates in different stages of maturation are present in lung adenocarcinoma. An increase in the number of mature lymphoid structures may be associated with progression free survival in patients with lung adenocarcinoma.