All samples were obtained from two independent clinical trials in which efficacy of Sanaria® PfSPZ Vaccine [radiation attenuated, aseptic, purified, cryopreserved P. falciparum (Pf) sporozoites (SPZ)] and PfSPZ-CVac (infectious, aseptic, purified, cryopreserved PfSPZ administered with an antimalarial drug) were assessed by CHMI (ClinicalTrials.gov identifiers NCT02858817 and NCT02704533). The studies were conducted at the Institute of Tropical Medicine, University Hospital Tübingen and full results of both trials will be reported elsewhere [Mordmüller et al. unpublished data and (53)].

For the present study on immunosuppressive cell populations, only samples during CHMI were used, regardless of previous vaccination. In trial NCT02858817, a PfSPZ-CVac study, volunteers received either PfSPZ or normal saline (placebo) and atovaquone/proguanil (1,000 /400 mg) (A/P) three times every 4 weeks. CHMI of these subjects immunized with PfSPZ-CVac (A/P) was performed 10 weeks following the last vaccination. Immunizations consisted of three doses of either 5.12 × 10⁴ or 15 × 10⁴ PfSPZ. The second trial (NCT02704533) used PfSPZ Vaccine, which has been described before (54), for immunization. Here, volunteers were vaccinated with either three doses of 9 × 10⁵ or two doses of 1.35 × 10⁶ or 2.7 × 10⁶ PfSPZ of PfSPZ Vaccine. CHMI was done 3 weeks following immunization.

CHMI was performed with 3.2 × 10³ sporozoites [Sanaria® PfSPZ Challenge (PfNF54)] by DVI. Volunteers had daily clinical visits, thick blood smear readings and reverse transcription quantitative PCR (RT-qPCR) for up to 3 weeks. RT-qPCR was performed as described previously, achieving a lower limit of detection of six parasites/ml (55). The end-point for treatment initiation with atovaquone/proguanil was met when a volunteer became parasitemic, defined as three consecutive positive RT-qPCR results with at least one parasitemia above 100 parasites per ml or any parasitemia detected by microscopy. Peripheral blood mononuclear cells (PBMC) were prepared in the following sampling days during CHMI (Figure 1A). (i) Baseline, before the injection of fully infectious PfSPZ Challenge (C); (ii) C+7, generally the day of first detectable parasitemia by RT-qPCR; (iii) C Malaria, when parasitemic participants fulfilled the treatment initiation end-point criteria, being followed by two more samples at the 2nd (C Treat2) and 3rd (C Treat3) day of treatment; (iv) C+14, in protected aparasitemic participants as comparable control for malaria positive cases; (v) C+21, at the end of CHMI. As per-protocol, participants received a 3 days course treatment with atovaquone/proguanil, administered always after blood sampling at C Malaria, C Treat2 and C Treat3; and C+21 if they were not positive for parasitemia during CHMI.

In brief, whole blood was collected in Na-heparin tubes (S-Monovette, Sarstedt) from all volunteers. All samples for analysis were acquired before participants received the first dose of rescue treatment except for those on 2nd and 3rd day of malaria-specific treatment (Figure 1A). As previously described, MDSC and T_reg were promptly quantified (56–58) from peripheral blood mononuclear cells (PBMC) freshly isolated by density gradient centrifugation (Ficoll-Paque^TM PLUS; GE Healthcare).

MDSC were characterized by flow cytometry (FACScalibur Flow cytometer, BD) after staining PBMC with: mouse anti-human CD14-FITC (BD Biosciences, Clone MϕP9), mouse anti-human HLA-DR-PerCP (BD Biosciences, Clone L243), mouse anti-human CD33-PE (Miltenyi Biotec, Clone AC104.3E3) and rat anti-human CD11b-APC (Miltenyi Biotec) to identify Monocytic-MDSC (M-MDSC) from the monocyte population as CD11b⁺CD33⁺HLA-DR^−/loCD14⁺; while PBMC in the SSC^hi area were defined as PMN-MDSC by the staining with mouse anti-human CD66b-FITC (BD Pharmingen). The percentage of PMN-MDSC was verified from the SSC^hi region of the M-MDSC sample by gating the CD11b⁺CD33⁺HLA-DR^−/loCD14⁻ population thereof (Supplementary Figures 1, 2A,B).

The MDSC counts were normalized to the initial values within participant, defined as the MDSC fold change for every measured time point in relation to the baseline MDSC levels for each individual participant.

