Precursor Frequency Research Articles

IMMU-50. GLIOBLASTOMA MANIPULATES HEMATOPOIETIC STEM CELL DIFFERENTIATION OUTCOMES AND DRIVES ALTERED IMMUNE RECONSTITUTION

Abstract INTRODUCTION Bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) give rise to the cellular components of the immune system. Unfortunately, immune reconstitution from HSPCs are negatively impacted by solid cancers, including high-grade gliomas. For example, an expansion of myeloid progenitor cells has been previously described across several cancers that originate outside the CNS. A similar expansion of MDSCs coupled with diminished T cell function has also been described in the peripheral blood of patients with newly-diagnosed GBM. Alterations in both lymphoid and myeloid compartments due to CNS malignancy led us to determine how intracranial gliomas impact HSPCs in both their capacity to reconstitute the immune compartment and in their cell fate determination. This is important to better understand the impact of gliomas on immunity and how we can leverage these findings to better develop cellular immunotherapeutics. METHODS HSPCs were isolated from bone marrow of C57BL/6 mice with orthotopic KR158B glioma, or age-matched naïve mice. Experiments were conducted to compare relative changes in: gene expression (RNA-sequencing), precursor frequencies, cell fate determination, and cellular function of cells derived from HSPCs of glioma-bearing mice. RESULTS RNA-sequencing revealed 700+ genes whose expression was significantly up- or downregulated in HSPCs from glioma-bearing mice, particularly those involved with stemness and metabolic activity. Importantly, HSPCs from glioma-bearing mice expressed upregulation of genes involved in myelopoiesis relative to naïve mice. This was coupled with an expansion of granulocyte macrophage precursors (GMPs), the progenitors to gMDSCs. Next, differentiation assays revealed that HSPCs from glioma-bearing mice had higher propensity of differentiating into MDSC under homeostatic conditions relative to controls both in vitro and in vivo. Furthermore, mice bearing intracranial gliomas possess an expansion of MDSCs which are more suppressive on T cell proliferation and hinders T cell-mediated tumor cell killing relative to MDSCs derived from naïve control mice.

Behavioral Measures of Cochlear Gain Reduction Depend on Precursor Frequency, Bandwidth, and Level.

Sensory systems adjust to the environment to maintain sensitivity to change. In the auditory system, the medial olivocochlear reflex (MOCR) is a known physiological mechanism capable of such adjustment. The MOCR provides efferent feedback between the brainstem and cochlea, reducing cochlear gain in response to sound. The perceptual effects of the MOCR are not well understood, such as how gain reduction depends on elicitor characteristics in human listeners. Physiological and behavioral data suggest that ipsilateral MOCR tuning is only slightly broader than it is for afferent fibers, and that the fibers feed back to the frequency region of the cochlea that stimulated them. However, some otoacoustic emission (OAE) data suggest that noise is a more effective elicitor than would be consistent with sharp tuning, and that a broad region of the cochlea may be involved in elicitation. If the elicitor is processed in a cochlear channel centered at the signal frequency, the growth of gain reduction with elicitor level would be expected to depend on the frequency content of the elicitor. In the current study, the effects of the frequency content and level of a preceding sound (called a precursor) on signal threshold was examined. The results show that signal threshold increased with increasing precursor level at a shallower slope for a tonal precursor at the signal frequency than for a tonal precursor nearly an octave below the signal frequency. A broadband noise was only slightly more effective than a tone at the signal frequency, with a relatively shallow slope similar to that of the tonal precursor at the signal frequency. Overall, these results suggest that the excitation at the signal cochlear place, regardless of elicitor frequency, determines the magnitude of ipsilateral cochlear gain reduction, and that it increases with elicitor level.

Vaccine genetics of IGHV1-2 VRC01-class broadly neutralizing antibody precursor na\xefve human B cells

A successful HIV vaccine eliciting broadly neutralizing antibodies (bnAbs) must overcome the hurdle of being able to activate naive precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether bnAb precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single-cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naive VRC01-class B cells. Further, we analyzed IGHV1-2 allelic usage among three different cohorts; we find that IGHV1-2 alleles traditionally thought to be incompatible with VRC01-class responses are relatively common in various human populations and that germline variation within IGHV1-2 associates with gene usage frequencies in the naive BCR repertoire.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

