Antigen Re-encounter Research Articles

Polarization of the memory B cell response.

Memory B cells are long-lived cells that are induced following infection or vaccination. Upon antigen re-encounter, memory B cells rapidly differentiate into antibody-secreting or germinal center B cells. While memory B cells are an important component of long-term protective immunity following vaccination, they also contribute to the progression of diseases such as autoimmunity and allergy. Numerous subsets of memory B cells have been identified in mice and humans that possess important phenotypic and functional differences. Here, we review the transcriptional circuitry governing memory B cell differentiation and function. We then summarize emerging evidence that the inflammatory environment in which memory B cells develop has an important role in shaping their phenotype and examine the pathways regulating the development of memory B cells during a type 1-skewed and type-2 skewed immune response.

Alarm functions of PD-1+ brain resident memory T cells.

Resident memory T cells (T RM ) have been described in barrier tissues as having a 'sensing and alarm' function where, upon sensing cognate antigen, they alarm the surrounding tissue and orchestrate local recruitment and activation of immune cells. In the immunologically unique and tightly restricted CNS, it remains unclear if and how brain T RM , which express the inhibitory receptor PD-1, alarm the surrounding tissue during antigen re-encounter. Here, we reveal that T RM are sufficient to drive the rapid remodeling of the brain immune landscape through activation of microglia, DCs, NK cells, and B cells, expansion of Tregs, and recruitment of macrophages and monocytic dendritic cells. Moreover, we report that while PD-1 restrains granzyme B expression by reactivated brain T RM , it has no effect on cytotoxicity or downstream alarm responses. We conclude that T RM are sufficient to trigger rapid immune activation and recruitment in the CNS and may have an unappreciated role in driving neuroinflammation.

Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells

Abstract Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I. Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4586.

Four-Color ImmunoSpot® Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory Bcell (Bmem) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter. Here, we describe how the frequency and Ig class and IgG subclass use of an antigen-specific Bmem repertoire can be determined with relatively little labor and cost, requiring only 8×105 freshly isolated peripheral blood mononuclear cells (PBMC), or if additional cryopreservation and polyclonal stimulation is necessary, 3 × 106 PBMC per antigen. To experimentally validate such cell saving assays, we have documented that frequency measurements of antibody-secreting cells (ASC) yield results indistinguishable from those of enzymatic (ELISPOT) or fluorescent (FluoroSpot) versions of the ImmunoSpot® assay, including when the latter are detected in alternative fluorescent channels. Moreover, we have shown that frequency calculations that are based on linear regression analysis of serial PBMC dilutions using a single well per dilution step are as accurate as those performed using replicate wells. Collectively, our data highlight the capacity of multiplexed Bcell FluoroSpot assays in conjunction with serial dilutions to significantly reduce the PBMC requirement for detailed assessment of antigen-specific B cells. The protocols presented here allow GLP-compliant high-throughput measurements which should help to introduce high-dimensional Bmem characterization into the standard immune monitoring repertoire.

Microbiota regulation of thymic microbiota-specific T cell development

Abstract While exposure to microbial antigens in peripheral sites causes expansion and differentiation of antigen-specific T cells, a role for antigen-specific expansion had not been identified during T cell development in the thymus. We recently found that intestinal colonization induced migration of dendritic cells carrying bacterial antigens to the thymus, driving thymic expansion of microbiota-specific T cells early in life. In contrast, colonization in adulthood led to expansion of peripheral but not thymic microbiota-specific T cells. Thymic microbiota-specific T cells expanded early in life could cause intestinal inflammation in colitis models upon antigen reencounter. We next sought to understand microbiota regulation of this developmental window and define if thymic expansion of microbiota-specific T cells could be restarted in adulthood. Remarkably, we found that microbiota manipulation reestablished expansion of microbiota-specific T cells in the adult thymus. In contrast to early life, adult thymic microbiota-specific T cells ameliorated intestinal inflammation in colitis models. In both early life and adulthood, development of thymic microbiota-specific T cells was driven by the same intestinal dendritic cell population. Our data suggests that intestinal colonization controls the development of thymic microbiota-specific T cells which can modulate inflammatory processes. NIH (K99AR080207)

Viral infection in the brain induces priming and maintenance of a novel antigen specific CD8 T cell population within the bone marrow that dysregulates the stem cell niche