Regulatory T cells were defined as surface CD4⁺, CD25^+/high and intracellular FoxP3⁺ (Supplementary Figure 2C). Antibody clones used for the staining were mouse anti-human CD4-FITC (BD, Clone SK3), mouse anti-human CD25-PE (BD Pharmingen, Clone M-A251) and anti-human FoxP3-Alexa Fluor 647 (Biolegend, Clone MOPC-21). To control for unspecific staining, the respective isotype control was used (Biolegend). Intracellular staining was performed using a commercial kit following the respective protocol (Cytofix/Cytoperm, BD).

Peripheral blood mononuclear cells (PBMC) from a healthy donor were stained with arboxyfluorescein diacetate succinimidyl ester [Vybrant® CFDA SE (CFSE) Cell Tracer Kit, #V12883, Molecular probes by life technologies] according to the manufacturer's manual allowing lymphocyte proliferation traceability by their simultaneous stimulation with 100 U/ml IL-2 (R&D Systems) and 1 μg/ml purified NA/LE Mouse anti-human CD3 (BD Pharmingen, #555336, Clone HIT3a).

PMN-MDSC were freshly isolated from parasitemic participant's PBMC (autoMACS®Pro Separator, Miltenyi Biotec), gathered by positive selection after mouse anti-human CD66b-FITC (BD Pharmingen, #555724) and anti-FITC MicroBeads (Miltenyi Biotec, #130-048-701) staining, as described before (59). Subsequently, the highly purified PMN-MDSC were seeded in a round bottom 96-well plate in different proportions to a fixed amount of 6 × 10⁴ CFSE labeled PBMC per well (1:2, 1:4, 1:8, 1:16) and incubated at 37°C and 5% CO₂. Complete media (RPMI1640 supplemented with 10% autologous serum, 1% L-glutamine, and 1% Penicillin-Streptomycin) without additional cells was added to stimulated and non-stimulated labeled PBMC as positive and negative control, respectively. As controls for the contribution of polymorphonuclear cells (PMN) to suppression, PMN from the participants isolated with an erythrocyte lysis step from the high-density fraction of the Ficoll were seeded in the same manner as PMN-MDSC.

After 4 days of incubation, cells were harvested and stained with mouse IgG1, κ anti-human CD4-PE (Biolegend, #300508, Clone RPA-T4) and mouse IgG1, κ anti-human CD8a-APC (Biolegend, #300912, Clone HIT8a). Shortly before measurement, Propidium Iodide (BD Pharmingen, #51-66211E) was added to determine cell viability. CFSE fluorescence intensity was analyzed by flow cytometry to determine proliferation of CD4⁺ and CD8⁺ T cells (Supplementary Figures 2D,E). Proliferation was defined as the ratio of percentage of T cell proliferated following addition of PMN-MDSC or PMN to percentage of T cell proliferation without co-culture (58).

The hematological parameters from participants' blood samples were measured with the Sysmex XN-series hematology analyzer. Lymphocytopenia was defined according to the central laboratory reference range (lymphocyte cell count ≤ 1.2 × 10³/μl of blood).

Flow Cytometry data was analyzed using FlowJo v10.6.1. Statistical tests were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Figures were created using ggplot2 package version 3.3.0 in R software (R-project, https://www.R-project.org/) version 3.6.3.

The area under the curve was computed by using the trapezoid rule.

Multiple comparisons on cellular kinetics were calculated by mixed effects (Model III) ANOVA where factors are both fixed and random. This approach represents a normal ANOVA when some random missing values are presented. As the experiment design is repeated measures, sphericity was not assumed, following the recommendation of Maxwell and Delaney. The mixed model uses a compound symmetry covariance matrix, and is fit using Restricted Maximum Likelihood (REML). Variation analysis was corrected for multiple comparisons by the “two-stage” Benjamini et al. procedure for controlling the false discovery rate (FDR) (60).

Circulating MDSC kinetics were quantified during CHMI, done in a subset of participants of the two clinical malaria vaccine trials (trial identifiers: NCT02704533, NCT02858817) in healthy naïve volunteers that will be reported elsewhere [Mordmüller et al. unpublished data and (53)]. A total of 44 participants underwent CHMI as described (20). Ten to 3 weeks before CHMI, volunteers completed immunizations with either placebo (normal saline; n = 9), PfSPZ Vaccine (n = 17) or PfSPZ-CVac (A/P) (n = 18) (Figure 1B). PfSPZ Vaccine and PfSPZ-CVac (A/P) lead to a complete arrest of parasite development during liver stage. As expected, none of the participants developed blood stage parasitemia detectable by ultra-sensitive RT-qPCR during the immunization phase.

