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SARS-CoV-2-specific T cell response has been proven essential for viral clearance, COVID-19 outcome and long-term memory. Impaired early T cell-driven immunity leads to a severe form of the disease associated with lymphopenia, hyperinflammation and imbalanced humoral response. Analyses of acute SARS-CoV-2 infection have revealed that mild COVID-19 course is characterized by an early induction of specific T cells within the first 7 days of symptoms, coordinately followed by antibody production for an effective control of viral infection. In contrast, patients who do not develop an early specific cellular response and initiate a humoral immune response with subsequent production of high levels of antibodies, develop severe symptoms. Yet, delayed and persistent bystander CD8+ T cell activation has been also reported in hospitalized patients and could be a driver of lung pathology. Literature supports that long-term maintenance of T cell response appears more stable than antibody titters. Up to date, virus-specific T cell memory has been detected 22 months post-symptom onset, with a predominant IL-2 memory response compared to IFN-γ. Furthermore, T cell responses are conserved against the emerging variants of concern (VoCs) while these variants are mostly able to evade humoral responses. This could be partly explained by the high HLA polymorphism whereby the viral epitope repertoire recognized could differ among individuals, greatly decreasing the likelihood of immune escape. Current COVID-19-vaccination has been shown to elicit Th1-driven spike-specific T cell response, as does natural infection, which provides substantial protection against severe COVID-19 and death. In addition, mucosal vaccination has been reported to induce strong adaptive responses both locally and systemically and to protect against VoCs in animal models. The optimization of vaccine formulations by including a variety of viral regions, innovative adjuvants or diverse administration routes could result in a desirable enhanced cellular response and memory, and help to prevent breakthrough infections. In summary, the increasing evidence highlights the relevance of monitoring SARS-CoV-2-specific cellular immune response, and not only antibody levels, as a correlate for protection after infection and/or vaccination. Moreover, it may help to better identify target populations that could benefit most from booster doses and to personalize vaccination strategies.

Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and/or a defective antibody response to T-dependent and T-independent antigens. CVID response to immunization depends on the antigen type, the vaccine mechanism, and the specific patient immune defect. In CVID patients, humoral and cellular responses to the currently used COVID-19 vaccines remain unexplored. Eighteen CVID subjects receiving 2-dose anti-SARS-CoV-2 vaccines were prospectively studied. S1-antibodies and S1-specific IFN-γ T cell response were determined by ELISA and FluoroSpot, respectively. The immune response was measured before the administration and after each dose of the vaccine, and it was compared to the response of 50 healthy controls (HC). The development of humoral and cellular responses was slower in CVID patients compared with HC. After completing vaccination, 83% of CVID patients had S1-specific antibodies and 83% had S1-specific T cells compared with 100% and 98% of HC (p = 0.014 and p = 0.062, respectively), but neutralizing antibodies were detected only in 50% of the patients. The strength of both humoral and cellular responses was significantly lower in CVID compared with HC, after the first and second doses of the vaccine. Absent or discordant humoral and cellular responses were associated with previous history of autoimmunity and/or lymphoproliferation. Among the three patients lacking humoral response, two had received recent therapy with anti-B cell antibodies. Further studies are needed to understand if the response to COVID-19 vaccination in CVID patients is protective enough. The 2-dose vaccine schedule and possibly a third dose might be especially necessary to achieve full immune response in these patients.

