Faculty Bibliography

Memory CD4 T cells are critical to human immunity, yet it is unclear whether viral inflammation during memory formation has long-term consequences. Here, we compared transcriptional and epigenetic landscapes of Spike (S)-specific memory CD4 T cells in 24 individuals whose first exposure to S was via SARS-CoV-2 infection or mRNA vaccination. Nearly 2 years after memory formation, S-specific CD4 T cells established by infection remained enriched for transcripts related to cytotoxicity and for interferon-stimulated genes, likely because of a chromatin accessibility landscape altered by inflammation. Moreover, S-specific CD4 T cells primed by infection had reduced proliferative capacity in vitro relative to vaccine-primed cells. Furthermore, the transcriptional state of S-specific memory CD4 T cells was minimally altered by booster immunization and/or breakthrough infection. Thus, infection-associated inflammation durably imprints CD4 T cell memory, which affects the function of these cells and may have consequences for long-term immunity.

Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.

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The 2022 global outbreak of clade IIb mpox represented a turning point in public health's handling of poxviruses. The primary vaccine available for the prevention of mpox is modified vaccinia Ankara from Bavarian-Nordic (MVA-BN). We previously reported a nondurable and low-avidity antibody response against mpox elicited by MVA-BN. In this study, we expanded upon this knowledge by employing a microneutralization assay to measure monkeypox virus (MPXV) neutralizing titers and a multiplexed immunoassay to assess IgG titers and avidity against eight MPXV antigens and two vaccinia antigens. Through a machine learning analysis, we uncovered that MVA-BN vaccinees without prior smallpox vaccination largely return to a baseline seroprofile within a year of immunization. Notably, we identified a discrete population within this group that mounted a robust neutralizing antibody response associated with a longer dosing interval during the MVA-BN primary series. Furthermore, we found that boosting with a third dose of MVA-BN increases IgG avidity against certain MPXV antigens, as part of a booster-specific seroprofile. These findings provide critical insights into optimizing immune responses through MVA-BN boosters, presenting a potential approach to addressing the limited MPXV-specific immunity observed following the primary series.

Xenotransplantation of genetically-modified pig kidneys offers a solution to the scarcity of organs for end-stage renal disease patients.¹ We performed a 61-day alpha-Gal knock-out pig kidney and thymic autograft transplant into a nephrectomized brain-dead human using clinically approved immunosuppression, without CD40 blockade or additional genetic modification. Hemodynamic and electrolyte stability and dialysis independence were achieved. Post-operative day (POD) 10 biopsies revealed glomerular IgM and IgA deposition, activation of early complement components and mesangiolysis with stable renal function without proteinuria, a phenotype not seen in allotransplantation. On POD 33, an abrupt increase in serum creatinine was associated with antibody-mediated rejection and increased donor-specific IgG. Plasma exchange, C3/C3b inhibition and rabbit anti-thymocyte globulin (rATG), completely reversed xenograft rejection. Pre-existing donor-reactive T cell clones expanded progressively in the circulation post-transplant, acquired an effector transcriptional profile and were detected in the POD 33 rejecting xenograft prior to rATG treatment. This study provides the first long-term physiologic, immunologic, and infectious disease monitoring of a pig-to-human kidney xenotransplant and indicates that pre-existing xenoreactive T cells and induced antibodies to unknown epitope(s) present a major challenge, despite significant immunosuppression. It also demonstrates that a minimally gene-edited pig kidney can support long-term life-sustaining physiologic functions in a human.

Organ shortage remains a major challenge in transplantation, and gene-edited pig organs offer a promising solution^1-3. Despite gene-editing, the immune reactions following xenotransplantation can still cause transplant failure⁴. To understand the immunological response of a pig-to-human kidney xenotransplantation, we conducted large-scale multi-omics profiling of the xenograft and the host's blood over a 61-day procedure in a brain-dead human (decedent) recipient. Blood plasmablasts, natural killer (NK) cells, and dendritic cells increased between postoperative day (POD)10 and 28, concordant with expansion of IgG/IgA B-cell clonotypes, and subsequent biopsy-confirmed antibody-mediated rejection (AbMR) at POD33. Human T-cell frequencies increased from POD21 and peaked between POD33-49 in the blood and xenograft, coinciding with T-cell receptor diversification, expansion of a restricted TRBV2/J1 clonotype and histological evidence of a combined AbMR and cell-mediated rejection at POD49. At POD33, the most abundant human immune population in the graft was CXCL9+ macrophages, aligning with IFN-γ-driven inflammation and a Type I immune response. In addition, we see evidence of interactions between activated pig-resident macrophages and infiltrating human immune cells. Xenograft tissue showed pro-fibrotic tubular and interstitial injury, marked by S100A6⁵, SPP1⁶ (Osteopontin), and COLEC11⁷, at POD21-POD33. Proteomics profiling revealed human and pig complement activation, with decreased human component after AbMR therapy with complement inhibition. Collectively, these data delineate the molecular orchestration of human immune responses to a porcine kidney, revealing potential immunomodulatory targets for improving xenograft survival.