A Glimpse into the Diverse Cellular Immunity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific cellular immune response has been shown to play a critical role in preventing severe illness or death in patients infected with SARS-CoV-2 or its variants. Given the multiple T-cell epitopes shared by wild-type virus and its variants, we hypothesized that vaccines that target multiple T-cell epitopes of SARS-CoV-2 may provide a “universal protection” against the wild-type virus as well as its variants, even the heavily mutated ones. To test this, we assessed SARS-CoV-2-specific T-cell precursors in healthy individuals using overlapping peptide pools of SARS-CoV-2 structural and functional proteins, including spike (S), membrane (M), envelope (E), nucleocapsid (N), and protease (P) proteins as target antigens. Diverse T-cell precursor frequencies specific to these viral antigens were detected in healthy individuals, including high, medium, low, and no responders. This was further confirmed by efficient induction of anti-SARS-CoV-2 T-cell immune responses using ex vivo dendritic cell (DC)/T cell coculture. The results demonstrated T-cell responses consistent with the precursor frequencies of each of the individuals tested. Importantly, the combination of all five viral peptide pools induced the strongest cellular immune response, and further, after a DC-peptides re-stimulation, even the no responders developed an increased anti-viral T-cell response. These analyses recapitulate the presence of a broad anti-SARS-CoV-2 cellular immunity even in an immune naïve population, which could be enhanced by antigen presenting cells presenting the overlapping antigenic peptides. Given the critical role of cellular immunity in COVID-19 protection, these results have important implications for vaccine design and immunotherapy in fighting SARS-CoV-2 and its variants.

Abstract 1878: Fc-enhanced anti-CTLA-4 antibody, AGEN1181: new mechanistic insights for potent antitumor immunity and combination potential in treatment-resistant solid tumors

Abstract The advent of immune checkpoint inhibitors (ICI) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1) transformed the treatment landscape in a variety of human cancers. However, responses to currently approved ICI agents occur in small subsets of patients with immunogenic tumors, and resistance to ICI therapy continues to be a limiting factor to achieving optimal clinical benefits. AGEN1181 is a Fc-enhanced, clinical-stage anti-CTLA-4 antibody designed to expand therapeutic benefit to a broader patient population, including tumor types that are refractory to conventional ICI approaches. Here we show that AGEN1181 exploits a novel Fcy receptor-related mechanism of action to promote T cell-mediated antitumor immunity. In primary in vitro T cell and antigen presenting cell (APC) co-culture stimulation assays, AGEN1181 demonstrated superior T cell priming and activation that was independent of Treg depletion, and across all CD16 allele variants - in contrast to conventional IgG1 agents that showed limited activity. In PD-1 refractory tumor-bearing mouse models, a murine AGEN1181 surrogate demonstrated superior antitumor activity compared to an Fc-unmodified anti-CTLA-4 antibody. This enhanced activity was associated with improved T cell priming, intratumoral Treg depletion, increased frequency of CD8 effector memory precursor T cells, enhanced peripheral TCR clonality, and expansion of tumor-associated T cell clones. Next we evaluated the combination potential of the AGEN1181 surrogate with targeted therapies, cell therapy or checkpoint blockade in a series of treatment-resistant preclinical tumor models. Notably, this antibody induced robust tumor control and antitumor immunity when administered alongside anti-PD-1 and focal radiation, adoptive T cell therapy, or iNKT-activating therapy in the refractory B16F10 or B16F1.OVA melanoma models. In treatment-resistant KPC pancreatic ductal tumor-bearing mice, curative responses were seen in combination with chemotherapy (gemcitabine, nab-paclitaxel and cisplatin). Moreover, combinations with other ICI (e.g. anti-PD-1 or anti-TIGIT antibodies) deepened responsiveness and overcame limited activity seen with conventional ICI therapy. Importantly, antitumor immune responses could be further improved with optimal sequencing of combination therapies with AGEN1181. Together, these data demonstrate that the novel Fcy receptor-related mechanism of action employed by AGEN1181 enhances antitumor immunity and confers the potential to expand therapeutic benefit to tumors refractory to ICI therapy. Clinical responses support these findings and a phase II trial is underway in patients with a range of solid tumor types, including angiosarcoma and microsatellite stable tumors. Citation Format: Antoine J. Tanne, Claire Galand, Abdo Abou-Slaybi, Margaret Wilkens, Marilyn Marques, Serina Ng, Haiyong Han, Sylvia Dietrich, Jeremy Waight, Bishnu Joshi, Burcu Yigit, Xavier Michelet, Daniel Levey, David Savitsky, Jennifer Buell, Dhan Chand. Fc-enhanced anti-CTLA-4 antibody, AGEN1181: new mechanistic insights for potent antitumor immunity and combination potential in treatment-resistant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1878.

SARS-CoV-2-specific CD8+ T-cell responses and TCR signatures in the context of a prominent HLA-A*24:02 allomorph.

In‐depth understanding of human T‐cell‐mediated immunity in coronavirus disease 2019 (COVID‐19) is needed if we are to optimize vaccine strategies and immunotherapies. Identification of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) T‐cell epitopes and generation of peptide–human leukocyte antigen (peptide–HLA) tetramers facilitate direct ex vivo analyses of SARS‐CoV‐2‐specific T cells and their T‐cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS‐CoV‐2 epitopes restricted by HLA‐A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike‐derived peptides generated CD8+IFNγ+ responses above background, S1208–1216 (QYIKWPWYI), S448–456 (NYNYLYRLF) and S193–201 (VFKNIDGYF), with S1208 generating immunodominant CD8+IFNγ+ responses. Using peptide–HLA‐I tetramers, we performed direct ex vivo tetramer enrichment for HLA‐A*24:02‐restricted CD8+ T cells in COVID‐19 patients and prepandemic controls. The precursor frequencies for HLA‐A*24:02‐restricted epitopes were within the range previously observed for other SARS‐CoV‐2 epitopes for both COVID‐19 patients and prepandemic individuals. Naïve A24/SARS‐CoV‐2‐specific CD8+ T cells increased nearly 7.5‐fold above the average precursor frequency during COVID‐19, gaining effector and memory phenotypes. Ex vivo single‐cell analyses of TCRαβ repertoires found that the A24/S448 +CD8+ T‐cell TCRαβ repertoire was driven by a common TCRβ chain motif, whereas the A24/S1208 +CD8+ TCRαβ repertoire was diverse across COVID‐19 patients. Our study provides an in depth characterization and important insights into SARS‐CoV‐2‐specific CD8+ T‐cell responses associated with a prominent HLA‐A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID‐19 and could be exploited in vaccine or immunotherapeutic approaches.

Highly Variable Frequencies of Donor Antigen-Specific Naive Cells May Dictate the Immunogenicity of Minor Histocompatibility Antigens after Hematopoietic Stem Cell Transplantation

In unrelated HLA-matched hematopoietic stem cell transplantation there are thousands of mismatches of non-synonymous SNPs between donor and patient, some of which result in polymorphic peptides presented in MHC context, known as Minor Histocompatibility Antigens (MiHAs). As these peptides are foreign to the donor immune cells they may drive allogeneic immune response, causing both graft-versus-host disease and beneficial graft-versus-tumor effect. But due to the phenomenon of immunodominance some of MiHAs are much more immunogenic than the others. We propose that antigenic dominance could be at lest partially explained by the frequencies of precursor T cells in the donor immune system and thus in the graft. As donor lymphocytes have never encountered polymorphic human peptides, before transplantation MiHA specific T cells reside exclusively in naïve T cell (TN) subpopulation. To test the proposed hypothesis we compared the frequencies of naïve T cells, specific for highly immunogenic minor antigen HA-2 and less immunogenic ACC-1Y in individuals negative for these antigens.We have selected a panel of healthy volunteers bearing HLA-A*02:01 and HLA-A*24:02 which can present HA-2 and ACC-1Y respectively. The panel was genotyped for SNP encoding these antigens using rapid multiplex MiHA genotyping approach developed in our laboratory. We have chosen donors having HA-2 -/- and ACC1Y -/- genotypes as sources of MiHA specific TN cells and HA-2 +/- and ACC1Y +/- donors as negative controls. Dendritic cells were derived from donor peripheral blood monocytes, loaded with appropriate peptides and co-cultured with autologous TN cells in extreme limiting dilution assay, which allowed us to calculate TN frequencies.We have determined that frequency of specific TN was substantially higher for immunodominant HA-2 antigen than for subdominant ACC-1Y (5 x 10-6 vs. 2 x 10-7, p=3 x 10-9), which correlates with immunodominance of HA-2 and falls in line with the initial hypothesis. The variation of frequencies of TN specific to various MiHAs could be explained either by the peptide properties or by the influence of the negative selection on initial T cell repertoire. ACC-1Y has the alternative allelic variant (ACC-1C) presented in the thymus, which leads to elimination of all cross-reactive clones, whereas alternative allelic variant of HA-2 is cleaved by proteasome and is not presented to the immune system. Further studies of antigen-specific TN frequencies would facilitate creating a complex model of MiHA immunogenicity, which could be used to predict allogeneic immune response after transplantation. Funding was provided by Russian Science Foundation grant 17-15-01512 DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Methylome and Transcriptome High-Resolution Maps Define the Effects of Epigenetic Modifiers on the Landscape of Hematopoietic Differentiation