A high frequency of regulatory B (Breg) cells, generally transitional B cells, has been associated with long-term kidney allograft survival and operational tolerance. However, circulating follicular helper T cells (cTfh) correlate with graft rejection. In order to better understand the interplay between these cell subsets and to determine their association with graft outcome we studied transitional and IL10+ Breg cells, as well as cTfh, pre- and post-transplantation in a prospective cohort of 200 kidney transplant recipients and in healthy volunteers. Patients with end-stage kidney disease had higher frequencies of transitional and IL10+ Breg cells compared to controls, and these subsets decreased during the one-year post-transplant follow-up. Higher frequencies of pre-transplant IL10+ Breg cells, and a larger reduction in these cells early post-transplantation, predicted acute rejection and graft failure. Moreover, IL10+ Breg cells correlated with cTfh pre-transplantation, and a post-transplant increase in the cTfh/IL10+Breg ratio preceded acute rejection. Thus, evaluation of pre-transplant IL10+ Breg cells and the regular monitoring of the cTfh/IL10+Breg ratio may be useful to assess post-transplant risk. Hence, our observations suggest the need to develop therapeutic strategies aimed at preserving regulatory B cells, and depleting Tfh, post-transplantation.

Despite the growing number of patients with persistent symptoms after acute SARS-CoV-2 infection, the pathophysiology underlying long-COVID is not yet well characterized, and there is no established therapy. We performed a deep immune profiling in nine patients with persistent symptoms (PSP), before and after a 4-day prednisone course, and five post-COVID-19 patients without persistent symptoms (NSP). PSP showed a perturbed distribution of circulating mononuclear cell populations. Symptoms in PSP were accompanied by a pro-inflammatory phenotype characterized by increased conventional dendritic cells and augmented expression of antigen presentation, co-stimulation, migration, and activation markers in monocytes. The adaptive immunity compartment in PSP showed a Th1-predominance, decreased naïve and regulatory T cells, and augmentation of the PD-1 exhaustion marker. These immune alterations reverted after the corticosteroid treatment and were maintained during the 4-month follow-up, and their normalization correlated with clinical amelioration. The current work highlights an immunopathogenic basis together with a possible role for steroids in the treatment for long-COVID.

An early analysis of circulating monocytes may be critical for predicting COVID-19 course and its sequelae. In 131 untreated, acute COVID-19 patients at emergency room arrival, monocytes showed decreased surface molecule expression, including low HLA-DR, in association with an inflammatory cytokine status and limited anti-SARS-CoV-2-specific T cell response. Most of these alterations had normalized in post-COVID-19 patients 6 months after discharge. Acute COVID-19 monocytes transcriptome showed upregulation of anti-inflammatory tissue repair genes such as BCL6, AREG and IL-10 and increased accessibility of chromatin. Some of these transcriptomic and epigenetic features still remained in post-COVID-19 monocytes. Importantly, a poorer expression of surface molecules and low IRF1 gene transcription in circulating monocytes at admission defined a COVID-19 patient group with impaired SARS-CoV-2-specific T cell response and increased risk of requiring intensive care or dying. An early analysis of monocytes may be useful for COVID-19 patient stratification and for designing innate immunity-focused therapies.

Background In patients with immune-mediated rheumatic diseases (RMD), the development of T-cell responses against SARS-CoV-2 may be impaired by either the immune disturbances associated with the disease, or by the effects of immunosuppressive therapies. We aimed at determining the magnitude of SARS-CoV-2-specific interferon (IFN)-γ-producing T-cell response after COVID-19 recovery in a cohort of patients with RMD on different immunosuppressive therapies. Patients and methods 53 adult patients with inflammatory or autoimmune RMD and 61 sex and age-matched non-RMD patients with confirmed COVID-19 were included. Peripheral blood mononuclear cells were obtained and T-cell-IFN-γ antigen-specific responses against the S1 domain of the spike glycoprotein, the nucleoprotein (N) and the membrane (M) protein from SARS-CoV-2 were assessed by FluoroSpot assay. Results Patients with RMD and COVID-19 showed positive T-cells-IFN-γ responses to SARS-COV-2 antigens, in a similar proportion and magnitude as non-RMD patients at a median of 298 [151–316] and 165 [162–167] days after COVID-19 respectively. Among RMD patients 83%, 87% and 90%, and among non-RMD patients, 95%, 87% and 93% responded to S1, N and M protein respectively. Similar responses were observed in the different diagnostic and therapeutic groups, including conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs), TNF-α inhibitors, IL-17 inhibitors, rituximab, JAK inhibitors or other immunosuppressants. Conclusion T-cell responses to the main SARS-CoV-2 antigens are present after COVID-19 recovery in most patients with RMD and are not impaired by immunosuppressive therapies.

IntroductionThe rapid development of vaccines to prevent COVID-19 has raised the need to compare the capacity of different vaccines in terms of developing a protective humoral response. Previous studies have shown inconsistent results in this area, highlighting the importance of further research to evaluate the efficacy of different vaccines.MethodsThis study utilized a highly sensitive and reliable flow cytometry method to measure the titers of IgG1 isotype antibodies in the blood of healthy volunteers after receiving one or two doses of various vaccines administered in Spain. The method was also used to simultaneously measure the reactivity of antibodies to the S protein of the original Wuhan strain and variants B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.617.1 (Kappa).ResultsSignificant differences were observed in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech, and Ad26.COV.S/Janssen. Furthermore, a relative reduction in the reactivity of the sera with the Alpha, Delta, and Kappa variants, compared to the Wuhan strain, was observed after the second boosting immunization.DiscussionThe findings of this study provide a comparison of different vaccines in terms of anti-S antibody generation and cast doubts on the convenience of repeated immunization with the same S protein sequence. The multiplexed capacity of the flow cytometry method utilized in this study allowed for a comprehensive evaluation of the efficacy of various vaccines in generating a protective humoral response. Future research could focus on the implications of these findings for the development of effective COVID-19 vaccination strategies.

Whether immunosuppression impairs severe acute respiratory syndrome coronavirus 2‐specific T‐cell‐mediated immunity (SARS‐CoV‐2‐CMI) after liver transplantation (LT) remains unknown. We included 31 LT recipients in whom SARS‐CoV‐2‐CMI was assessed by intracellular cytokine staining (ICS) and interferon (IFN)‐γ FluoroSpot assay after a median of 103 days from COVID‐19 diagnosis. Serum SARS‐CoV‐2 IgG antibodies were measured by ELISA. A control group of non‐transplant immunocompetent patients were matched (1:1 ratio) by age and time from diagnosis. Post‐transplant SARS‐CoV‐2‐CMI was detected by ICS in 90.3% (28/31) of recipients, with higher proportions for IFN‐γ‐producing CD4+ than CD8+ responses (93.5% versus 83.9%). Positive spike‐specific and nucleoprotein‐specific responses were found by FluoroSpot in 86.7% (26/30) of recipients each, whereas membrane protein‐specific response was present in 83.3% (25/30). An inverse correlation was observed between the number of spike‐specific IFN‐γ‐producing SFUs and time from diagnosis (Spearman's rho: ‐0.418; P‐value = 0.024). Two recipients (6.5%) failed to mount either T‐cell‐mediated or IgG responses. There were no significant differences between LT recipients and non‐transplant patients in the magnitude of responses by FluoroSpot to any of the antigens. Most LT recipients mount detectable —but declining over time— SARS‐CoV‐2‐CMI after a median of 3 months from COVID‐19, with no meaningful differences with immunocompetent patients.