UNLABELLED:The 2022 global outbreak of clade IIb mpox was the first major outbreak of mpox outside of African nations. To control the outbreak, public health officials began vaccination campaigns using the third-generation orthopoxvirus vaccine modified vaccinia Ankara from Bavarian Nordic (MVA-BN). Prior to this outbreak, the durability of monkeypox virus (MPXV)-specific immunity induced by MVA-BN was poorly understood. In 2022, we launched the New York City Observational Study of Mpox Immunity (NYC OSMI, NCT05654883), a longitudinal study of 171 participants comprising MVA-BN vaccines and mpox convalescent individuals. Peripheral blood sampling was performed at intervals including prior to vaccination, after one dose, and after the second dose. MVA-BN vaccinees with and without a history of smallpox vaccination demonstrated detectable MPXV-specific memory B cells at 1-year post-vaccination. Additionally, MVA-BN increased MPXV neutralizing titers in smallpox vaccine-naïve vaccinees, with a comparable maximum titer reached in naïve and smallpox vaccine-experienced vaccinees. However, neutralizing titers returned to baseline within 5-7 months for naïve individuals, while remaining elevated in those with prior smallpox vaccination. Both naïve and experienced individuals generated robust IgG responses against MPXV H3 and A35, but naïve vaccinees' IgG responses showed lower avidity than experienced vaccinees. These data highlight a low avidity antibody response elicited by MVA-BN that is short-lived in naïve vaccinees. This work supports the need for long-term studies on protection induced by MVA-BN, including the potential need for booster doses as well as the development of next-generation orthopoxvirus vaccines. IMPORTANCE/OBJECTIVE:The ongoing outbreaks of mpox demonstrate the continuing threat of orthopoxviruses to global health. While previous orthopoxvirus vaccines generated lifelong antibody and cellular immunity, we show here that the current mpox vaccine, MVA-BN or JYNNEOS, fails to induce durable antibody immunity in individuals with no prior smallpox vaccination. This raises the important question of whether MVA-BN vaccinees have long-term protection from mpox. Our work highlights the need for further studies into the durability of protection generated by MVA-BN as well as whether subsequent booster doses are necessary to maintain protection.

The mechanisms underlying the efficacy of anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) therapy are incompletely understood. Here, by immune profiling responding PD-1⁺CD8⁺ T (T_Resp) cell populations from patients with advanced melanoma, we identified differential programming of T_Resp cells in response to combination therapy, from an exhausted toward a more cytotoxic effector program. This effect does not occur with anti-PD-1 monotherapy. Single-cell transcriptome and T cell receptor repertoire analysis was used to identify altered effector programming of expanding PD-1⁺CD8⁺ T cell clones with distinct regulon usage, STAT1 and STAT3 utilization and antitumor specificity connected to interleukin (IL)-21 signaling in combination and anti-CTLA-4 monotherapy. Therapeutic efficacy of CTLA-4 blockade was lost in B16F10 melanoma models with either Il21r^- deficiency or anti-IL-21 receptor blockade. Together, these results show how IL-21 signaling to T_Resp is critical for anti-CTLA-4-based checkpoint therapies and highlight major signaling differences to anti-PD-1 monotherapy.

Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. Here, we perform paired single-cell RNA and T cell receptor (TCR) sequencing across time in 36 patients with stage IV melanoma treated with anti-PD-1, anti-CTLA-4, or combination therapy. We develop the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induces waves of clonal T cell responses that peak at distinct time points. Combination therapy results in greater magnitude of clonal responses at 6 and 9 weeks compared to single-agent therapies, including melanoma-specific CD8⁺ T cells and exhausted CD8⁺ T cell (T_EX) clones. Focused analyses of T_EX identify that anti-CTLA-4 induces robust expansion and proliferation of progenitor T_EX, which synergizes with anti-PD-1 to reinvigorate T_EX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies.