key: cord-0740913-ov3n3kp4
authors: Soni, Mithil; Migliori, Edoardo; Assal, Amer; Chan, Hei T; Ciubotariu, Rodica; Pan, Jian B.; Cicero, Kara; Pereira, Marcus; Mapara, Markus Y.; Muranski, Pawel
title: Development of T cell immunity in a recipient of liver and hematopoietic stem cell transplant following COVID-19 infection.
date: 2021-05-31
journal: Cytotherapy
DOI: 10.1016/j.jcyt.2021.05.005
sha: 8d04e8cf9ae5bf5902e3a649071d478c644aeea0
doc_id: 740913
cord_uid: ov3n3kp4

nan

The outbreak of coronavirus disease 2019 has disproportionately affected patients with comorbidities, including recipients of solid organ and hematopoietic stem cell transplants 

The ongoing COVID-19 pandemic has disproportionately affected older adults and individuals with co-morbidities including the solid organ (SOT) and hematopoietic stem cell transplant (SCT) recipients. Those patients typically have complex underlying medical conditions affecting multiple organs and, in the case of SOT recipients, life-long immunosuppression is required to prevent graft loss. Consequently, COVID-19 has had devastating yet highly variable outcomes in reported series of SOT recipients, including subjects over 10 years post-transplant [1, 2] . Among liver transplant recipients, a high hospitalization rates and overall case fatality ratio of 12% to 30% has been reported in European cohorts [3, 4] . While the severe and fatal cases of COVID-19 attract the most attention, the spectrum of this disease predominantly includes fully recovered survivors with relatively mild infection courses even among highly vulnerable subjects with comorbidities, including transplant recipients. Healthy survivors of COVID-19 develop variable degrees of post-infection humoral immunity evidenced by the presence of antibodies against SARS-CoV2 antigens upon serological testing [5] .

Although cellular adaptive immunity against COVID-19 mediated by T cells is less understood, emerging data suggest that T cell responses may be long-lived and crucial in mediating protection against re-infection, possibly well beyond the period of seropositivity [6] [7] [8] [9] [10] . The extent and magnitude of humoral and cellular immunity in vulnerable transplant recipients who survived COVID-19 remains poorly characterized [11] . Here we report the case of a solid organ and bone marrow transplant recipient with evidence of SARS-CoV2 infection. We performed a comprehensive analysis of in vitro T cell responses to SARS-CoV2 and other viruses via microculture prime/expansion methodology, previously developed in our laboratory as a measure of immunocompetence in subjects with JC virus-associated progressive multifocal leukoencephalopathy (PML) [12, 13] . This technique expands even the rare memory precursors from seropositive subjects and has been originally used at a large scale for generation of clinicalgrade virus-specific T cells targeting simultaneously multiple epitopes for adoptive immunotherapy application. The ability to target common latent viruses including CMV, EBV, AdV and BK polyomavirus was evaluated, as compared to the responses against the immunodominant antigens from SARS-CoV2 to understand the COVID-19 induced postinfection immunological memory in the context of overall anti-viral immunity in this life-long immunosuppressed patient. Furthermore, we also studied the responses against structurally related counterpart antigens from non-SARS hCoVs 229E, OC43, NL63 and HKU1, which are commonplace respiratory pathogens associated with the self-limiting upper respiratory tract infections ("common cold") in healthy individuals, but capable of causing serious lower respiratory tract infections and prolonged shedding in vulnerable subjects with comorbidities [14] [15] [16] . T cell responses against those viruses have not been well characterized.

Sample collection and cryopreservation: Peripheral blood was collected after obtaining the informed consent from healthcare workers (healthy donors) and patients with hematological disorders with and without documented history of COVID-19 under the CUMC IRB approved study of T cell immunity against CoVs. PBMCs were isolated using gradient separation and cryopreserved Cryostor 10 freezing media. For this analysis samples from the three randomly selected sero-negative healthcare workers were used as control. 

The patient is a 33-year-old male with EPP, originally diagnosed at the age of 5. EPP is an inborn deficiency of ferrochelatase in which protoporphyrin accumulates within the bone marrow and in the hepatocytes, causing direct hepatobiliary toxicity [17] . The patient developed liver cirrhosis at the age of 18 despite the medical management with hemin and plasmapheresis.

Consequently, in 2010 he underwent liver transplantation at the age of 20, but the hepatic damage from EPP recurred. Two years later, the patient underwent a double cord allo-SCT with the intention to prevent further liver injury. However, SCT was unsuccessful due to primary graft failure. Over the next three years he experienced the gradual progression of liver disease with the development of portal hypertension and hypersplenism further exacerbating pancytopenia. In 2013, the patient received a second OLT with splenectomy followed by a second allo-SCT 5 and IE-1, BK large T (LT) and viral protein 1 (VP1), EBV BZLF1 and EBNA1, and ADV penton (Ad5). Antigen specific polyfunctional (TNF-α and IL-2 are shown) cytokine release was observed within both the CD4 + and CD8 + compartments depending on the viral target in what is likely a donor-specific pattern (Figure 1) . We found robust reactivity against both CMV antigens, BK LT and VP1 and Ad5. However, less potent reactivity specific for the two tested EBV pepmixes was seen. Overall, we concluded that this complex patient with history of SOT, splenectomy and allogeneic SCT retains a remarkable degree of immunocompetence.

