Children treated against lymphoid malignancies display diminished IFN-gamma producing T cells after polyclonal and Varicella zoster virus peptide activation

doi:10.21203/rs.3.rs-4136953/v1

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Children treated against lymphoid malignancies display diminished IFN-gamma producing T cells after polyclonal and Varicella zoster virus peptide activation

https://doi.org/10.21203/rs.3.rs-4136953/v1

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Knowledge on the impact of hematological cancers and their treatment on children's memory T cells is limited. Memory T cells play a crucial role in defending against herpesviruses, particularly relevant in pediatric cancer care. We examined 40 children undergoing cancer or hematological disorder treatment and 13 healthy controls, focusing on memory T-cell subsets using flow cytometry and analyzed cytokine-secreting T cells in response to polyclonal and varicella-zoster virus (VZV) peptides. Children with lymphoid malignancies or post-allo-HSCT showed an accumulation of CD4 + T effector memory (TEM)/ T effector (TEFF) cells among CD3 + cells as follows; [51% (3.8–68.8%)] versus 5.5% (1.3–40.4%), p < 0.001]. Similarly, CD8 + TEM/TEFF proportions were elevated in patients treated for lymphoid malignancies. Following VZV stimulation, these children displayed a significantly lower number of cytokine-secreting cells (183 (30–3181) vs 47 (9–368), p < 0.05) compared to children with other cancer diagnosis/healthy controls. The former group also exhibited a diminished IFN-γ response upon VZV stimulation compared to healthy controls [2 (0–308) vs 53 (5–351), p < 0.001] also noted after polyclonal stimulation. This suggests qualitative differences in T-cell memory among children treated for lymphoid cancers, potentially increasing their susceptibility to severe viral infections, and impacting immunotherapy.

memory T cell

varicella

IFN-𝛾

children

lymphoid malignancy

It is well established that T-cell memory is generated throughout childhood and that, upon birth, the T-cell compartment in peripheral blood consists primarily of naïve T cells. The T-cell compartment then gradually shifts to contain a larger proportion of memory T cells with age and exposure to antigens¹. Memory T cells are crudely divided into central memory (TCM), effector memory (TEM) T cells and terminally differentiated effectors (TEFF). These subsets differ by means of proliferative and tissue homing ability. Along with antigen exposure, memory T cells can eventually differentiate to TEFF, which is accompanied by a loss of proliferative potential and gain of specific tissue-homing and functional abilities^1–3.

It has been shown through comparisons of cell populations, as well as cytokine responses and expression, that our immune system is largely shaped by non-hereditary factors^{1, 4, 5}, an important factor being herpesvirus infections^5–9. Following a self-limiting primary infection in a healthy host, herpesviruses become latent and persist for life. Intermittent reactivation, which is often subclinical, ensures viral spread. Though latency in immunocompetent carriers is mostly asymptomatic, the immune system is highly active in controlling the latency, resulting in the emergence of a marked pool of memory CD4⁺ and CD8⁺ T cells in the host¹⁰. However, with immunosuppression, immune control can be perturbed and lead to reactivation of herpesviruses that can become life-threatening. For instance, among children with oncological or haematological disorders, varicella zoster virus (VZV), which causes chickenpox as primary infection and herpes zoster (HZ) upon reactivation, can lead to treatment-delay, secondary bacterial infections and, in rare cases, severe life-threatening infections^{11, 12}. It has been shown that a third of children treated for acute lymphoblastic leukaemia (ALL) suffer from reactivation of VZV¹². A similar incidence rate of VZV reactivation has also been reported in children after haematopoietic stem cell transplantation (HSCT)¹³.

Most of the studies regarding T-cell responses against herpesviruses in humans have been conducted in adults whereas those on T-cell responses to VZV, in healthy and immunosuppressed children are few or lacking. By measuring cell proliferation, Haining et al reported preserved proliferative memory T-cell responses against VZV in children treated for ALL¹⁴. However, whether additional functional readouts, including cytokine release, also were preserved was not examined in that setting.