Abstract Bone marrow is a primary immune organ responsible for stem cell maintenance and hematopoiesis. While the bone marrow niche is mainly studied in the context of hematopoiesis, aging, and mechanisms underlying stem cell and plasma cell survival, changes of the niche during brain viral infections and primary T cell responses is unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared 30 days post infection due to generation of an antigen specific CD8 T cell response raised against the immunodominant epitope VP2 121–130. We determined that 7–10% of CD8 T cells present in the sternal and femoral bone marrows and 15–20% in the cranial bone marrow were antigen specific 7 days post intracranial TMEV infection. Over 70% of these CD8 T cells were located outside of the vasculature as determined by IV labeling. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen specific CD8 T cells in any bone marrow compartment. CD8 T cells in the bone marrow established durable memory following viral clearance and were reactivated upon cognate antigen reencounter. Both acute TMEV infection and antigen specific reactivation caused an increase in lineage −, Sca-1 +, ckit +(LSK) cells in the bone marrow. This LSK dysregulation was abrogated upon CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Both acute and memory recall CD8 T cell responses cause stem cell dysregulation. Our data hence paves the way for crucial studies of bone marrow resident antigen specific CD8 T cells in health and disease. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Depleting CD103+ resident memory T cells in vivo reveals immunostimulatory functions in oral mucosa.

The oral mucosa is a frontline for microbial exposure and juxtaposes several unique tissues and mechanical structures. Based on parabiotic surgery of mice receiving systemic viral infections or co-housing with microbially diverse pet shop mice, we report that the oral mucosa harbors CD8+ CD103+ resident memory T cells (TRM), which locally survey tissues without recirculating. Oral antigen re-encounter during the effector phase of immune responses potentiated TRM establishment within tongue, gums, palate, and cheek. Upon reactivation, oral TRM triggered changes in somatosensory and innate immune gene expression. We developed in vivo methods for depleting CD103+ TRM while sparing CD103neg TRM and recirculating cells. This revealed that CD103+ TRM were responsible for inducing local gene expression changes. Oral TRM putatively protected against local viral infection. This study provides methods for generating, assessing, and in vivo depleting oral TRM, documents their distribution throughout the oral mucosa, and provides evidence that TRM confer protection and trigger responses in oral physiology and innate immunity.

Functional heterogeneity in the memory B-cell response.

Most vaccines induce robust antibody and memory B-cell (MBC) responses that are capable of mediating protective immunity. However, antibody titers wane following vaccination necessitating the administration of booster vaccines to maintain a protective antibody titer. MBCs are stably maintained following vaccination and can rapidly give rise to antibody-secreting cells or undergo further affinity maturation upon antigen re-encounter. Repeated antigen encounter results in the development of MBCs that encode antibodies capable of mediating broadly protective immunity against viruses such as SARS-CoV-2 and influenza. Here, we summarize emerging evidence that MBCs are a heterogeneous population composed of transcriptionally and phenotypically distinct subsets that have discrete roles in mediating protective immunity upon antigen re-encounter and examine the implications of these findings for the development of vaccines capable of eliciting broadly protective immunity.

Tissue-resident memory T cells in renal autoimmune diseases.

The discovery of tissue-resident memory T cells (TRM cells) reinterpreted the potential of human tissue-specific immunity. Following T cell receptor (TCR) activation and clonal expansion, effector T cells migrate to peripheral tissues where they remain long-term and differentiate to TRM cells after antigen clearance. This allows for prompt immunological responses upon antigen re-encounter. In addition to their protective properties in acute infections, recent studies have revealed that TRM cells might lead to aggravation of autoimmune diseases, such as lupus nephritis (LN) and anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (GN). These diseases present as proliferative and crescentic glomerulonephritis (cGN), which is a life-threatening condition leading to end-stage renal disease (ESRD) if left untreated. A better understanding of renal TRM cells might lead to identifying new therapeutic targets for relapsing autoimmune diseases of the kidney. In this review, we summarize the current knowledge of renal TRM cells and discuss their potential pathophysiological roles in renal autoimmune diseases.

