Lack of a prompt normalization of immunological parameters is associated with long-term care and poor prognosis in COVID-19 affected patients receiving convalescent plasma: a single center experience

Introduction

In December 2019, a new member of the coronavirus family emerged in Wuhan, China [1]. This pathogen, named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused coronavirus disease 2019 (COVID-19), which rapidly spread across the globe leading to a still ongoing pandemic [2]. A number of treatments, including anti-viral, anticoagulant and anti-inflammatory agents have been tried in COVID-19 patients with controversial results [3]. The passive transfer of anti-SARS-CoV-2 neutralizing antibodies from plasma of recently recovered individuals to patients with severe COVID-19 was among the first therapy used [3], [4], [5].

Although the results of clinical trials are controversial, there is current evidence of the potential clinical benefit of COVID-19 convalescent plasma (CCP) when early transfused (within three days of symptom onset) and at high anti-SARS-CoV-2 neutralizing titer (>1:160) in COVID-19 patients [5], [6], [7]. Although the effect of CCP is supposed to be mainly driven by neutralizing antibodies, its exact mechanism of action is not completely understood [8]. Specifically, there are very few data on the immunomodulatory effect of CCP, which could be non-trivial considering the COVID-19-associated dysregulation and impairment of the immune system [9, 10].

To better characterize the immune response changes after CCP transfusion in different groups of patients, we have monitored post-treatment modulation in biochemical and immune markers associated with disease severity and recovery in a cohort of 28 adult COVID-19 patients.

Materials and methods

This prospective observational cohort study stems from the collaboration between the Transfusion Center of the City Hospital of Mantua and the Laboratory of the Spedali Civili of Brescia. The study was approved by the Local Ethical Committee of Valpadana. Treated patients were enrolled at the Divisions of Infectious Diseases and Pneumology of the City Hospital of Mantua during an eight-month period (October 2020-May 2021). All patients were hospitalized for COVID-19 (confirmed by a positive SARS-CoV-2 PCR test result from nasopharyngeal swab) and diagnosed for moderate to severe acute respiratory distress syndrome (ARDS), characterized by a ratio of arterial partial pressure of oxygen (PaO₂) and fraction of inspired oxygen (FiO₂) of less than 200. The selection of CCP donors was performed in agreement with national CCP regulation [11]. CCP was collected through a plasmapheresis procedure using a cell separator, processed, biologically validated and stored at the Transfusion Center of Mantua according to the Italian regulation and the indications of the Italian National Blood Center [11]. Each CCP unit had a 300 mL volume and an anti–SARS-CoV-2 neutralizing titer of 1:80 or higher. The neutralization test for the identification of anti–SARS-CoV-2-neutralizing antibodies was performed at the Molecular Virology Unit of the University Hospital of Pavia and was based on the determination of cytopathic effect, as previously described [12]. COVID-19 patients were transfused with 1–3 units of ABO type compatible CCP, according to the clinical response, in agreement with the routine procedures previously described [11, 12].

Patients were regularly followed clinically, according to our previous protocol [12, 13], and a detailed flow cytometry evaluation of immunological markers was performed at baseline and on days +3 and +7 after CCP infusion. Multiparametric flow cytometry analyses were performed using appropriate mixtures of monoclonal antibodies (mAbs) according to manufacturer’s protocols on blood samples drawn in the previous 24 h and shipped at room temperature. A standard TBNK panel was used to identify and quantify main lymphocyte populations (CD3+, CD4+, CD8+ T lymphocytes and their HLA-DR+ activated fraction, CD19+ B lymphocytes and CD3-CD56/CD16+ NK cells). T-cell differentiation was assessed by using anti-CD3, CD4, CD8, CD45RA, CCR7, CD31 mAbs, which in particular allow to identify CD45RA+CCR7+CD31+ recent thymic emigrants (RTEs), CD45RA-CCR7-effector memory and CD45RA+CCR7-terminal differentiated cells. Analyses of B-cell differentiation was assessed by using anti-CD19, CD20, IgM, IgD, CD38, CD27 mAbs which in particular allow to identify IgD-IgM-CD27+ switched memory and CD38++CD27+CD20-terminal differentiated cells. Plasmacytoid (pDC) and myeloid (mDC) dendritic cells were identified as 45dimCD4dimCD123+BDCA2+ cells and 45dimCD4dimCD1c+CD14-cells, respectively.