To this end, we assessed a cohort of paediatric haematology-oncology patients for T-cell subset composition and polyclonal and VZV-specific responses by quantifying IL-22, IFN-g, IL-10, and IL-17A producing T cells through FluoroSpot. Data herein show that memory T-cell populations are enriched in peripheral blood in children with oncological or severe haematological disease compared to children without malignancies. In addition, our results indicate a markedly lower VZV-specific IFN-g response in children treated for lymphoid malignancies and/or post-HSCT.

Cohort description

This study was part of a prospective, single-centre longitudinal cohort study with the primary aim to examine adaptive immune responses to COVID-19 in children between 0 and 18 years of age with cancer¹⁵. The current study addresses the secondary aim; to investigate memory responses to previously encountered pathogens in immunosuppressed children. Ethical approval was granted by the Swedish Ethical Review Authority (reference 2020–02154, approved 2020/05/22; amendment 2020–04672 approved 2020/11/23). A total of 151 patients were included as of June 2020 to October 2022 at the Department of Children’s Oncology and Haematology, Uppsala University Children’s Hospital, Sweden. Furthermore, data from 14 children without malignancies was used to establish a control group (previously partly described in ¹⁶). Ethical approval for this cohort was granted (Dnr 2020–05664 and Dnr 2014-1164-31/1). Patient inclusion for this cohort is described in detail in Nilsson et al¹⁶.

Peripheral venous blood was collected from included patients at a maximum of six different time-points. Clinical characteristics, such as age, gender, diagnosis, immunosuppressive treatment and previous allo-HSCT, were retrieved from electronic patient charts. A more detailed description of the cohort can be found in Sundberg et al¹⁵. Of the total 151 recruited patients, 103 had available blood samples for this sub-study and 62 of these had sufficient peripheral-blood mononuclear cells (PBMCs ≥ 4 million), required for the immunological analyses.

Two experienced paediatric oncologists (AN and AHA) categorised patients based on their diagnosis (Table 1). This resulted in three groups; group I with controls and children with anaemias having never been treated with chemotherapy (n = 17), group II with solid tumours, CNS tumours and Hodgkin’s lymphoma undergoing or having undergone mild/moderate immunosuppressive treatment (n = 17) and finally group III, consisting of 17 children with leukaemia or lymphoblastic lymphomas as well as two children with severe haematological disorders necessitating allo-HSCT, all undergoing or having undergone intense immunosuppressive treatment (n = 19).

Table 1

**Clinical characteristics of the Varicella zoster virus (VZV) seropositive children (n = 53) in the different subgroups.** *Abbreviations*: ALL, acute lymphoblastic leukaemia; Allo-HSCT, allogeneic haematopoietic stem cell transplantation; CAR, chimeric antigen receptor; CNS, central nervous system.
Group	Diagnosis	Sex	Age	Ongoing therapy (months)	Follow up (months)	Details
1	Control	F	17	N/A	N/A
1	Control	F	1	N/A	N/A
1	Control	F	3	N/A	N/A
1	Control	F	4	N/A	N/A
1	Control	F	4	N/A	N/A
1	Control	F	4	N/A	N/A
1	Control	F	4	N/A	N/A
1	Control	F	10	N/A	N/A
1	Control	F	10	N/A	N/A
1	Control	F	15	N/A	N/A
1	Control	F	16	N/A	N/A
1	Control	M	5	N/A	N/A
1	Control	M	5	N/A	N/A
1	Control	M	6	N/A	N/A
1	Anaemia, pyruvate kinase deficiency	M	18	N/A	N/A
1	Thalassemia major	F	18	N/A	N/A
1	Thalassemia major	M	9	N/A	N/A
2	A-Rhabdomyosarcoma, relapse	M	14	> 6	N/A
2	Anaplastic ependymoma	M	5	ongoing radiotherapy	N/A
2	CNS germinoma	M	17	1	N/A
2	Ewing sarcoma	M	14	at diagnosis	N/A
2	Ewing sarcoma	M	16	> 3	N/A
2	Ganglioneuroma	M	5	N/A	> 1
2	Hodgkin lymphoma	F	16	> 3	N/A
2	Hodgkin lymphoma	F	16	N/A	> 12
2	Kaposiform lymphangiomatosis	M	17	> 9	N/A
2	Osteosarcoma	M	8	N/A	> 1
2	Osteosarcoma	M	15	1	N/A
2	Pilocytic astrocytoma	F	5	N/A	> 6
2	Pilocytic astrocytoma	F	8	> 6	N/A
2	Retinoblastoma	F	2	N/A	> 6
2	Synovial sarcoma	F	8	> 3	N/A
2	Wilm's tumour	F	12	1	N/A
2	Wilm's tumour, relapse	M	5	3	N/A
3	B-ALL	F	2	> 9	N/A
3	B-ALL	F	4	> 9	N/A
3	B-ALL	F	6	No	> 3	HSCT
3	B-ALL	F	7	> 3	N/A
3	B-ALL	F	11	> 3	N/A
3	B-ALL	M	6	> 24	N/A
3	B-ALL	M	11	No	> 2	HSCT
3	B-ALL	M	12	No	> 6	HSCT
3	B-ALL	M	15	No	> 3	CAR-T
3	B-ALL	M	15	No+	> 3	CAR-T, HSCT
3	B-ALL	M	15	at diagnosis	N/A
3	Chronic Myeloid Leukaemia	F	18	> 6	N/A
3	Haematological disorder	F	15	No	> 12	HSCT
3	Haematological disorder	M	14	No	> 3	HSCT
3	Leukaemia	M	3	No	> 12	HSCT
3	Mixed-Phenotype Acute Leukaemia	F	17	No	> 3	HSCT
3	T-ALL	M	15	No	> 1	HSCT
3	T-ALL	M	10	> 12	N/A
3	T-Lymphoblastic Lymphoma	M	14	> 3	N/A

Handling of PBMCs and cryopreservation

Blood samples were collected in CPT™ Mononuclear Cell Preparation Tubes with sodium heparin (BD Biosciences) during planned clinical sampling. PBMCs and plasma were separated through centrifugation after which plasma was aliquoted and frozen at -80°C. Next, PBMCs were washed twice with phosphate-buffered saline (PBS, Gibco) before being counted with 0.4% Trypan blue (Thermo Fisher Scientific). The PBMCs were then resuspended in freezing medium, 10% dimethyl sulfoxide (DMSO, Invitrogen) in fetal bovine serum (FBS, Sigma-Aldrich), at a concentration of 10⁷ viable cells/mL and frozen in liquid nitrogen. For immunophenotyping of T-cell populations and FluoroSpot, PBMCs were thawed and washed. Viable cell counts were once again determined with trypan blue staining and resuspended to a concentration of 2x10⁶ cells/mL in cell culture medium [(RPMI 1640) supplemented with 10% FCS, L-glutamine (2mmol/L), penicillin G sodium (100U/mL) and streptomycin sulphate (100g/mL, all from Thermo Fisher Scientific)]. A portion of PBMCs, 0.3–1.0 million, were assessed for flow cytometric analysis. The remaining were rested overnight at 37°C and 5% CO₂ ahead of FluoroSpot (described below). For the control cohort, the same procedure was carried out apart from using Ficoll-Hypaque gradient centrifugation (GE Healthcare Bio-Sciences AB) instead of CPT tubes.

Assessment of VZV IgG in plasma through enzyme-linked immunosorbent assay (ELISA)

To determine seropositivity for VZV, the Platelia VZV IgG kit assay (Bio-Rad Laboratories) was carried out on all collected plasma according to the manufacturer’s instructions. Optical densities were measured using the Multiskan FC microplate photometer (Thermo Scientific) at the appropriate wavelength.

Immunophenotyping of T-cell subsets by Flow Cytometry

PBMCs underwent PBS washing and staining with a dead cell marker (DCM, Fixable Dead Cell Stain kit, Invitrogen) for 10 minutes at 4°C in the dark. After washing, cells were labeled with fluorescent-conjugated antibodies against CD3, CD4, CD8, CD25, CD57, CD95, CD127, CCR7, FOXP3, and PD-1 (Biolegend, Invitrogen, and BD Biosciences; full panel in supplementary table 1). For the memory T-cell panel, samples were fixated with 4% paraformaldehyde for 5 minutes (Thermo Fischer Scientific), washed, and analyzed. The control cohort received additional CD45RO staining. Regulatory T-cell immunostaining was exclusively performed on the oncological and haematological cohort. For regulatory T-cell staining, PBMCs were fixated with True-Nuclear™ Fix buffer (Biolegend) after surface staining with CD4, CD25, and CD127. True-Nuclear™ Perm Buffer (Biolegend) enabled permeabilization for intracellular FOXP3 labeling, followed by final wash steps. Data was acquired with the Novocyte 3000 (ACEA Biosciences) and analysed through the FlowJo software (FlowJo LLC). For statistical analysis, a threshold of 1 000 live CD3⁺ single cells were established.