Unique features of the TCR repertoire of reactivated memory T cells in the experimental mouse tumor model

T cell engineering with T cell receptors (TCR) specific to tumor antigens has become a breakthrough towards personalized cancer adoptive cell immunotherapy. However, the search for therapeutic TCRs is often challenging, and effective strategies are strongly required for the identification and enrichment of tumor-specific T cells that express TCRs with superior functional characteristics. Using an experimental mouse tumor model, we studied sequential changes in TCR repertoire features of T cells involved in the primary and secondary immune responses to allogeneic tumor antigens. In-depth bioinformatics analysis of TCR repertoires showed differences in reactivated memory T cells compared to primarily activated effectors. After cognate antigen re-encounter, memory cells were enriched with clonotypes that express α-chain TCR with high potential cross-reactivity and enhanced strength of interaction with both MHC and docked peptides. Our findings suggest that functionally true memory T cells could be a better source of therapeutic TCRs for adoptive cell therapy. No marked changes were observed in the physicochemical characteristics of TCRβ in reactivated memory clonotypes, indicative of the dominant role of TCRα in the secondary allogeneic immune response. The results of this study could further contribute to the development of TCR-modified T cell products based on the phenomenon of TCR chain centricity.

416.5: Pregnancy-Induced Sensitization in Female Transplant Candidates: HLA-Specific Memory B Cells and Paternal HLA Epitope Mismatch

Background: Access to kidney transplantation (KT) may be limited by HLA antibodies (HLA-Ab) induced by sensitizing events. Pregnancy is a frequent sensitizing event in female candidates, through which they are exposed to foreign paternal HLA antigens. Consequently, they can generate specific HLA-Ab and memory B cells (mBc), which can be re-stimulated upon antigen reencounter after KT. We aimed to study the existence of mBc able to produce specific HLA-Ab against paternal antigens in female KT candidates sensitised by previous pregnancies, to compare them with HLA-Ab found in their serum and to analyse these HLA-Ab in the context of the molecular HLA mismatch between mother and father. Methods: We selected 8 HLA sensitised women awaiting KT with at least one successful pregnancy from a single partner, detectable serum HLA-Ab (MFI≥1000) and available paternal DNA for full HLA typing. All women and their partners were typed by next-generation sequencing for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1, DPB1. To study mBc, we used peripheral blood mononuclear cells cryopreserved before KT and cultured them with a polyclonal stimulation cocktail consisting of a toll-like receptor agonist and an interleukin for ten days. We examined concentrated culture supernatants (SN) derived from activated mBc, purified IgG and identified HLA-Abs using single antigen beads on Luminex®. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA mismatch with HLAMatchmaker® software. Results: After 10-day polyclonal stimulation, mBc increased from 29.9% (day 0) to 65.3% (day- 10), and antibody-secreting cells (ASC) from 0.1% (day-0) to 29.4% (day-10) (Figure 1). SAB analysis revealed 211 HLA-Abs in serum and 116 in SN: 98/211 (46,5%) HLA-Ab were serum-exclusive (79,6% class I and 20,4% class II) and only 3/116 (2,3%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34,6%) HLA-Ab shared by serum and SN: 65/113 (57,5%) class I and 48/113 (42,5%) class II.Pregnancy-Induced Antibodies (PIA) were present in serum in 6/8 women (21 HLA-Abs: 10 class I, 11 class II) and in 4/8 women in SN (8 HLA-Abs: 2 class I, 6 class II). The HLAMatchmaker analysis showed that those specific HLA-ab were reactive against incompatible paternal epitopes: in 3/6 women for class I & 3/6 women for class II in serum; in 2/4 women for class I & 3/4 women for class II in SN (Table 1).Conclusion: We describe the study of the memory B cell compartment and the HLA-Abs produced with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by memory B cells followed a restricted pattern compared with serum HLA-Ab. The study of paternal HLA molecular mismatch explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitised patients awaiting TK is currently a matter of study.

Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

Novel H7N9 influenza immunogen design enhances mobilization of seasonal influenza T cell memory in H3N2 pre-immune mice

ABSTRACT Strategies that improve influenza vaccine immunogenicity are critical for the development of vaccines for pandemic preparedness. Hemagglutinin (HA)-specific CD4+ T cell epitopes support protective B cell responses against seasonal influenza. However, in the case of avian H7N9, which poses a pandemic threat, HA elicits only weak neutralizing antibody responses in infection and vaccination without adjuvant. We hypothesized that an immune-engineered H7N9 HA incorporating a broadly reactive H3N2 HA-specific memory CD4+ T cell epitope that replaces a regulatory T cell-inducing epitope at the corresponding position in H7N9 HA could harness preexisting influenza T cell immunity to increase CD4+ T cells that are needed for protective antibody development. We designed and produced a virus-like particle (VLP) vaccine that carries the epitope augmented H7N9 HA (OPT1) and immunized HLA-DR3 transgenic mice with established H3N2 immunity. OPT1-VLPs stimulated higher stem cell, central, and effector memory CD4+ T cell levels over wild type VLP immunization. In addition, activated, IL-21-producing follicular helper T cell frequencies were enhanced. This novel immunogen design strategy illustrates that site-specific modifications aimed to augment T cell epitope content enhance CD4+ T cell responses among critical subpopulations capable of aiding protective immune responses upon antigen re-encounter and that mobilization of immune memory can be used to overcome the poor immunogenicity of avian influenza viruses.