Expression of CD4 and HLA-DR on monocytes was evaluated by Mean Fluorescence Intensity (MFI) and compared to their expression on resting CD4+ T helper cells, which is expected to remain constant. Thus, CD4 and HLA-DR MFI values on T helper cells were used to normalize the variable expression of these markers on monocytes. This approach, based on CD4 T helper/monocyte MFI ratio and HLA-DR monocyte/T helper MFI ratio, was chosen because it guarantees a better reproducibility of data compared to direct measurement of MFI values and establishes reference values independent from the immunofenotype panels and instrument settings used.

Expression of CD21 and HLA-DR on B lymphocytes was also evaluated. To normalize their variability, we measured the expression of CD20 on B lymphocytes and HLA-DR on non-activated T helper cells, respectively, and we calculated CD20/CD21 and HLA-DR B Lymphocyte/T helper MFI ratios.

Immunofenotypic stainings were acquired on FACSCanto II (BD Biosciences) flow cytometers, while data analyses were performed using FACSDiva software (BD Biosciences).

The main demographic features of each group were summarized as medians (with minimum and maximum) or ratios, and were compared by the Kruskall-Wallis tests or the Fisher’s exact test, respectively. Comparisons between immunological parameters were performed by two-factor ANOVA using mixed models with a random intercept, time point as a repeated measure, and a group-by-time point interaction. Only when the interaction term was significant, post-hoc Bonferroni-corrected comparisons between single time points of distinct groups were made. If the main effect of group was significant but not the interaction term, Bonferroni-corrected comparisons between pairs of groups were made (Moderate vs. Severe, Severe vs. Critical and Moderate vs. Critical), independent of time. If the main effect of time point was significant but not the interaction term, tests on the overall effect of time were made by comparing T7 with T0, in order to demonstrate an increasing or decreasing trend in all groups. Statistical significance threshold was p<0.05.

Results

Our cohort comprised 28 patients, ten of which manifested a milder respiratory distress allowing an oxygen therapy with Venturi masks. Based on this feature, they were categorized as “Moderate” group. On the opposite, the remaining 18 patients required non-invasive (12) or invasive (6) ventilator support and were initially all together considered as severe. Among the “Severe” patients, ten progressed to more critical clinical conditions leading to death in six cases or, in the remaining four patients, to a long-term hospitalization (34–147 days) (Table 1) and for this reason they were categorized as “Critical”. Notably, three of the deceased patients presented with comorbidities, while five of them subsequently developed superinfections. Pharmacological treatment was the same for all the patients, with only few exceptions (Table 1 and Supplementary Table S1).

Comparison of age and gender did not evidence any significant difference between the three groups. We obtained the same result when we compared PaO₂/FiO₂ ratios at the time of CCP transfusion. On the opposite, at day +7 from transfusion PaO₂/FiO₂ ratios significantly diverge between the three groups, with a worsening of critical patients that contrasts with the improvement of moderate and severe patients (Table 1). Notably, effects of CCP transfusion among the three groups was not conditioned neither by the dose of CCP nor by the time of infusion in term of days after diagnosis (Table 1).

All the COVID-19 patients were monitored at T0 in order to evaluate their immunological status by multiparemetric flow cytometry assays before CCP transfusion. Analyses were directly performed aiming to quantify the main lymphocyte populations and to evaluate T-and B-cell subsets [14]. As expected, lymphocyte counts of most patients were below the lower reference limits. Specifically, T-cell lymphopenia was observed in 25/28 patients, and to lesser extent on CD4+ and CD8+ subsets (Figure 1 and Supplementary Figure S1). Reduction of B cells characterized 16/28 patients, while NK cell counts were generally normal (Figure 1 and Supplementary S1). On the other hand, the increased number of neutrophils made the neutrophil-lymphocyte ratio (NLR) generally exceeding normal values (26/28 patients) (Figure 1). As presented in Figure 1 and Supplementary Figure S1, none of these immunological parameters displayed significant differences between the three groups of patients.

Figure 1:

Immunological and biochemical data of the three groups of patients (moderate: white, severe: light grey and critical: dark grey) plotted at T0, T3 and T7 and compared to normal values (filled light grey areas) for the following parameters: (A) Neutrophils/lymphocytes ratio, (B) CD3+ cell counts, (D) CD19+ cell counts, (E) percentages of plasmablasts within the B cell compartment, (G) HLA-DR expression on monocytes presented as monocytes/T-helper lymphocytes MFI ratio, (H) CD4 expression on monocytes presented as T-helper lymphocytes/monocytes MFI ratio, (L) serum C-reactive protein and (M) D-Dimer values. Regression lines of the relations between A and B, D and E, G and H at 7 days post-transfusion are presented in (C), (F), (I) respectively. When three lines are shown (C, I), a significant difference was present between the slopes and between the intercepts; when only one line is shown (F), no statistically significant differences were observed. Curly brackets indicate statistical significance applying post-hoc Bonferroni-corrected comparisons between single timepoints of distinct groups. Square brackets indicate a time-independent statistical significance performing a comparison of Moderate vs. Severe, Severe vs. Critical and Moderate vs. Critical groups. Single dashed lines indicate statistically significant increasing or decreasing trends comparing T7 with T0 of all groups. Legend: *p<0.05, **p<0.01, ***p<0.001.

