Memory Tfh Research Articles

Dynamics of T follicular helper cells in patients with rheumatic diseases and subsequent antibody responses in a three-dose immunization regimen of CoronaVac.

Given increased acceptance of the CoronaVac, there is an unmet need to assess the safety and immunogenic changes of CoronaVac in patients with rheumatic diseases (RD). Here we comprehensively analysed humoral and cellular responses in patient with RD after a three-dose immunization regimen of CoronaVac. RD patients with stable condition and/or low disease activity (n=40) or healthy controls (n=40) were assigned in a 1:1 ratio to receive CoronaVac (Sinovac). The prevalence of anti-receptor binding domain (RBD) antibodies and neutralizing antibodies was similar between healthy control (HC) and RD patients after the second and the third vaccination. However, the titers of anti-RBD IgG and neutralizing antibodies were significantly lower in RD patients compared to HCs (p < 0.05), which was associated with an impaired T follicular helper (Tfh) cell response. Among RD patients, those who generated an antibody response displayed a significantly higher Tfh cells compared to those who failed after the first and the second vaccination (p < 0.05). Interestingly, subjects with a negative serological response displayed a similar Tfh memory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides as their anti-RBD IgG positive counterpart, and all (4/4) of the non-responders in HCs, and 62.5% (5/8) of the non-responders in patients with RD displayed a positive serological response following the third dose. No serious adverse events were observed. In conclusion, our findings support SARS-CoV-2 vaccination in patients with RD with stable and/or low disease activity. The impaired ability in generating vaccine-specific antibodies in patients with RD was associated with a reduction in Tfh cells induction. The window of vaccination times still needs to be explored in future studies. Clinical trial registration: This trial was registered with ChiCTR2100049138.

Abstract A19: Integrated single cell analysis reveals co-evolution of malignant B cells and the tumor microenvironment in transformed follicular lymphoma

Abstract Introduction: Histological transformation from follicular lymphoma (FL) to aggressive B-cell lymphoma (tFL) is a disease course altering event linked to poor prognosis for affected patients. From a biological point of view, it is paradigmatic of disease dynamics with distinct clinical stages that project onto genetically and phenotypically divergent states. Aim: By applying a series of high-dimensional single cell (sc)RNA and DNA profiling techniques, we aimed to characterize the clonal and phenotypic evolution of tumor B cells and to reveal dynamic interactions with components of the tumor microenvironment (TME) during transformation. Methods: We included 11 tFL patients with paired FL (tFL-FL) and DLBCL (tFL-DLBCL) timepoint biopsies, and 11 indolent FL controls (with &amp;gt; 6y of follow-up without evidence of progression or transformation). Single cell whole transcriptome (scWTS) and BCR sequencing was performed for all samples and single cell whole genome sequencing (scWGS) for transformation pairs. Results: In each transformation pair, BCR sequencing confirmed the clonal relationship between FL and DLBCL timepoints. Clustering of scRNA data from each pair showed an inverse correlation between transcriptional similarity and time between the two biopsies. Some tFL-FL cells could always be found within the tFL-DLBCL clusters. Therefore, we labeled these cells as presumed “early-DLBCL cells”. Phylogenetic analysis using scWGS data showed distinct FL and DLBCL clones, and “mixed-clones” composed of cells from both timepoints in most pairs. FL cells in DLBCL clones were favored to represent precursor cells of transformation. DLBCL cells in FL clones likely represent residual FL cells after transformation, and could be found in the majority of the pairs. Divergent evolution from FL to DLBCL with specific copy number abnormalities unique to each timepoint was the most common mode of evolution during transformation, and only one pair showed linear evolution. Integrative analysis of scRNA and scDNA data highlighted that samples with the fewest genomic changes showed the least transcriptomic changes and vice versa. Differential expression and gene set enrichment analysis of malignant cells identified “MYC targets V1“ as the main pathway enriched in tFL-DLBCL cells in comparison to tFL-FL cells. Cells from the indolent control FL cases had a significantly lower MYC score than cells from pre-transformed FL. In parallel to the insights into tumor cell evolution, scRNAseq analysis also revealed significant shifts in TME composition, from T cells with a TFH and central memory phenotype in tFL-FL samples, to cells with an exhausted cytotoxic phenotype in tFL-DLBCL samples. Conclusion: Applying high-dimensional scRNA and DNA profiling techniques we identified precursor cell populations of transformation at the genomic and phenotypic level and linked genomic and phenotypic evolution with shifting TME composition in a comprehensive disease evolution model of transformation. Citation Format: Clementine Sarkozy, Shaocheng Wu, Katsuyoshi Takata, Tomohiro Aoki, Susana B Neriah, Katy Milne, Brad Nelson, Andrew Weng, David Scott, Jeffrey W Craig, Christian Steidl, Andrew Roth. Integrated single cell analysis reveals co-evolution of malignant B cells and the tumor microenvironment in transformed follicular lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A19.