Epigenetic modifications are critical determinants of cell identity and play a central role in normal hematopoietic differentiation. Moreover, mutations in epigenetic modifiers including TET2 and IDH2 mutations, which result in a decreased ability to de-methylate DNA, are frequent in myeloid leukemias and clonal hematopoiesis of indeterminate potential. This raises a central question in normal and pathologic hematopoiesis - how do epigenetic modifiers shape the differentiation landscape?To address this question, we applied high-throughput single-cell RNA-seq (scRNAseq) to reconstruct high-resolution maps of hematopoietic differentiation, with and without disruption of central epigenetic modifiers (Tet2 and Idh2). Briefly, FACS-sorted lineage-negative bone marrow cells were isolated from sixteen-week old Mx1 -cre wildtype (Wt, n=6), Mx1 -cre Tet2fl/fl (n= 6) or Mx1 -cre Idh2R140Q/Wt (n=3) mice, 4 weeks following recombination. In total, 45,100 cells underwent droplet-based scRNAseq. Clustering of Wt cells identified 25 distinct progenitor populations, recapitulating previous reports and providing a map of normal mouse hematopoietic differentiation.Tet2knockout (KO) resulted in a 1.5-fold increase in uncommitted cluster HSC4 (Flt3hi, c-Kithi, Cd34low) (Figure A-B), consistent with a potential differentiation block resulting from Tet2-KO. Notably, the HSC differentiation block did not uniformly affect differentiation into committed progenitors, but resulted in a 2-fold decrease in erythroid precursors and a 1.5-fold increase in monocyte precursors. These findings were evident by scRNA-seq four months before this phenotype was observed by flow cytometry. While the expression profiles of these committed erythroid and monocytic progenitors were not different from Wt progenitors, the degree of priming to these fates within uncommitted Tet2-KO HSCs (HSC clusters 1-4) showed reduced priming towards the erythroid fate and increased priming towards the monocytic fate (Figure C). In contrast, mutant Idh2 resulted in different skews of hematopoiesis than Tet2-KO, including an increase in neutrophil precursors, while there was no significant change in the frequency of erythroid precursors (Figure D). These data demonstrate that Tet2 and Idh2 mutations, both of which disrupt de-methylation, have distinct effects on hematopoietic differentiation.To explore the link between aberrant DNA methylation upon Tet2 - KO and the observed differentiation skews, we performed joint single-cell methylation and RNAseq, as well as ATAC-bisulfite sequencing. As expected, Tet2-KO resulted in higher global methylation. However, the increase in methylation was stochastically distributed across the genome, resulting in higher methylation heterogeneity (Figure E), and associated with higher cell-to-cell variation in transcriptional profiles. Importantly, we found that stochastic methylation changes result in deterministic differentiation skews due to differential sensitivity to methylation of binding motifs of lineage-defining transcription factors (TFs). For instance, motifs of erythroid-related TFs (including Nrf1, E2f4 and E2f1), but not monocytic-related TFs, showed a marked enrichment of CpGs and methylation-sensitive binding (Figure F).These data demonstrate the ability of high-resolution single-cell mapping of hematopoiesis to identify the exact point of disruption of HSC differentiation due to alterations in epigenetic modifiers. Specifically, these data show that Tet2-KO results in reduced erythroid but increased monocytic priming of HSCs, leading to skewed differentiation. Moreover, single-cell epigenomic analyses identified that while the hypermethylation effect of Tet2-KO is distributed widely across the genome, the skews in lineage priming result from biases in the sensitivity of lineage-defining TFs to CpG methylation at their motifs. The skews may therefore arise not from a change in methylation of a particular locus, but rather through broad lack of demethylation in targets of erythroid priming TFs, resulting from inherent enrichment of these motifs in CpGs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by “redirecting” polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

CD8+ T cell self-tolerance permits responsiveness but limits tissue damage.

Self-specific CD8+T cells can escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice expressing or lacking this enzyme (due to deficiency in Dct, which encodes Trp2). Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the size of this population was only slightly reduced in wild-type (WT) compared to Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype, or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

Ptpn2 and KLRG1 regulate the generation and function of tissue-resident memory CD8+ T cells in skin.