A total of 28 out of the 44 (64 %) participants undergoing CHMI by DVI administration of PfSPZ Challenge developed parasitemia and received a 3-day course treatment with atovaquone/proguanil. Of those 28 unprotected volunteers, five were part of the PfSPZ Vaccine study and 23, including the nine placebos, were part of PfSPZ-CVac (A/P) study. The geometric mean of days from DVI to rescue treatment in participants with circulating parasites was 11.7 days (range 10–14 days). None of the protected participants had parasites detectable in the circulation.

The highest detectable parasitemia in parasitemic participants during CHMI was 12 parasites per μl. The most frequent adverse event (AE) reported at this stage was headache (Supplementary Table 1). Notably one participant did not report any AE when study treatment end-point was reached. Elevated temperature of 38°C was observed in only one infected participant and occurred on the second treatment day. Furthermore, during the CHMI phase the severity of the AE was not dependent on infection on the days the AE were detected (Chi-square independence test, p = 0.8823). However, the number of reported AE increased substantially during the 2nd and 3rd day of treatment. Among the most common AE, lymphocytopenia stood out in parasitemic participants during the treatment days, which is described in more detail in a separate section below. In summary, the CHMI led to a very early state of malaria infection with no or few mild symptoms and signs.

Circulating suppressor cells were phenotypically characterized in participants' blood during CHMI. Cellular variation of circulating suppressor cells in the PBMC of the participants was controlled by adjusting all levels to the baseline levels within the same subjects prior to injection of the PfSPZ Challenge (Figure 2). The resulting area under the PMN-MDSC fold change increase (FC) to baseline - day curve (AUC) for each participant reflects the kinetic expansion of PMN-MDSC over the CHMI days, given in the unit of FC-days. Therefore, the median for the AUC was 35 [interquartile range (IQR), 15–64] FC-days in parasitemic participants, which was significantly higher than the 13 (IQR, 8–18) FC-days from the aparasitemic group (Figure 2A, p < 0.0001). This represents a general increase in circulating PMN-MDSC in those participants developing blood stage parasitemia. PMN-MDSC kinetics are similar between the different doses of parasites received during the previous immunization phase (p = 0.07; Supplementary Figure 3A), nor by being allocated to placebo or vaccine (p = 0.37; Supplementary Figure 3B), but by the development of detectable parasites in the circulation.

The kinetics of the other measured suppressor cells did not change to the same extent. The median AUC of 12 (IQR, 7–20) FC-days for the M-MDSC kinetics in the aparasitemic group was not significantly different from the 8 (IQR, 5–17) FC-days of the parasitemic group (Figure 2B, p = 0.4552). In the same fashion, the regulatory T cells (T_reg) median AUC was 13 (IQR, 12–19) T_reg FC-days for the parasitemic group and 14 (IQR, 8–16) FC-days for the aparasitemic individuals (Figure 2C, p = 0.36). Although the FC increase in Treg was not found to be significantly different between parasitemic and aparasitemic participants, a significant increase of Treg in C Malaria could be detected when the increase of Treg in the non-protected individuals from baseline to day of treatment was analyzed (One-way ANOVA, p-value < 0.001; mixed-effect model Benjamini, Krieger and Yekutieli post-hoc test Baseline vs. C Malaria p = 0.006, Supplementary Figure 4).

To more specifically determine the exact time point at which the PMN-MDSC are significantly higher in the parasitemic individuals, a mixed effect approach (Model III - following a two-way ANOVA approach when some random values are missing) in combination with the respective post-hoc test was done (Figure 3). The fold changes increase of PMN-MDSC to baseline were tested by using the study days and protection status (defined by the growth of parasites in the blood) as factors for the multiple comparisons. PMN-MDSC kinetics were explained by being unprotected (p < 0.001) and by their respective timing (study days) of parasitemia (p < 0.001), but the overall change on PMN-MDSC kinetics was not explained by the study days as the standalone factor (p = 0.8). The respective post-hoc test (Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli) revealed that circulating PMN-MDSC levels were 4 to 5-fold higher at C Malaria and the two subsequent treatment days in the parasitemic group compared to the aparasitemic counterpart at the respective sampling day C+14 (q < 0.001, Figure 3, Table 1). Over time, PMN-MDSC levels increased 1.9–2.5 times compared to baseline in the unprotected individuals (p < 0.001, Figure 3, right panel) compared to no change within the group of protected individuals who developed no parasitemia over time (Figure 3, left panel).