Antibody-mediated rejection is responsible for 30%-50% of renal graft failures. Differentiation of B cells into antibody-producing plasmablasts depends on the collaboration of follicular helper T cells (Tfh). We analyzed circulating Tfh (cTfh) in kidney recipients and studied cTfh relationship with anti-HLA antibody production and graft outcome. cTfh were longitudinally analyzed in a prospective cohort of patients (n = 206), pre- and posttransplantation. Clinical data, HLA sensitization, and cTfh function were recorded. Both pretransplant and 6-month posttransplant cTfh were able to derive IgG-producing plasmablasts. Pretransplant cTfh was decreased in patients, especially in those who received dialysis. However, these cells were increased in patients with previous allograft or transfusions and in HLA-sensitized recipients. After transplantation cTfh expanded, significantly more in patients who developed de novo anti-HLA antibodies than in patients who remained unsensitized. Augmented pretransplant cTfh positively correlated with higher intensity of pretransplant anti-HLA class I and with de novo anti-HLA class I and anti-HLA class II antibodies. Consistently, pretransplantation cTfh were higher in patients who experienced acute rejection (HR = 1.14 [1.04-1.25]). Thus, we show a role for Tfh in anti-HLA sensitization and rejection. Multicenter studies with additional patient cohorts are needed to validate these results. Immunosuppressive drugs targeting Tfh could be useful to improve outcomes.

Primary infection and/or reactivation of cytomegalovirus (CMV) in kidney transplant recipients (KTR) favour rejection and mortality. T follicular helper cells (TFH) could contribute to protection against CMV. Circulatory TFH (cTFH) were studied pre-transplantation and early post-transplantation in 90 CMV seropositive KTR not receiving antithymocyte globulin or antiviral prophylaxis, followed-up for one year. Patients who presented CMV infection had significantly lower cTFH and activated cTFH pre-transplant and early post-transplantation. Pre-transplantation activated cTFH were also lower within patients who developed CMV disease. Pre- and 14 days post-transplant activated cTFH were an independent protective factor for CMV infection (HR 0.41, p=0.01; and 0.52, p=0.02, respectively). KTR with low cTFH 7 days post-transplantation (

Background Coronavirus disease 2019 (COVID-19) is a highly variable condition. Validated tools to assist in the early detection of patients at high risk of mortality can help guide medical decisions. Objective To validate externally, as well as in patients from the second pandemic wave in Europe, our previously developed mortality prediction model for hospitalized COVID-19 patients. Methods Three validation cohorts were generated: two external with 185 and 730 patients from the first wave and one internal with 119 patients from the second wave. The probability of death was calculated for all subjects using our prediction model, which includes SpO2/FiO2 ratio, neutrophil-to-lymphocyte ratio, LDH, interleukin-6 and age. Discrimination and calibration were evaluated in the validation cohorts. The prediction model was updated by re-estimating individual risk factor effects in the overall cohort (N=1477). Results The mortality prediction model showed good performance in the external validation cohorts 1 and 2, and in the second wave validation cohort 3 (AUC 0.94, 0.86 and 0.86, respectively), with excellent calibration (calibration slope 0.86, 0.94 and 0.79; intercept 0.05, 0.03 and 0.10, respectively). The updated model accurately predicted mortality in the overall cohort (AUC 0.91), which included patients from both the first and second COVID-19 waves. The updated model was also useful to predict fatal outcome in patients without respiratory distress at the time of evaluation. Conclusions This is the first COVID-19 mortality prediction model validated in patients from the first and second pandemic waves. The COR+12 online calculator is freely available to facilitate its implementation (https://utrero-rico.shinyapps.io/COR12_Score/), We externally validated a mortality prediction model for hospitalized COVID-19 patients, which included early one-time measurements of SpO2/FiO2, N/L ratio, LDH, IL-6 and age. The model has been updated with 1477 patients from the first and second waves.