Escherichia coli-Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer.

In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli-specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221.

The Differentiation and Maintenance of SARS-CoV-2-Specific Follicular Helper T Cells.

Upon acute viral infection, virus-specific CD4+ T cells differentiate into either TH1 cells or follicular helper T (TFH) cells. The molecular pathways governing such bimodal cell fate commitment remain elusive. Additionally, effector virus-specific TFH cells further differentiate into corresponding memory population, which confer long-term protection against re-infection of same viruses by providing immediate help to virus-specific memory B cells. Currently, the molecular mechanisms underlying the long-term maintenance of memory TFH cells are largely unknown. In this review, we discuss current understanding of early differentiation of virus-specific effector TFH cells and long-term maintenance of virus-specific memory TFH cells in mouse models of viral infection and patients of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Bupi Yichang Pill alleviates dextran sulfate sodium-induced ulcerative colitis in mice by regulating the homeostasis of follicular helper T cells

BackgroundFollicular helper T (Tfh) cells-based therapy represents a new treatment option for inflammatory bowel disease. Bupi Yichang Pill (BPYCP), a traditional Chinese formula for the treatment of dysentery and diarrhea, exhibits potential anti-inflammatory activities in treating various kinds of inflammation. However, its anti-inflammatory effect on colitis and the underlying mechanisms remain unknown. PurposeTo explore the protective role and underlying immunomodulatory effects of BPYCP in the treatment of UC. MethodsThe dextran sodium sulfate (DSS) free-drinking method induced UC in C57BL/6 mice, and BPYCP was orally administrated at a dosage of 1.5, 3.0, or 6.0 g/kg/day. Throughout the experimental period, the effects of BPYCP on DSS-induced clinical symptoms and disease activity index (DAI) were monitored and analyzed. Hematoxylin-eosin staining was used to observe the histopathological injury of the colon. Flow cytometry was used to detect the levels of Tfh cells, Tfh cell subpopulations, and memory Tfh cells. ELISA, Western blot, and qRT-PCR were used to detect the expression of inflammatory cytokines and Tfh cell-related biomarkers. ResultsMedium and high dosages of BPYCP effectively alleviated DSS-induced experimental colitis with increased body weight, survival rate and colonic length, and decreased DAI, colonic weight, and colonic index, as well as less ulcer formation and inflammatory cell infiltration, increased anti-inflammatory cytokine IL-10, and decreased pro-inflammatory cytokines IL-17A, IL-6, and TNF-ɑ. Moreover, BPYCP administration significantly decreased the percentage of Tfh cells and the expression of Tfh markers ICOS, PD-1 and Bcl-6 in the mesenteric lymph nodes of colitis mice. In addition, BPYCP treatment obviously decreased the percentages of Tfh1, Tfh17 and Tem-Tfh cells and upregulated Tfr cells in colitis mice. However, there were no significant regulatory effects of BPYCP on Tfh cell response in normal mice. ConclusionTaken together, these results demonstrated a protective effect of BPYCP against DSS-induced experimental colitis by regulating Tfh cell homeostasis.

Antiviral CD19+CD27+ Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpesvirus Infection.

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4+ Tfh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19+CD27+ memory B cells, and (ii) high frequencies of HSV-specific switched IgG+CD19+CD27+ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0+CXCR5+CD4+ memory Tfh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD+CD138+ B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice, there were increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory Tfh cells. Moreover, following UV-B-induced reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Protein Immunization Induces Memory CD4+ T Cells That Lack Th Lineage Commitment.

Acute viral infection generates lineage-committed Th1 and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory Th cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation in mice (Mus musculus) following adjuvanted glycoprotein immunization compared with acute lymphocytic choriomeningitis virus infection. Glycoprotein immunization induced CXCR5- non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular Th cell lineage but had undergone memory differentiation. However, upon challenge with virus, these Th lineage-nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.

IL-21 is necessary to both induce and sustain humoral immunity to Plasmodium

Abstract Resistance to malaria depends on the generation of antibodies derived from germinal center (GC) responses. T follicular helper (Tfh) cells play a critical role in the selection of high-affinity GC B cells that can differentiate into either memory B cells or antibody secreting plasma cells. Mice deficient in IL-21 or its receptor fail to mount GC responses, secrete parasite-specific IgG and control primary Plasmodium infections. However, whether IL-21 exerts temporally distinct contributions to either the maintenance of GC reactions or memory, during primary anti-Plasmodium humoral immunity or memory recall responses, is not known. We hypothesize that CD4+ T cell derived IL-21 acts to support the maintenance of GC B cells and is critical for secondary humoral immune reactions orchestrated by memory Tfh. To test this hypothesis, we utilized IL-21-VFP reporters and developed cell type-specific and conditional IL-21 knockout systems. Our data show that Tfh-derived IL-21 expression peaks during the first week of infection as GC are initially forming. Following tamoxifen mediated deletion of CD4 derived IL-21 during an established GC reaction (days 16–18 p.i.), we observed significant reductions in GC B cells and loss of memory B cell-derived, parasite-specific IgG production following challenge. Thus, in addition to inducing GC responses, our experiments show that IL-21 is critical for sustaining GC responses and the formation of functional memory B cell responses.

Protein immunization induces T follicular helper memory cells and nonpolarized T helper (Tnp) memory cells

Abstract Acute viral infection generates lineage-committed T helper 1 (Th1) and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory T helper cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation following adjuvanted glycoprotein immunization compared to acute lymphocytic choriomeningitis virus (LCMV) infection. Glycoprotein immunization-induced CXCR5-non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular T helper cell lineage but had undergone memory differentiation. However, upon challenge with virus, these T helper lineage-nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.

Stepwise Tfh cell differentiation revisited: new advances and long-standing questions

T follicular helper (Tfh) cells play an essential role in germinal center formation and the generation of high-affinity antibodies. Studies have proposed that Tfh cell differentiation is a multi-step process. However, it is still not fully understood how a subset of activated CD4+ T cells begin to express CXCR5 during the early stage of the response and, shortly after, how some CXCR5+ precursor Tfh (pre-Tfh) cells enter B cell follicles and differentiate further into germinal center Tfh (GC-Tfh) cells while others have a different fate. In this mini-review, we summarize the recent advances surrounding these two aspects of Tfh cell differentiation and discuss related long-standing questions, including Tfh memory.

Conversion of T Follicular Helper Cells to T Follicular Regulatory Cells by Interleukin-2 Through Transcriptional Regulation in Systemic Lupus Erythematosus.

This study was undertaken to identify characteristics of follicular regulatory T (Tfr) cells and elucidate the mechanisms by which follicular helper T (Tfh) cells convert to Tfr cells. We probed the phenotype of T helper cells in patients with systemic lupus erythematosus (SLE) and underlying transcriptional regulation using cytokine-induced STAT family factors. Peripheral blood mononuclear cells from 41 patients with SLE and 26 healthy donors were used to sort out the memory Tfh cell subset, and Tfh cells were cultured under various conditions. The phenotype of T helper cells and underlying mechanisms of transcriptional regulation were probed using flow cytometry and quantitative polymerase chain reaction analyses. These analyses evaluated the expression of characteristic markers and phosphorylation of STATs. Chromatin immunoprecipitation was used to evaluate histone modifications. In patients with SLE, the proportion of CD4+CXCR5+FoxP3-PD-1high Tfh cells was increased (P < 0.01), whereas the proportion of CD4+CXCR5+CD45RA-FoxP3high activated Tfr cells was decreased (P < 0.05). Serum interleukin-2 (IL-2) levels were also reduced in patients with SLE. IL-2 induced conversion of memory Tfh cells to functional Tfr cells, which was characterized by CXCR5+Bcl-6+FoxP3high pSTAT3+pSTAT5+ cells. The loci of FOXP3 and BCL6 at STAT binding sites were marked by bivalent histone modifications. Following IL-2 stimulation, STAT3 and STAT5 selectively bound to FOXP3 and BCL6 gene loci accompanied by suppression of H3K27me3. Finally, IL-2 stimulation suppressed the generation of CD38+CD27high plasmablasts in Tfh and B cell coculture assays ex vivo. Impaired function of Tfr cellsmight beattributed to defective IL-2 production. ExogenousIL-2 restores the function of Tfr cells through the conversion of Tfh cells to Tfr cells in patients with SLE. Thus, restoring balance between Tfh and Tfr cells may provide new therapeutic approaches in SLE.

Transcriptome dynamics of CD4+ T cells during malaria maps gradual transit from effector to memory.

The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

Deficiency of the methylcytosine dioxygenase Tet2 enhances CD4 T cell memory generation

Abstract In response to viral infection, antigen-specific CD4 T cells differentiate into Th1 and T follicular helper (Tfh) cell subsets that acquire distinct DNA demethylation changes which act to enforce their lineage commitment and functions. Following viral clearance, the populations of Th1 and Tfh cells contract and form distinct memory populations that maintain their specific methylation programming. Previously, we found that deficiency of Tet2, an oxygenase that mediates CpG demethylation, skews the balance of Th1 and Tfh cells at 7 days post infection leading to the formation of significantly more germinal center (GC) Tfh cells. Here we examined whether Tet2 impacts CD4 T cell memory formation and the recall of lineage specific functions upon re-challenge. At days 15, 30, and 45 post infection with lymphocytic choriomeningitis virus (LCMV) there was a greater frequency of Tet2 deficient cells positive for memory markers in the blood when compared to WT cells. To assess the recall ability of Tet2 deficient memory CD4 T cells, Tet2KO and WT memory cells were purified from spleens of recipient mice at 90 days post-infection and equivalent numbers of WT and Tet2KO cells were then re-transferred into naive C57BL6/J recipient mice followed by LCMV infection. At 7 days post re-challenge, we observed a greater secondary expansion of Tet2KO cells compared to WT. Tet2KO memory cells also retained the ability to generate higher frequencies of GC Tfh cells in response to re-challenge. Ongoing work will identify whether Tet2 deficient memory Tfh cells can provide enhanced help for B cells during secondary infection and improve the antibody response. Taken together, these data suggest that Tet2 can act to restrict memory differentiation in CD4 T cells.

1IFN-α Modulates Memory Tfh Cells and Memory B Cells in Mice, Following Recombinant FMDV Adenoviral Challenge.

Follicular helper T (Tfh) cells regulate high-affinity antibody production. Some findings have indicated that Tfh cells could be differentiated into memory cells. Here we have investigated the effects of IFN-α, as an adjuvant, on the generation of memory Tfh cell and memory B cell responses. The data showed that adenoviral vectors expressing: (i) foot-and-mouth disease virus (FMDV) VP1 proteins and porcine IFN-α, or (ii) porcine IFN-α alone, potently enhanced the generation of memory Tfh cells, especially the CCR7lo memory Tfh subset. Upon rechallenge with FMD recombinant adenoviral vaccines, IFN-α enhances Tfh cells activity, rapidly upregulating their signature Bcl-6, CXCR5, and IL-21 markers. The results suggest that IFN-α enhances the levels of the transcription factor Bcl-6 within Tfh cells, potentially by regulating STAT1. Additionally, IFN-α substantially increased the number of IgG1+ and CD86+ memory B cells, which are responsible for inducing the rapid effector functions of memory Tfh cells after vaccine reactivation, establishing the close relationship between memory B cell and memory Tfh cell subsets. In brief, IFN-α enhances the potency of FMD recombinant adenoviral vaccines to induce memory Tfh and memory B cell responses, thus elevating serum antibody titers. IFN-α administration therefore represents an attractive strategy for enhancing responses to vaccination.