FluoroSpot for simultaneous detection of IL-22, IFN-γ, IL-10 and IL-17A secreting cells

The number of IL-22, IFN-γ, IL-10 and IL17-A secreting cells (from now on referred to as spot-forming cells, SFC) was assessed with a human IL-22/IFN-γ/IL-10/IL1-7A FluoroSpot^PLUS kit (Mabtech AB) according to the manufacturer’s instruction. Carried out in a sterile environment, pre-coated FluoroSpot plates were washed with PBS and blocked with cell culture medium for one hour. Following removal of the blocking medium, the T-cell mitogen Staphylococcal enterotoxin A (SEA, m Staphylococcus aureus, Sigma Aldrich), serving as polyclonal activator, was transferred to adequate wells at a final concentration of 20ng/mL. Mixed VZV peptide pools, representing acute and latency MHC class I and II restricted epitopes, were used to assess specific T-cell responses to VZV. The VZV PepMix™ was aliquoted to a 1mg/mL final concentration for each of the IE63 and gE antigens per well (JPT Peptide Technologies GmbH). Furthermore, a co-stimulatory anti-CD28 monoclonal antibody (Mabtech AB) was added, according to the manufacturer’s instructions, in all wells.

Following thawing and overnight rest, PBMCs were washed with PBS, counted, and resuspended in cell culture medium. A total of 80,000–100,000 live PBMCs were thereafter transferred to each well for T-cell stimulation with SEA. 200,000–300,000 live PBMCs were then transferred to appropriate wells, either for VZV-specific T-cell stimulation or to wells containing cell culture medium supplemented with the peptide pool diluent (DMSO, serving as background control). Not all donors were assessed for all four cytokines due to limited number of PBMCs after thawing. The plate was incubated for 48 hours in 37°C, humidified 5% CO₂ atmosphere. Following this, plates were sequentially washed and reagents for the detection of cytokine producing SFCs were added. Plates were read using the Mabtech IRIS™ ELISpot/FluoroSpot reader (Mabtech AB) equipped with wavelength specific filters corresponding to DAPI, FITC, Cy3 and Cy5 spectrum for detection of each cytokine separately. Each fluorescent spot represents the secretory footprint of a single cell. The frequency of spots was assessed with the Apex software version 1.1 (Mabtech AB). Data in figures are presented as SFCs per 10⁶ PBMCs, in mitogen or VZV antigen-stimulated wells, after subtraction of SFC in the background control wells. Furthermore, relative spot volumes (RSV) were also used to quantify secretion through the Mabtech software Apex™.

Statistics

For clinical characteristics, a Kruskal-Wallis test and Fisher’s exact test were used to compare age and gender between the groups, respectively. The Kruskal-Wallis test was also used for both flow cytometric and FluoroSpot data together with a Dunn’s multiple comparisons test. Data is presented as median (min - max), if not otherwise specified. X/Y tables for flow cytometric data, with a simple linear regression, were used solely as an illustrative method with age as a continuous variable. For donors with a set of complete FluoroSpot data (i.e. for all four cytokines examined), the proportion of SFCs for each cytokine among total SFCs per donor was calculated. The medians of these proportions, within each group, were then plotted into pie charts and statistically tested using the Kruskal-Wallis test with Dunn’s multiple comparisons test. A p-value of < 0.05 was deemed statistically significant. All statistical analyses were carried out in Prism (v. 9, GraphPad Software, LLC).

Demographic and clinical cohort characteristics

From the original cohort, 40 patients (66.1%) were IgG seropositive for VZV and, among them, 39 had sufficient PBMCs following thawing for further downstream assays. Data from 14 children without malignancy were included as controls, resulting in a total of 53 included children. Details on diagnosis and stage of treatment are found in Table 1.