Tissue-resident memory T cells (TRM cells) are key elements of tissue immunity. Here, we investigated the role of the regulator of T cell receptor and cytokine signaling, Ptpn2, in the formation and function of TRM cells in skin. Ptpn2-deficient CD8+ T cells displayed a marked defect in generating CD69+ CD103+ TRM cells in response to herpes simplex virus type 1 (HSV-1) skin infection. This was accompanied by a reduction in the proportion of KLRG1- memory precursor cells and a transcriptional bias toward terminal differentiation. Of note, forced expression of KLRG1 was sufficient to impede TRM cell formation. Normalizing memory precursor frequencies by transferring equal numbers of KLRG1- cells restored TRM generation, demonstrating that Ptpn2 impacted skin seeding with precursors rather than downstream TRM cell differentiation. Importantly, Ptpn2-deficient TRM cells augmented skin autoimmunity but also afforded superior protection from HSV-1 infection. Our results emphasize that KLRG1 repression is required for optimal TRM cell formation in skin and reveal an important role of Ptpn2 in regulating TRM cell functionality.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.

Patients with myelodysplastic syndromes (MDS) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in MDS del(5q) patients. Here, we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2f/+Vav1cre) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2f/+Vav1cre erythroid precursors suggested immune system activation and transcriptomic analysis revealed an increase in p53-dependent interleukin-22 (IL-22) in Riok2f/+Vav1cre CD4+ T cells (TH22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2f/+Vav1cre mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of del(5q) MDS patients as well as patients with anemia secondary to chronic kidney disease (CKD). This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.

Highly Mutated Antibodies Capable of Neutralizing N276-Glycan Deficient HIV after a Single Immunization with an Env Trimer

Elicitation of broadly neutralizing antibodies (bnAbs) is a major challenge in HIV vaccine development, because unmutated precursors to bnAbs are rare and typically do not bind the wildtype HIV Envelope glycoprotein (Env) trimers. One strategy to initiate bnAb type responses is to use germline-targeting immunogens that have high affinity to bnAb-class precursor B cells, then shepherd affinity maturation with booster immunogens that successively look more similar to native Env. Here, we demonstrate in a rare VRC01gHL (inferred germline of bnAb VRC01) B cell precursor model in mouse, starting from a physiological 1 in 1 million VRC01 gHL precursor frequency, a single immunization with a germline targeting soluble Env trimer can generate a significant number of high affinity bnAb-class somatic hypermutations (SHM). Notably, deletions in the light chain complementarity determining region 1 were observed in multiple germinal center (GC) B cell clones after a single immunization. Many of the GC-derived VRC01gHL antibodies developed binding to the wildtype Env trimer without an N276 glycan. Antibodies elicited after a single priming immunization neutralized several N276 glycan-deficient tier-2 HIV strains. These results are encouraging for germline-targeting Env trimer vaccine designs, demonstrating it can be possible to accumulate substantial SHM, including indels, and functional bnAb features after a single immunization.

Development of a method for the identification of receptor activator of nuclear factor-κB+ populations in vivo

ObjectivesOsteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK+ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK+ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). MethodsWe sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro. ResultsThe frequency of the RANK+ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK+ cells were detected in both the B220+ and B220− populations, the macrophage lineage was present only in B220−. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK−CSF-1R+ cells than in RANK+CSF-1R+ cells contained in the B220− population. In contrast, the PB-derived B220−RANK+CSF-1R+ population contained a significantly higher frequency of osteoclast precursors than the B220−RANK−CSF-1R+ population. ConclusionsThese results suggest that GST-RANKL-biotin is useful for the detection of RANK+ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.

Negative Feedback by NUR77/ <i>Nr4a1</i> Restrains B Cell Clonal Dominance During Early T-Dependent Immune Responses

B cell clones compete for entry into and dominance within germinal centers (GC), where the highest affinity BCRs are selected. However, diverse and low affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesized that a negative feedback loop may operate within B cells to preferentially restrain high affinity clones from monopolizing the early GC niche. Here we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We previously showed that NUR77 expression scales with antigen stimulation and restrains B cell expansion when T cell help is limiting. Here we show that, although NUR77 is dispensable for regulating GC size when GC are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. Moreover, this is independent of B cell precursor frequency and reflects, at least in part, a B cell-intrinsic role for NUR77. We propose that this is important to preserve early B cell clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

Modulating the quantity of HIV Env-specific CD4 T cell help promotes rare B cell responses in germinal centers.