Next, we evaluated the T cell responses against the immunodominant structural proteins from SARS-CoV2 including S1, S2, M and NP. Surprisingly, we observed remarkable activity against all studied antigenic targets, especially within the CD4 + T cell compartment (Figure 2A and   2B ), while very little activity was seen in randomly selected otherwise healthy seronegative healthcare workers (Figure 2C , representative example shown). The highest reactivity was seen against S2 (26.9%) followed by NP (13.53%), M (12.47%) and S1 (11.73%), thus the magnitude of the observed reactivity was comparable to the well documented immunodominant components of CMV and BK viruses (compare with Figure 1 ), indicating development of robust multiepitope specific post-infection cellular immunity, despite active immunosuppression. Since SARS-CoV2 is a beta-CoV, we hypothesized that the post-COVID-19 immunity might extend to the other related members of the hCoV family. Therefore, we evaluated the immune responses against beta-hCoVs OC43 and HKU1 as well as alpha-hCoVs 229E and NL63. Indeed, upon testing against the custom-synthesized panel of peptide libraries, a marked immunological reactivity was observed against S1, S2, NP and M antigens from all evaluated hCoVs, with the most profound responses revealed in the CD4 + T cell compartment and targeting the NP antigen (range 9.64 to 14.1%) followed by S1 (range 10.1 to 6.31%) (Figure 2A, 2B, 2D) , while relatively lower activity was seen in seronegative healthy donors (Figure 2C and 2D) . Activity within the CD8 + T cell compartment was seen against NP and M antigens derived from alpha-hCoVs 229E and NL63, but minimally against beta-hCoVs, pointing to the inherent differences in immunogenicity between those pathogens. Furthermore, the SARS-CoV2 specific CD4 + T cells from this patient retained significant polyfunctionality, including robust ability to secrete IL-2, a feature associated with long-term persistence and lack of exhaustion. While the subject appeared to be highly capable of mounting immune responses against all tested hCoVs the magnitude of reactivity was lower than the responses against SARS-CoV2, suggesting that the recent COVID-19 infection played the most significant role in shaping his immune repertoire.

Post-COVID-19 humoral immunity is thought to induce neutralizing antibodies that provide a period of relative protection in convalescent subjects. However, antibody titers and half-lives are highly variable, and some subjects might never mount a detectable antibody response [5] . The role of T cell immunity in host defenses against SARS-CoV2 and other hCoVs is less understood, but otherwise healthy COVID-19 and SARS-CoV1 infection survivors develop potent long-lived antigen-specific immunological memory against the immunodominant antigens [7, 9, 18] . It is therefore imperative to characterize these responses in vulnerable subjects with immunodeficiencies as a tool to gauge the overall immunocompetence and possible degree of long-term protection. It is also possible to exploit the in vitro generated SARS-CoV2 T cells for adoptive immunotherapy of COVID-19, as proposed recently by Keller et. al [19] .

Patients with a history of SOT and SCT are a highly heterogeneous group with various degrees of immune dysfunction and baseline inflammation, depending on the interval from transplant, conditioning regimen used, other on-going organ dysfunction and concurrent immunosuppressive therapies. Furthermore, some immunosuppressive pharmacological agents, such as steroids have been shown to ameliorate COVID-19 symptoms and prevent progression to severe respiratory distress syndrome; however, they may also blunt the development of postinfection immunity or abrogate the responses to vaccination. Overall, the patient described here is considered as high risk in terms of risk for infectious complications [20] . Nevertheless, despite severe comorbidities, compounded risk factors and lifelong immunosuppression the subject survived uncomplicated COVID-19 and was incidentally found to be seropositive upon routine screening.

We estimated the T cell immunity against viral antigens using a highly sensitive functional method of measuring broad T cell memory responses [12] . Our analysis revealed development of a highly active cellular immunity against multiple antigens derived from SARS-CoV2.

Strikingly, the magnitude of the SARS-CoV2-specific T cell responses was comparable to the well characterized immunity against CMV, BK, AdV and EBV and much higher than in healthy unexposed controls. Moreover, this COVID-19 survivor also had the capability of recognizing antigens from all four non-SARS hCoVs, albeit at the lower magnitude/frequency. Importantly, our analysis extended beyond the S1 and S2 antigens, commonly examined in the context of the currently approved anti-COVID-19 vaccines, but also includes the far less studied M and NP proteins from SARS-CoV2 and all four common hCoVs suggesting possible heterologic crossprotection against future infections including emerging mutant variants of SARS-CoV2 and other members of CoV family [6] . Recent evidence suggests that there might be a tendency for a milder course of the disease in COVID-19 patients who had recently experienced hCoV-related infections with some cross-reactive antibodies increasing post-COVID [10, [21] [22] [23] .

Unfortunately, in the patient described here magnitude and breath of the pre-existing T cell memory specific for hCoV antigens is unknown. We are exploring this question in other healthy and immunodeficient individuals.

Overall, this case reveals that at least some SOT and SCT recipients maintained on long-term calcineurin inhibitor retain robust T cell immunocompetence and develop potent cellular immunity against SARS-CoV2. Our strategy allows to measure these responses in the context of broader anti-viral immunity and use the data for risk stratification, as well as to forecast the outcomes of vaccination and provide the rationale for possible use of in vitro expanded SARS-CoV2 T cells for adoptive immunotherapy. Specific TNF-α secretion in cultured primed with S1 and S2 antigens from the indicated beta and alpha CoVs is shown. S-CoV2 = SARS-CoV2. B. Reactivity of cultures primed with M and NP pepmixes from the indicated CoVs is shown. C. SARS-CoV2-specific reactivity of in vitro expanded T cells from a healthy COVID-19 seronegative donor (representative data shown). D.

Summary of antigen-specific reactivity against indicated viral antigens for SARS-CoV2 and related hCoVs within the CD4 + and CD8 + T cell (CD3 + ) compartment in the described patient, as compared to activity in three seronegative healthy donors is shown. E. Summary of polyfunctionality among the SARS-CoV2 antigen reactive (defined as TNF-α + ) CD4 + T cells specific for indicated antigens.

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