Two doses of mRNA vaccine elicit cross-neutralizing memory B-cells against SARS-CoV-2 Omicron variant

SARS-CoV-2 Beta and Omicron variants have multiple mutations in the receptor-binding domain (RBD) allowing antibody evasion. Despite the resistance to circulating antibodies in those who received two doses of mRNA vaccine, the third dose prominently recalls cross-neutralizing antibodies with expanded breadth to these variants. Herein, we longitudinally profiled the cellular composition of persistent memory B-cell subsets and their antibody reactivity against these variants following the second vaccine dose. The vaccination elicited a memory B-cell subset with resting phenotype that dominated the other subsets at 4.9 months. Notably, most of the resting memory subset retained the ability to bind the Beta variant, and the memory-derived antibodies cross-neutralized the Beta and Omicron variants at frequencies of 59% and 29%, respectively. The preservation of cross-neutralizing antibody repertoires in the durable memory B-cell subset likely contributes to the prominent recall of cross-neutralizing antibodies following the third dose of the vaccine.

In-Depth Profiling of T-Cell Responsiveness to Commonly Recognized CMV Antigens in Older People Reveals Important Sex Differences

The impact of biological sex on T-cell immunity to Cytomegalovirus (CMV) has not been investigated in detail with only one published study comparing CMV-specific T-cell responses in men and women. Many studies, however, have shown an association between CMV infection and immunosenescence, with broad effects on peripheral blood lymphocyte subsets as well as the T and B-cell repertoires. Here, we provide a detailed analysis of CMV-specific T-cell responses in (n=94) CMV+ older people, including 47 women and 47 men aged between 60 and 93 years. We explore sex differences with respect to 16 different CMV proteins arranged in 14 peptide pools (overlapping peptides). Following ex vivo stimulation, CD4 and CD8 T-cells producing IFN-γ, TNF, and IL-2 were enumerated by flow-cytometry (intracellular cytokine staining). T-cell responses were evaluated in terms of each cytokine separately or in terms of cytokines produced simultaneously (polyfunctionality). Surface memory phenotype and CD3 downmodulation were assessed in parallel. The polyfunctionality index and a memory subset differentiation score were used to identify associations between response size, cytokine production, polyfunctionality, and memory subset distribution. While no significant sex differences were found with respect to overall CMV target protein selection, the T-cell response in men appeared more focused and accompanied by a more prominent accumulation of CMV-specific memory CD4 and CD8 T-cells. T-cell polyfunctionality and differentiation were similar in the sexes, however, CMV-specific T-cells in men produced more pro-inflammatory cytokines. Particularly, TNF production by CD4 T-cells was stronger in men than in women. Also, compared with women, men had larger responses to CMV proteins with immediate-early/early kinetics than women, which might have been driven by CMV reactivation. In conclusion, the CMV-specific T-cell response in men was larger and more pro-inflammatory than in women. Our findings may help explain sex differences in CMV-associated pathologies.

548 CD8+ tissue-resident memory T cells are tumour reactive and increase after immunotherapy in a case of metastatic mucosal melanoma

BackgroundMucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors (ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment.MethodsWe investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1).ResultsCD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets.Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanomaConclusionsIn this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets.AcknowledgementsThe authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding.Ethics ApprovalPatients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively.ReferencesCarvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. [cited 2020 Apr 1]; Available from: https://www.uptodate.com/contents/mucosal-melanomaDel Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7.Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32.D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35.Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. Antitumour activity of pembrolizumab in advanced mucosal melanoma: a post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer 2018;119(6):670–4.Boddupalli CS, Bar N, Kadaveru K, Krauthammer M, Pornputtapong N, Mai Z, et al. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells. JCI Insight [Internet]. 2016 Dec 22 [cited 2019 Apr 24];1(21). Available from: https://insight.jci.org/articles/view/88955Edwards J, Wilmott JS, Madore J, Gide TN, Quek C, Tasker A, et al. CD103+ Tumor-resident CD8+ T cells are associated with improved survival in immunotherapy-naïve melanoma patients and expand significantly during anti-PD-1 treatment. Clin Cancer Res Off J Am Assoc Cancer Res 2018 Jul 1;24(13):3036–45.Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018 Jul;24(7):986–93.

Fc receptor-like 4 and 5 define human atypical memory B cells

Atypical memory B cells accumulate in chronic infections and autoimmune conditions, and commonly express FCRL4 and FCRL5, respective IgA and IgG receptors. We characterized memory cells from tonsils on the basis of both FCRL4 and FCRL5 expression, defining three subsets with distinct surface proteins and gene expression. Atypical FCRL4+FCRL5+ memory cells had the most discrete surface protein expression and were enriched in cell adhesion pathways, consistent with functioning as tissue-resident cells. Atypical FCRL4−FCRL5+ memory cells expressed transcription factors and immunoglobulin genes that suggest poised differentiation into plasma cells. Accordingly, the FCRL4−FCRL5+ memory subset was enriched in pathways responding to endoplasmic reticulum stress and IFN-γ. We reconstructed ongoing B-cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. Classical FCRL4−FCRL5− memory cells were infrequently detected in lineage trees, suggesting the majority were in a quiescent state. FCRL4−FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Together, these differences suggest FCRL4 and FCRL5 are unlikely to be passive markers but rather active drivers of human memory B-cell development and function.

Relative Contribution of Naïve and Memory T Cells to Alloreactivity in Hematopoietic Cell Transplantation

Introduction: Graft-versus-host disease (GvHD) is a major impediment to the cure of blood disorders by hematopoietic cell transplantation (HCT). GvHD is mediated by alloreactive T cells recognizing histocompatibility antigen (HAg) mismatches between patient and donor. Naïve T cells are thought to be the main mediators of alloreactive responses since, theoretically, memory T cells would have never been exposed to and selected by alloantigens, except in multiparous women or transfused individuals. Accordingly, clinical trials using naïve T cell-depleted allografts are being conducted with the aim to reduce GvHD after human leukocyte antigen (HLA)-matched HCT. However, several groups have shown that memory T cells can also mediate alloreactive responses, in particular against mismatched HLA. We hypothesized that the relative importance of naïve vs. memory T cell alloreactivity depends on the matching status of the patient-donor pair. Specifically, we reasoned that naïve-depletion strategies will be most efficient in HLA-identical sibling HCT, where minor (m)HAg presented by self-HLA are the only targets of T cell alloreactivity, but less so in HLA-matched unrelated HCT, where HLA-DPB1 mismatches (mmDPB1) are frequent and potentially recognized through molecular mimicry by both naïve and memory T cells. Methods: In order to model T cell alloreactivity to mHAg and to major HLA mismatches post HCT, we used a quantitative in vitro assay based on co-culture of responder and stimulating cells. Naïve (CD45RA+CD45RO-) and memory (CD45RA-CD45RO+) CD4+ T cells were enriched from peripheral blood mononuclear cells from healthy individuals using microbead technology to >95% purity and used as responders. Irradiated transduced HeLa cells engineered to express single HLA-DP antigens and the necessary machinery for HLA class II antigen presentation were used to stimulate CD4+ T cells. HeLa transductants expressing the autologous (i.e. DP-matched, response restricted to mHAg) or an allogeneic (mmDPB1) DP antigen were used to challenge naïve and memory CD4+ cells from each responder. After 14 days of culture, T cells were restimulated overnight and the levels of T cell response were quantified by cell surface expression of the activation marker CD137. Results: In 36 independent T cell cultures from 8 different individuals, the overall levels of alloreactivity against mHAg were significantly lower than those against mmDPB1 (mean 50.3% vs 20.7%, p<0.0001) (Figure 1A). Consistent with current concepts, alloreactivity to mHAg was significantly higher in the naïve than in the memory subset (mean 27.7% vs 10.5%, p=0.015) (Figure 1B). This was most evident in 5/8 responders (mean 38.4% vs 13.3%, p=0.016), in particular in females under 40 years of age. In 3 of the 8 responders, mHAg alloreactivity was generally low and not significantly different between the naïve and the memory subsets (mean 10.3% vs 12.9%, p=0.73). In contrast, alloreactivity against mmDPB1 was evenly distributed between the naïve and the memory subset (mean 52.1% vs 48.5%, p=0.62) in all responders, independent of age, sex or cytomegalovirus serostatus of the responder (Figure 1C). Interestingly, naïve DPB1*04:01-restricted mHAg alloreactive CD4+ T cells were able to cross-recognize the structurally similar (i.e. permissive) DPB1*04:02 (mean 43.3%) but not the dissimilar (i.e. non-permissive) DPB1*09:01 (mean 14.1%) (Figure 1D). Moreover, when purified CD4+ cells from self-DPB1*04:01 homozygous donors were challenged with DPB1*04:02 or DPB1*09:01, naïve CD4+ T cells were the main source of alloreactive responses against the permissive mmDPB1 (mean 25.0% vs 7.4% for naïve and memory cells, respectively), while both memory (mean 50.0%) and naïve (mean 46.0%) CD4+ cells elicited strong alloresponses against the non-permissive mmDPB1. Conclusion: Our data provide the first direct experimental evidence that alloreactivity against mmDPB1 is stronger than against mHAg, and importantly that it is mediated equally by naïve and memory CD4+ T cells while the mHAg response is mediated mainly by the naïve subset. However, our data also suggests that some mmDPB1 involving structurally (and hence functionally) similar alleles (in general permissive) might behave similarly to DPB1 matches. These observations should be taken into account in clinical trials aimed at improving the outcome of unrelated HCT by selective depletion of naïve T cells. Disclosures Turki: Jazz Pharmaceuticals, CSL Behring, MSD.: Consultancy; Neovii Biotech, all outside the submitted work: Other: Travel subsidies. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria.

Longitudinal Changes in Cd4+, Cd8+ T Cell Phenotype and Activation Marker Expression Following Antiretroviral Therapy Initiation among Patients with Cryptococcal Meningitis

Despite improvement in the prognosis of HIV/AIDS (human immunodeficiency virus/acquired immune deficiency syndrome) patients on antiretroviral therapy (ART), cryptococcal meningitis (CM) still causes 10–15% mortality among HIV-infected patients. The immunological impact of ART on the CD4+ and CD8+ T cell repertoire during cryptococcal co-infection is unclear. We determined longitudinal phenotypic changes in T cell subsets among patients with CM after they initiated ART. We hypothesized that ART alters the clonotypic phenotype and structural composition of CD4+ and CD8+ T cells during CM co-infection. For this substudy, peripheral blood mononuclear cells (PBMC) were isolated at four time points from CM patients following ART initiation during the parent study (ClinicalTrials.gov number, NCT01075152). Phenotypic characterization of CD4+ and CD8+ T cells was done using T cell surface marker monoclonal antibodies by flow cytometry. There was variation in the expression of immunophenotypic markers defining central memory (CD27+CD45R0+), effector memory (CD45R0+CD27–), immune activation (CD38+ and Human Leucocyte Antigen DR (HLA-DR+), and exhaustion (Programmed cell death protein one (PD-1) in the CD4+ T cell subset. In comparison to the CD4+ T cell population, the CD8+ central memory subset declined gradually with minimal increase in the effector memory subset. Both CD4+ and CD8+ T cell immune exhaustion and activation markers remained elevated over 12 weeks. The relative surge and decline in the expression of T cell surface markers outlines a variation in the differentiation of CD4+ T cells during ART treatment during CM co-infection.