FC 112: HLA-Specific Memory B Cells and Paternal HLA Molecular Mismatch in Female Transplant Candidates Sensitized by Pregnancy

Abstract BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

CXCR6 is required for tissue resident memory T cell formation across diverse peripheral non-lymphoid tissues

Abstract CD8 T cell control of viral infections in peripheral tissues is largely dependent upon their spatial positioning. Clearance of pathogens requires direct contact between CD8 T cells and infected cells. Chemokine receptors play an essential role in this process by facilitating T cell entry into tissues, directing T cell positioning within tissues and by influencing egress out of tissues. Tissue resident memory T cells (TRM), which fail to egress after pathogen clearance and scan cells for signs of infection, are potent sources of immunity to secondary encounters with pathogens. Here we show that expression of the chemokine receptor CXCR6 is a conserved feature of TRM after viral infection in many non-lymphoid tissues. Using CXCR6KO TCR-tg T cells, we found that CXCR6 is necessary for proper formation of TRM in all non-lymphoid tissues examined. CXCR6 was necessary for early accumulation of effector T cells in several tissues such as the kidney and salivary gland but CXCR6 was dispensable for early accumulation in the skin. After vaccinia infection in the skin, CXCR6 expression is reinforced upon antigen re-encounter and promotes CD8 T cell accumulation in a migration-independent manner. Using single cell transcriptional analysis, we show that CXCR6KO TRM appear to exhibit a survival disadvantage and are outcompeted by wildtype T cells. This work reveals a critical requirement for generating TRM in diverse anatomical locations and may benefit strategies for vaccine design, anti-tumor immunity and reducing T-cell mediated autoimmunity. Supported by R01CA238163 T32Al100853-10

Microbiota regulation of development of thymic microbiota-specific T cells

Abstract While exposure to microbial antigens in peripheral sites causes expansion of antigen-specific T cells, a role for antigen-specific expansion had not been identified during T cell development in the thymus. We recently found that intestinal colonization induced intestinal dendritic cells migration to the thymus, driving thymic expansion of microbiota-specific T cells early in life. In contrast, colonization in adulthood leads to expansion of peripheral but not thymic microbiota-specific T cells. Thymic microbiota-specific T cells developed early in life were undifferentiated and caused intestinal inflammation in immunocompromised hosts upon antigen reencounter. We sought to understand regulation of this developmental window and define if thymic expansion of microbiota-specific T cells could be restarted in adult mice. We found that microbiota manipulation reestablished expansion of microbiota-specific T cells in the thymus of adult mice. We are in the process of characterizing these T cells in adulthood to assess their impact on intestinal inflammation. Our data suggests that intestinal colonization controls the generation of thymic microbiota-specific T cells which has the potential to modulate inflammatory processes.

Lung-resident memory CD4 T cells recognizing antigen in the airway enhance the kinetics of antigen presentation and naive T cell activation in draining lymph nodes

Abstract CD4+ tissue-resident memory (TRM) T cells act as sentinels at previous sites of infection and rapidly initiate protective inflammatory responses upon antigen re-encounter. Here, we tested if beyond local impacts in the lung, TRM responses lead to faster egress of antigen-loaded dendritic cells (DC) resulting in their more rapid accumulation in draining lymph nodes (DLN). To do so, we used a mouse model originally developed to examine CD4+ TRM-mediated protection against influenza virus infection. We established a TRM population in otherwise naïve mice by intranasal transfer of memory CD4 T cells specific for ovalbumin (OVA). The mice were then challenged with fluorescently-labelled OVA protein intranasally. Faster migration from the lung to the DLN by OVA+ DC was seen within 48 hours of protein administration in mice receiving TRM compared to controls without TRM transfer. We hypothesized that the enhanced kinetics of OVA presentation in DLN mediated by TRM antigen recognition would result in more rapid systemic activation of OVA-specific CD4 T cells. We thus transferred an indicator population of naïve OVA-specific CD4 T cells intravenously to mice receiving TRM transfer or not prior to OVA protein administration. Faster kinetics of naïve T cell activation were seen in recipients of TRM within 3 days as judged by expression of activation markers and proliferation. Most importantly, by day 4, responding naïve cells reached the lung in greater numbers and were more functional in mice receiving TRM. Our results reveal that CD4+ TRM activation can enhance the kinetics of antigen presentation in DLNs and suggest this mechanism can be harnessed by vaccines to fully integrate protective local and systemic CD4 T cell responses. Supported by grants from NIH (R21 R21AI148882)