Background Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic. Because the severity of the disease is highly variable, predictive models to stratify patients according to their mortality risk are needed. Objective Our aim was to develop a model able to predict the risk of fatal outcome in patients with COVID-19 that could be used easily at the time of patients' arrival at the hospital. Methods We constructed a prospective cohort with 611 adult patients in whom COVID-19 was diagnosed between March 10 and April 12, 2020, in a tertiary hospital in Madrid, Spain. The analysis included 501 patients who had been discharged or had died by April 20, 2020. The capacity of several biomarkers, measured at the beginning of hospitalization, to predict mortality was assessed individually. Those biomarkers that independently contributed to improve mortality prediction were included in a multivariable risk model. Results High IL-6 level, C-reactive protein level, lactate dehydrogenase (LDH) level, ferritin level, d -dimer level, neutrophil count, and neutrophil-to-lymphocyte ratio were all predictive of mortality (area under the curve >0.70), as were low albumin level, lymphocyte count, monocyte count, and ratio of peripheral blood oxygen saturation to fraction of inspired oxygen (SpO2/FiO2). A multivariable mortality risk model including the SpO2/FiO2 ratio, neutrophil-to-lymphocyte ratio, LDH level, IL-6 level, and age was developed and showed high accuracy for the prediction of fatal outcome (area under the curve 0.94). The optimal cutoff reliably classified patients (including patients with no initial respiratory distress) as survivors and nonsurvivors with 0.88 sensitivity and 0.89 specificity. Conclusion This mortality risk model allows early risk stratification of hospitalized patients with COVID-19 before the appearance of obvious signs of clinical deterioration, and it can be used as a tool to guide clinical decision making.

BACKGROUND Myeloid-derived suppressor cells (MDSCs) increase in patients with cancer and are associated with poor prognosis; however, their role in transplantation is not yet understood. Here we aimed to study the MDSC effects on the evolution of kidney transplant recipients (KTRs). METHODS A cohort of 229 KTRs was prospectively analyzed. Two myeloid cells subsets. CD11bCD33CD14CD15HLA-DR (monocytic MDSC [M-MDSC]) and CD11bCD33CD14CD15HLA-DR (monocytes), were defined by flow cytometry. The suppressive capacity of myeloid cells was tested in cocultures with autologous lymphocytes. Suppressive soluble factors, cytokines, anti-HLA antibodies, and total antioxidant capacity were quantified in plasma. RESULTS Pretransplant, M-MDSC, and monocytes were similar in KTRs and healthy volunteers. M-MDSCs increased immediately posttransplantation and suppressed CD4 and CD8 T cells proliferation. M-MDSCs remained high for 1 y posttransplantation. Higher M-MDSC counts at day 14 posttransplant were observed in patients who subsequently developed cancer, and KTRs with higher M-MDSC at day 14 had significantly lower malignancy-free survival. Day 14 M-MDSC >179.2 per microliter conferred 6.98 times (95% confidence interval, 1.28-37.69) more risk to develop cancer, independently from age, gender, and immunosuppression. Early posttransplant M-MDSCs were lower in patients with enhanced alloimmune response as represented by anti-HLA sensitization. M-MDSC counts correlated with higher circulatory suppressive factors arginase-1 and interleukin-10, and lower total antioxidant capacity. CONCLUSIONS Early posttransplant mobilization of M-MDSCs predicts cancer and adds risk as an independent factor. M-MDSC may favor an immunosuppressive environment that promotes tumoral development.

In transplanted intestines, depletion of T cells together with long-term persistence of ILC is observed, suggesting ILC insensitivity to immunosuppressive drugs. To further analyze helper ILC (hILC) apparent resistance to therapy, cytotoxic ILC (NK cells), hILC subsets (ILC1, ILC2, and ILC precursors (ILCP)), and their signature cytokines (IFNγ, IL4 + IL13, and IL22) were analyzed in peripheral blood of kidney and liver transplant recipients. Early after transplantation (posTx), transplanted patients showed significantly lower Lin + and NK cells, whereas total hILC, ILC1, ILC2, and ILCP numbers were similar in patients and controls. Between paired pre- and posTx samples, Lin + cell and NK cell counts significantly decreased, whereas all three hILC counts and their cytokine production remained similar. ILC1, ILC2, and ILCP numbers were also similar in patients under thymoglobulin or basiliximab (BAS), patients without induction (only maintenance therapy) and controls. hILC showed lower TMG binding comparing to Lin + cells, reduced expression of CD25 (BAS target), and diminished calcineurin activity with undetectable calcineurin and FKBP12 (tacrolimus target). hILC counts were not related to delayed graft function or biopsy-proven acute rejection. Thus, hILC remain stable early after transplantation and seem unaffected by immunosuppressors, which may be related to reduced targets expression and low calcineurin activity.