PMN-MDSC levels in the peripheral blood increased significantly and remained elevated for at least the treatment period, which coincides with the appearance of elevated parasite levels in the blood (Supplementary Figure 5). The geometric mean parasitemia at C Malaria, C Treat2 and C Treat3 were 649 parasites/ml (range 4–12,421), 852 parasites/ml (97–11,990) and 68 parasites/ml (3–667), respectively. At C+21, the geometric mean PMN-MDSC level in participants who developed parasites before in CHMI, was still 2.5 times higher (Wilcoxon rank sum test, p < 0.005) compared to the PMN-MDSC values on the same day from those who remained aparasitemic (Supplementary Figure 6A). However, at that time, all participants were considered healthy and parasite free, either because they cleared a patent parasitemia after administration of the treatment or because they never developed circulating parasites. As the percentage of PMN-MDSC in the peripheral blood at C+21 correlates with the respective values at C Malaria (Spearman's rank correlation test, p = 0.01, R = 0.5), the increase might be explained by the strong increase of PMN-MDSC achieved at C Malaria (Supplementary Figure 6B).

An ex-vivo suppression assay was performed when the end-point criteria for treatment was met at C Malaria in parasitemic participants: 20 out of the 23 participants from the PfSPZ-CVac and all five from the PfSPZ Vaccine study (Figure 4). Thus, the described PMN-MDSC can be distinguished from other non-functional MDSC-like cells expressing similar phenotypic markers. PMN were used in a subgroup of 15 of the 25 participants as controls for the suppression.

PMN-MDSC from parasitemic participants conferred a similar suppression on both CD4⁺- and CD8⁺- T cell-proliferation within the same ratio of PBMC to MDSC seeded (p-value > 0.05). The capability of PMN-MDSC to suppress stimulated CD4⁺ and CD8⁺ lymphocytes increased progressively with growing ratios as a linear trend (CD4⁺ & CD8⁺ p < 0.0001), reaching at a 1:2 cellular PMN-MDSC to T cell ratio a mean of 54% suppression of lymphoproliferation for both CD4⁺ and CD8⁺ T cells (Figure 4).

Altogether, these findings show that PMN-MDSC found in the circulation during P. falciparum parasitemia can suppress CD4⁺ and CD8⁺ T cells proliferation.

Lymphocytopenia was a common observation in parasitemic participants during the CHMI of both studies. At C Malaria before treatment initiation, the number of circulating lymphocytes in those who are about to develop lymphocytopenia was significantly lower compared to the ones not becoming lymphocytopenic (Figure 5A, Student's t-test, p < 0.05). However, on treatment initiation day, only four individuals were lymphocytopenic by definition ( ≤ 1.2 × 10³/μl). The majority of the parasitemic cases became lymphocytopenic over the second and third treatment days (16 out of 23 in PfSPZ CVac and four out of five in PfSPZ Vaccine).

Logistic regression was used to assess the odds ratio (OR) of becoming lymphocytopenic after treatment initiation with the number of PMN-MDSC at C Malaria. The analysis revealed that increased levels of PMN-MDSC at C Malaria was associated with an increased odds ratio to become lymphocytopenic (OR: 2.3; 95% confidence interval (CI): 1.2–5.5, p < 0.05). The PMN-MDSC levels correlated negatively at C Malaria with the subsequent lymphocyte circulating values at C Treat2 and reached borderline significance at C Treat3 (Spearman's rank correlation p < 0.05 and p = 0.056, respectively, Figures 5B,C), but did not correlate with the lymphocyte levels at C Malaria (p = 0.09, data not shown).

Change of lymphocyte counts could be a confounding factor for the presented changes of PMN-MDSC in the peripheral blood. PMN-MDSC are usually represented as the fraction of total PBMC (61). However, we additionally analyzed whether the absolute counts of PMN-MDSC were increased during the treatment period. The analysis revealed that the absolute counts of PMN-MDSC were significantly increased on C Malaria (median increase: 2.1-fold, p < 0.01, two-stage linear step-up procedure of Benjamini, Krieger & Yekutieli). After the onset of lymphocytopenia on C Treat2 and C Treat3, numbers of PMN-MDSC were still elevated, but the statistical test did not reach level of significance (median increase: 1.6-fold on both days, p > 0.05).