The histone methyltransferase EZH2 primes the early differentiation of follicular helper T cells during acute viral infection

Epigenetic modifications to histones dictate the differentiation of naïve CD4+ T cells into different subsets of effector T helper (TH) cells. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the mechanism regulating the differentiation of TH1, TH2 and regulatory T (Treg) cells. However, whether and how EZH2 regulates follicular helper T (TFH) cell differentiation remain unknown. Using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, we observed abundant EZH2 expression and associated H3K27me3 modifications preferentially in the early committed virus-specific TFH cells compared to those in TH1 cells. Ablation of EZH2 in LCMV-specific CD4+ T cells leads to a selective impairment of early TFH cell fate commitment, but not late TFH differentiation or memory TFH maintenance. Mechanistically, EZH2 specifically stabilizes the chromatin accessibility of a cluster of genes that are important for TFH fate commitment, particularly B cell lymphoma 6 (Bcl6), and thus directs TFH cell commitment. Therefore, we identified the chromatin-modifying enzyme EZH2 as a novel regulator of early TFH differentiation during acute viral infection.

Abstract P3-03-19: Tumor cell detection and immune profiling of lymph nodes from breast cancer patients by mass cytometry

Abstract Introduction: A sentinel lymph node (SN) is the primary node draining the tumor and is assumed to be affected early in the metastatic process. Detection of metastases in SN is a standard procedure in breast cancer diagnostics based on microscopic evaluation (morphology and immunohistochemistry), determining the need for removal of all axillary glands for inspection which again is crucial for tailoring adjuvant therapy. The identification by microscopy is time-consuming and has a risk for false negative results. We hypothesize that the immune profile of SN changes with the presence of tumor cells, even at very low frequencies (micrometastases). By using a multi marker approach to characterize millions of cells from sentinel lymph nodes with and without metastases we aimed at identifying both tumor cells but also characterize a tumor specific immune response. This dual approach might provide an opportunity for a more sensitive test for SN diagnostics. Material and Methods: We established a mass cytometry assay containing 38 markers (antibodies) using CyTOF technology to combine immune profiling with identification of breast cancer cells. Cell suspensions from 14 metastatic axillary lymph nodes (ALNmet), 16 metastatic sentinel lymph nodes (Snmet) and 14 non-metastatic sentinel lymph nodes (SN) from breast cancer patients from the clinical observational trial Oslo2 (early, operable breast cancer patients representing all subtypes) were successfully analyzed by the multimarker panel (single cell resolution). Results: By using mass cytometry, we detected tumor cells (gated as PanKeratin+/CD45- cells) in 86% (26/30) metastatic lymph nodes (ALNmet and Snmet) and in 14% (2/14) non-metastatic lymph nodes (SN). Further, the leukocyte population, identified as CD45+ cells, was gated into 15 subpopulations, mainly comprising different subsets of B and T cells, monocytes and NK cells. By comparing the leukocyte composition in the ALNmet with those in SN samples we identified a significant increase in the abundance of CD8+ memory phenotype, TFH and TCRγδ cells and a decrease in the CD4+ subpopulation in ALNmet compared to the SN samples. The Snmet samples had smaller deposits of tumor cells than the ALNmet samples, and we found no significant differences in leukocyte composition between Snmet and SN samples. Interestingly, when looking at the activation marker CD56, we observed a significant higher expression in the CD4RO, CD8RO, TFH and Treg subpopulations of Snmet samples compared to SN samples. Conclusion: In this study we identified a significant difference in immune cell composition in lymph nodes with and without metastases (ALNmet compared to SN samples). We also identified activation markers unique for subpopulations of lymphocytes in Snmet, but not in negative lymph nodes (SN). We were also able to detect and identify micrometastases in most lymph nodes where morphological examination had identified them, but in addition found tumor cells in two samples scored as negative. The results will be validated in a larger sample series. Citation Format: Russnes HG, Rye IH, Huse K, Schlichting E, Garred O, Mykelbust JH. Tumor cell detection and immune profiling of lymph nodes from breast cancer patients by mass cytometry [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-19.