Regarding T-and B-lymphocyte subsets, we observed an increased percentage of plasmablasts on B cells of most patients in all the three groups (Figure 1E), while T cells were characterized by a reduction of recent thymic emigrants and, on the opposite, by high percentages of terminal differentiated cells, in the absence of elevated activation (HLA-DR+ cells) (Supplementary Figure S1D–F). As for the main lymphocyte subsets, also comparison of T-and B-lymphocyte subset values among the three groups of patients did not show any statistically significant differences.

Amelioration of lymphocyte parameters began to be evident at day +3 and became more significant at day +7 post-CCP transfusion. Specifically, we observed a general normalization of T- and B-cells counts in recovering patients belonging to the “Moderate” and “Severe” groups (Figure 1A, B). This normalization is associated to a reduction of abnormal levels of NLR and plasmablasts at T0 (Figure 1D, E). On the opposite, lymphopenia persisted in a large fraction of “Critical” patients, as well as high NLR and plasmablasts percentages. Regression analysis showed a negative association between NLR and CD3, CD19 cells and plasmablasts. In particular, for the first set of parameters (Figure 1C), the slope of the regression line of “Critical” patients was significantly less steep than that of the other groups (p=0.029), indicating that in these patients the slight reduction of NLR drives to a poor T-cell recovery.

Regarding T-lymphocyte subsets we observed a significant increase of CD4+ T cells and RTEs limited to the “Moderate” and “Severe” groups (Supplementary Figure S1A, D). On the opposite, immunophenotipic changes related to CD8+ T cells showed an overall effect of time in all groups, indicating an increasing trend of total CD8+ cells, as well as of the effector memory and the activated CD8+ cell fractions, while exhibiting a decreasing trend in terminally differentiated cells (Supplementary Figure S1E–G). Among B-lymphocyte subsets we report a significant increase of switched memory cells from T0 onward, that was common to all groups (Supplementary Figure S1H).

Since decreased expression of HLA-DR and CD4 on monocytes has been associated to severe manifestation of COVID-19 [15], we evaluated the expression of these markers as ratios of mean fluorescence intensity (MFI) between T-helper cells and monocytes (Figure 2).

Figure 2:

Representative histograms showing CD4 (upper row) and HLA-DR expression (lower row) on T-helper cells (black line), monocytes (dark grey line) and B cells (light grey line) in a healthy control vs. a critical patient. T-helper lymphocytes/monocytes ratio, as well as B cell or monocytes/T-helper lymphocytes ratio were calculated using mean fluorescence intensity (MFI) values, as shown.

CD4 expression on monocytes, measured as T-helper MFI/monocyte MFI ratio, resulted significantly increased in the group of “Critical” patients at T0, compared to reference values and patients of the other two groups (Figure 1H); this discrepancy became even more pronounced at T3 and T7. Similarly, HLA-DR expression on monocytes, obtained dividing monocyte MFI by the one of non-activated T helper cells, was significantly reduced in the group of “Critical” patients from T0 onward (Figure 1G). Regression lines obtained by plotting ratios of monocyte markers at day +7 post CCP transfusion emphasized the differences between “Critical” patients and patients who started recovering (Figure 1I).

At T0, we also observed a general decrease of CD21 and HLA-DR expression on B lymphocytes of our patients, whose values, calculated as CD20/CD21 and HLA-DR B Lymphocyte/T helper MFI ratios respectively, were almost constantly below normal donors’ lower limits. Analyses of these markers at day +3 and +7 showed a slight amelioration of their expression in all three groups compared to T0, although only few patients ranged reference values (Supplementary Figure S1I–L).

Finally, we evaluated the percentage of plasmacytoid (pDC) and myeloid (mDC) dendritic cells on peripheral blood mononuclear cells (PBMCs), which in almost all patients were markedly reduced or undetectable at T0, independently from the severity of the disease manifestations. The subsequent time point analyses displayed a significant increase in mDC in all the three groups, while pDC remained stably low or absent for all the observational period (Supplementary Figure S1M, N).

From the biochemical point of view, we focused on the analyses of C-reactive protein (CRP) and D-Dimer, which are known to be important markers of COVID-19 severity. Their concentrations in “Critical” patients, although heterogeneous, were higher than in patients of the “Moderate” and “Severe” groups at presentation (Figure 1L, M). In the subsequent measurements CRP concentrations remained elevated in “Critical” patients, while in the other two groups CRP rapidly approached normality. D-Dimer concentrations remained stable for “Moderate” and “Severe” patients in the days following CCP transfusion, while they generally increased in “Critical” patient. Due to these trends, such biochemical parameters allowed to easily discriminate between “Critical” and recovering patients in a CRP vs. D-Dimer plot at day +7 from CCP transfusion (Figure 1N).

Discussion

Being the potential beneficial effects of CCP transfusions in COVID-19 patients already documented, our study evaluated post-CCP transfusions immunological changes in 28 hospitalized patients, keeping in mind that lack of a control group not receiving CCP does not allow us to make a clear dissection of the impact of CCP on the immunological and clinical course of the disease in our cohort of patients. For this reason, we particularly focused on immunological differences among patients who rapidly recovered after treatment and patients who did not show any clinical benefits or even worsened their condition after CCP transfusion, thus aiming to identify predictive hallmarks of prognosis of treated patients. Specifically, our 28 patients were categorized into three groups of progressive severity based on clinical parameters at presentation and on disease progression during hospitalization. Of note, the three groups of patients were considered homogenous in terms of personal data, clinical presentation and immunological parameters at T0.

During the seven days of observation, our patients displayed significant changes in T-and B-lymphocyte subset distribution. In particular, we observed a general increased proportion of activated and effector CD8+ T lymphocytes, as well as of switched memory B cells in all the three groups of patients, indicating that the presence of an immunological response against COVID-19 infection was not peculiar of a specific patients’ subgroup. On the opposite, only patients who displayed a more moderate respiratory distress at presentation or who recovered within a month from hospitalization showed a trend towards normalization of T-and B-lymphocyte counts, whose alteration is associated to the phase of uncontrolled inflammatory immune response of COVID-19 infection [9, 15]. No significant changes of T-and B-lymphocyte counts ware observed in “Critical” patients in the first week after CCP transfusion. Lymphocyte counts also conditioned the average reduction of NLR and plasmablast percentages, which were the poorest in “Critical” patients within a trend of general normalization.

Analyses of monocyte markers displayed a significant different expression among “Moderate/Severe” and “Critical” patients that emerged as independent from timing. In fact, CD4 and HLA-DR expression on monocytes resulted particularly impaired in “Critical” patients from the first analysis onwards. But, although a poorer expression of monocyte markers was already seen in “Critical” patients at the first time point, our study highlights that the combination of both HLA-DR and CD4 expression at day +7 could represent the main immunological hallmark of “Critical” patients, as illustrated in Figure 1I.

Conversely, impact of COVID-19 infection on the reduction of constitutively expressed markers on B cells did not allow discrimination among groups. Nor did the reduction of immune-related cell subsets such as dendritic cells, whose trend at the end of the observational period was similar in all the three groups.

Analysis of immunological parameters was supplemented by evaluation of biochemical parameters, such as CRP and D-Dimer, whose alterations are associated to severe COVID-19 infection. In our cohort D-Dimer showed altered values, which were significantly lower in “Critical” patients from T0 onwards. Moreover, the peculiar persistence of high CRP in “Critical” patients allowed to discriminate them from patients of the other two groups. As for monocyte markers, the combination of CRP and D-Dimer values at day +7 (Figure 1N) allowed to identify the most critical cases among patients who received CCP transfusion.

Taken together, these results show that, although it is not possible to identify a clear hallmark of prognosis before CCP transfusion, analysis of a combination of appropriate immunological and biochemical markers during the first week of treatment can help to presume which patients might be at higher risk of long-term care or medical complications. This possibility becomes crucial when thinking that prolongation of critical conditions associated to SARS-CoV-2 increases the risk of fatal superinfections caused by other pathogens. Our cohort well represent this risk, since all of the patients’ decease occurred subsequently to superimposed infection after the observation period of the study.

In conclusion, we believe that immunological analyses based on flow cytometry may be potentially useful for monitoring COVID-19 patients treated with convalescent plasma and that such approach could be also extended to other treatments based on antibody mediated neutralization of viral particles.