The Chemokine Receptor CCR5 Links Memory CD4+ T Cell Metabolism to T Cell Antigen Receptor Nanoclustering.

The inhibition of anabolic pathways, such as aerobic glycolysis, is a metabolic cornerstone of memory T cell differentiation and function. However, the signals that hamper these anabolic pathways are not completely known. Recent evidence pinpoints the chemokine receptor CCR5 as an important player in CD4+ T cell memory responses by regulating T cell antigen receptor (TCR) nanoclustering in an antigen-independent manner. This paper reports that CCR5 specifically restrains aerobic glycolysis in memory-like CD4+ T cells, but not in effector CD4+ T cells. CCR5-deficient memory CD4+ T cells thus show an abnormally high glycolytic/oxidative metabolism ratio. No CCR5-dependent change in glucose uptake nor in the expression of the main glucose transporters was detected in any of the examined cell types, although CCR5-deficient memory cells did show increased expression of the hexokinase 2 and pyruvate kinase M2 isoforms, plus the concomitant downregulation of Bcl-6, a transcriptional repressor of these key glycolytic enzymes. Further, the TCR nanoclustering defects observed in CCR5-deficient antigen-experienced CD4+ T cells were partially reversed by incubation with 2-deoxyglucose (2-DG), suggesting a link between inhibition of the glycolytic pathway and TCR nanoscopic organization. Indeed, the treatment of CCR5-deficient lymphoblasts with 2-DG enhanced IL-2 production after antigen re-stimulation. These results identify CCR5 as an important regulator of the metabolic fitness of memory CD4+ T cells, and reveal an unexpected link between T cell metabolism and TCR organization with potential influence on the response of memory T cells upon antigen re-encounter.

Young but not older adults exhibit an expansion of CD45RA+CCR7+CD95+ T follicular helper cells in response to tetanus vaccine.

A subset of CD4+ T cells, known as T follicular helper (Tfh), provides co-stimulating signals required to establish long-term humoral immunity. Recent studies have shown a reduced frequency and functionality of this population in older adults in comparison to young adults, in response to vaccination. To evaluate whether memory generation of circulating Tfh (cTfh) cells contributes to this phenomenon, the memory subpopulations of cTfh, and their activation degree, were evaluated both ex-vivo and in-vitro, in response to the model antigen tetanus toxoid (TT) after the first dose of tetanus vaccine. Here, we report a lower frequency of cTfh after vaccination in older adults compared to young adults. Moreover, whereas cTfh from older adults preferably expanded with an effector memory phenotype, young adults experienced a temporal increase of CCR7+CD45RA+ cTfh cells, which also displayed higher levels of CD95, CD40L, CXCR3, and Bcl-6 upon antigen re-encounter. This phenotype was confirmed using automatized algorithm. In conclusion, our results suggest that an age-related loss of heterogeneity and an expansion of more differentiated memory cells within the cTfh compartment could affect the responsiveness of older individuals to vaccines, making this phenotype a characteristic feature of immunosenescence.

Memory B Cells in Pregnancy Sensitization.

Memory B cells play an important role in immunity to pathogens as these cells are poised to rapidly differentiate into antibody-secreting cells upon antigen re-encounter. Memory B cells also develop over the course of HLA-sensitization during pregnancy and transplantation. In this review, we discuss the potential contribution of memory B cells to pregnancy sensitization as well as the impact of these cells on transplant candidacy and outcomes. We start by summarizing how B cell subsets are altered in pregnancy and discuss what is known about HLA-specific B cell responses given our current understanding of fetal antigen availability in maternal secondary lymphoid tissues. We then review the molecular mechanisms governing the generation and maintenance of memory B cells during infection – including the role of T follicular helper cells - and discuss the experimental evidence for the development of these cells during pregnancy. Finally, we discuss how memory B cells impact access to transplantation and transplant outcomes for a range of transplant recipients.