Molecular Basis of the Differentiation and Function of Virus Specific Follicular Helper CD4+ T Cells.

During viral infection, virus-specific follicular helper T cells provide important help to cognate B cells for their survival, consecutive proliferation and mutation and eventual differentiation into memory B cells and antibody-secreting plasma cells. Similar to Tfh cells generated in other conditions, the differentiation of virus-specific Tfh cells can also be characterized as a process involved multiple factors and stages, however, which also exhibits distinct features. Here, we mainly focus on the current understanding of Tfh fate commitment, functional maturation, lineage maintenance and memory transition and formation in the context of viral infection.

Increased circulating Tfh17 and PD-1+Tfh cells are associated with autoantibodies in Hashimoto’s thyroiditis

Objective: Hashimoto’s thyroiditis (HT) is characterized by autoantibodies targeting the thyroid. Abnormal CD4+CXCR5+T cell levels were previously shown to be associated with HT. However, Tfh cells consist of heterogeneous subpopulations, and which T follicular helper (Tfh) cell subpopulation participates in the pathogenesis of HT remains poorly understood.Methods: Thirty healthy controls (HCs) and 52 HT patients were enrolled in the study. The percentages of Tfh, ICOS+Tfh, PD1+Tfh, Tfh1, Tfh2, Tfh17, effector Tfh, resting Tfh, effector memory Tfh, central memory Tfh, and naïve Tfh cells in the peripheral blood were all determined via flow cytometry, and the associations between the percentages of these cells and thyroid function indices were also investigated.Results: The percentage of Tfh cells was significantly higher in HT patients than in HCs. Examination of the Tfh cell subsets revealed that the percentages of Tfh1, Tfh2, and resting Tfh cells were significantly decreased, while those of the ICOS+Tfh, PD1+Tfh, Tfh17, and effector Tfh cells were significantly increased in HT patients. No significant differences in effector memory, central memory or naïve Tfh cell percentages were noted between the HC and HT groups. Furthermore, the percentage of PD1+Tfh cells was positively correlated with anti-thyroglobulin antibody levels. Most importantly, only Tfh17 cell percentages were positively correlated with anti-thyroglobulin and anti-thyroid peroxidase antibody levels and were negatively correlated serum free T3 and free T4 levels in HT patients.Conclusions: Increased circulating Tfh17 cell and PD1+Tfh percentages are associated with higher autoantibody levels in HT patients, which imply that Tfh17 or PD1+Tfh cells may play a pathogenic role in the development of HT.

Longitudinal profile of circulating T follicular helper lymphocytes parallels anti-HLA sensitization in renal transplant recipients.

Antibody-mediated rejection is responsible for 30%-50% of renal graft failures. Differentiation of B cells into antibody-producing plasmablasts depends on the collaboration of follicular helper T cells (Tfh). We analyzed circulating Tfh (cTfh) in kidney recipients and studied cTfh relationship with anti-HLA antibody production and graft outcome. cTfh were longitudinally analyzed in a prospective cohort of patients (n=206), pre- and posttransplantation. Clinical data, HLA sensitization, and cTfh function were recorded. Both pretransplant and 6-month posttransplant cTfh were able to derive IgG-producing plasmablasts. Pretransplant cTfh was decreased in patients, especially in those who received dialysis. However, these cells were increased in patients with previous allograft or transfusions and in HLA-sensitized recipients. After transplantation cTfh expanded, significantly more in patients who developed de novo anti-HLA antibodies than in patients who remained unsensitized. Augmented pretransplant cTfh positively correlated with higher intensity of pretransplant anti-HLA class I and with de novo anti-HLA class I and anti-HLA class II antibodies. Consistently, pretransplantation cTfh were higher in patients who experienced acute rejection (HR=1.14 [1.04-1.25]). Thus, we show a role for Tfh in anti-HLA sensitization and rejection. Multicenter studies with additional patient cohorts are needed to validate these results. Immunosuppressive drugs targeting Tfh could be useful to improve outcomes.