Immunodominance to nonneutralizing epitopes is a roadblock in designing vaccines against several diseases of high interest. One hypothetical possibility is that limited CD4 T cell help to B cells in a normal germinal center (GC) response results in selective recruitment of abundant, immunodominant B cells. This is a central issue in HIV envelope glycoprotein (Env) vaccine designs, because precursors to broadly neutralizing epitopes are rare. Here, we sought to elucidate whether modulating the quantity of T cell help can influence recruitment and competition of broadly neutralizing antibody precursor B cells at a physiological precursor frequency in response to Env trimer immunization. To do so, two new Env-specific CD4 transgenic (Tg) T cell receptor (TCR) mouse lines were generated, carrying TCR pairs derived from Env-protein immunization. Our results suggest that CD4 T cell help quantitatively regulates early recruitment of rare B cells to GCs.

High Precursor Frequency and Promiscuity in Αβ T Cell Receptor Pairing Underpin CD8+ T-Cell Responses to an Immunodominant SARS-CoV-2 Nucleocapsid Epitope

T-cell responses to SARS-CoV-2-derived peptide pools have been documented, however it remains largely unclear whether prominent SARS-CoV-2-specific T cell populations originate from naïve or pre-existing memory sets. As HLA-B*07:02-restricted N105-113 epitope (B7/N105) is the most dominant SARS-CoV-2 CD8+ T-cell specificity to date, we dissected CD8+ T-cell responses directed at this epitope by direct ex vivo analyses in peripheral blood mononuclear cells (PBMCs) from acute and convalescent COVID-19 patients, and pre-pandemic PBMCs, tonsils, lungs and spleens. Using peptide-HLA tetramers, immunodominant B7/N105+CD8+ T-cells were detected at a high frequency (∼2.18x10-4) in COVID-19 patients, comparable to the well-established influenza-specific A2/M158+CD8+ T-cell population. Remarkably, frequencies of B7/N105+CD8+ T-cells were also readily detectable in pre-pandemic PBMCs and tonsils (at 6.55x10-5 and 2.76x10-4, respectively), although they mainly displayed a naïve phenotype, indicating a lack of previous cross-reactive exposures. Ex vivo TCRαβ analyses revealed that a diverse TCRαβ repertoire and promiscuity in αβ TCR pairing underlie such high naïve precursor frequencies of B7/N105+CD8+ T-cells. Overall, our study demonstrates that high precursor frequency and plasticity of TCRα-TCRβ pairing underpin immunodominance of SARS-CoV-2-specific B7/N105+CD8+ T-cell responses and advocates for vaccine strategies which include the nucleocapsid protein to elicit immunodominant CD8+ T-cell responses in HLA-B*07:02 individuals.Funding: This work was supported by theClifford Craig Foundation to KLF and KK, NHMRC Leadership Investigator Grant to KK (1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of theHong Kong Special Administrative Region, China (#T11-712/19-N) to KK, the Victorian Government (SJK, AKW), MRFF award (#2002073) to SJK and AKW, MRFFAward (#1202445) to KK, NHMRC program grant 1149990 (SJK) and NHMRC project grant 1162760 (AKW). AKW is supported by Emerging Leadership 1 Investigator Grant (#1173433), JAJ by an NHMRC Early Career Fellowship (ECF) (#1123673), KK by NHMRC SeniorResearch Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship(#1137285) and SJK by NHMRC Senior Principal Research Fellowship (#1136322). CES has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532). JR is supported by an ARC Laureate fellowship. JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. LH is supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne.Ethical Approval: Experiments conformed to the Declaration of Helsinki Principles and theAustralian National Health and Medical Research Council Code of Practice. Written informed consents were obtained from all blood donors prior to the study. Lung and spleen tissues were obtained from deceased organ donors after written informed consents from the next of kin.Written informed consents were obtained from participants’ parental or guardians for underage tonsil tissue donors. The study was approved by the Alfred Hospital (#280/14), Austin Health (HREC/63201/Austin-2020); the University of Melbourne (#2057366.1, #2056901.1,#2056689, #2056761, #1442952, #1955465, and #1443389), the Australian Red CrossLifeblood (ID 2015#8), the Tasmanian Health and Medical (ID H0017479) and the James Cook University (H7886) Human Research Ethics Committees.