Antibody mediated rejection has been recognized as an important contributor to long-term graft loss in most solid organ transplants. Current immunosuppressive regimes are not capable of preventing anti-HLA antibody formation and eventual damage to the graft, and there is a need to develop drugs directed against novel targets to avoid graft allorecognition. In this review we introduce follicular helper T cells (Tfh), a subtype of lymphocyte specialized in helping B cells to differentiate into plasmablasts and produce class-switched antibodies. We focus on the role of Tfh in solid organ transplantation, what is known about Tfh and the production of alloantibodies, how current immunosuppressive therapies affect Tfh and what new molecules could be used to target Tfh in transplantation, with the goal of improving graft survival.

BACKGROUND We previously reported that preformed anti-MHC class I-related chain A (MICA) antibodies increase the risk for renal graft rejection and enhance the deleterious effect of PRA(+) status early after transplantation. METHODS We studied 727 kidney recipients. Days to reach optimal serum creatinine level, estimated glomerular filtration rate (eGFR) at Month 3 and chronic kidney disease (CKD) stages were recorded. Anti-MICA specificities and C1q binding were tested by solid-phase assay. Complement-dependent cytotoxicity (CDC) and flow cytometry (FC) cross-matches with HeLa and PMA/CD28-T-blasts were performed. RESULTS PRA(+)MICA(+) recipients exhibited longer time to reach optimal serum creatinine level after transplantation (P = 0.005) and had the lowest eGFR at Month 3 (P = 0.006). PRA(+)MICA(+) status independently increased the risk for CKDT stage 5 at Month 3 [hazard ratio (HR) 4.92, P = 0.030]. Pre-transplant anti-MICA antibodies were polyspecific and showed stronger reactions when coexisting with anti-HLA antibodies (mean standard fluorescent intensity 112 157 ± 44 426 in HLA(+)MICA(+) sera versus 49 680 ± 33 116 in HLA(-)MICA(+) sera, P = 0.0006). Anti-AYVE supereplet reactivity was significantly higher in HLA(+)MICA(+) versus HLA(-)MICA(+) patients (P < 0.001) and significantly superior than anti-CMGWS supereplet within HLA(+)MICA(+) patients (P = 0.001). Three of 13 anti-MICA(+) pre-transplant sera were positive for the C1q binding assay; one of them (serum 3) exclusively recognized AYVE supereplet with a strong reactivity against MICA*027 antigen (same as MICA*008). Anti-MICA antibodies in anti-HLA-absorbed serum 3 bound native MICA molecules in MICA*008(+) HeLa and PMA/CD28-T-blasts and mediated cell death by activating complement. CONCLUSION Preformed anti-MICA antibodies may occasionally be cytotoxic by fixing and activating complement. This way they might contribute to worse early kidney graft function.

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103(+) cells) principally due to reduced CD3(+) CD8(+) cells, while CD103(+) CD3(-) cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3(-) IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3(-) IELs of the grafts expressed CD56, NKp44, interleukin (IL)-23 receptor, retinoid-related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3(-) CD127(+) cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3(-) IELs in grafts produced significantly more interferon (IFN)-γ and IL-22, and a double IFNγ(+) IL-22(+) population was observed. Epithelial cell-depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3(-) IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56(+) , NKp44(+) , CCR6(+) , CD127(-) , cytotoxicity, and IFNγ secretion), ILC3 (CD56(+) , NKp44(+) , IL-23R(+) , CCR6(+) , RORγt(+) , and IL-22 secretion), and intermediate ILC1-ILC3 phenotypes (IFNγ(+) IL-22(+) ). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.