No significant differences were found between the groups with regards to demography. However, though not significant, group I had a lower median age compared to group II and III (6 vs 12 vs 12 years old). Group I consisted of an insignificantly larger proportion of females compared to group II and III (70.5% females vs 41.2% and 42.1% respectively). Finally, almost half (47.3%) of the patients in group III had undergone allo-HSCT, where two patients in the same group had also received chimeric antigen receptor (CAR) T-cell treatment.

Memory T-cell subsets are enriched whereas the naïve T-cell populations are diminished in children with lymphoid malignancies

The different T-cell populations were first examined through flow cytometry (Fig. 1A) according to diagnostic groups I-III (Fig. 1B). To illustrate the impact of age, the proportions of T-cell subsets in each group were also plotted against age as a continuous variable (Fig. 1B).

No significant differences were found in bulk CD4⁺ and CD8⁺ T-cell proportions between the groups. Both CD4⁺ and CD8⁺ naïve T-cell proportions were significantly lower in group III compared to group I (70.5% (23.2–84.5%) vs 22.3% (2.0–73.8%), p < 0.01, and 59.2% (38.4–90.6%) vs 11.8% (0.5–86.9%), p < 0.01, respectively). The X/Y graphs illustrate how this effect may partly be explained by increasing age in all groups. In this context, a more pronounced decrease in the proportion of naïve CD8⁺ T cells with increasing age in group III compared to the remaining groups is seen (Fig. 1B). Furthermore, the proportions of TEM/TEFF cells among CD4⁺ T cells were significantly lower in group I compared to group II and III (respectively as follows 5.5% (1.3–40.4%) vs 28.9% (3.2–67.6%) for group II, p < 0.05; and vs 51.0% (3.8–68.8%) for group III, p < 0.001). CD8⁺ TEM/TEFF proportions were also significantly lower in group I compared to group III (7.6% (1.9–24.1%) vs 43.5% (2.3–70.7%), p < 0.001). Once again, the X/Y plots reflected the impact of age, where higher proportions of TEM/TEFF populations were seen with increasing age. Nonetheless, the linear regression also suggested that group III had a more noticeable increase in CD8⁺ TEM/TEFF cells compared to group I. Proportions of CD4⁺ TCMs were significantly higher in group III compared to group I (18.0% (7.3–35.7%) vs 8.6% (4.6–29.6%), p < 0.01). The influence of age on TCM CD4⁺ proportions within the groups was not as clear as for the naïve and TEM/TEFF subsets (Fig. 1B). There were no differences for CD8⁺ TCM cell proportions between the groups, or in relation to age (Fig. 1B). Finally, no differences were seen between the groups regarding proportions of regulatory T cells (data not shown). Two subjects within group III had received CAR-T-cell therapy, which did not allow stratification of data for statistical analysis. Overall, the T-cell populations of these subjects did not deviate from the remaining individuals in group III (data not shown).

Depleted IFN- γ secretion following stimulation with SEA and VZV peptides in children treated for lymphoid malignancies

The functional status of T cells was assessed through simultaneous quantification of IL-22, IFN-γ, IL-10 and IL17-A secreting cells upon polyclonal SEA or VZV-specific stimulation (Fig. 2A). First, SFC counts for all cytokines were pooled for individuals with a complete assay including all four cytokines. The number of total SFCs differed between the groups for both polyclonal and antigen-specific activation (Fig. 2B). Group I had significantly more total SFCs upon SEA stimulation compared to group III (6245 (490–11945) vs 1080 (11–12800), p < 0.05). Upon VZV stimulation, group I and II had a similar total SFC count, whereas group III had a much lower response. This shift became significant when comparing the total SFCs following VZV stimuli in group II with group III (183 (30–3181) vs 47 (9–368), p < 0.05, Fig. 2B).

Next, the cytokine profiles upon polyclonal activation of PBMCs with SEA were generated using SFC data for donors with complete assays (Fig. 2C, upper panel). The overall cytokine responses to SEA were dominated by IFN-γ in all groups, with the remaining cytokines accounting for only 16–23% of secreting cells. However, a greater variation in cytokine response was found between the groups following stimulation with VZV peptides (Fig. 2C, lower panel). The proportions of IFN-γ secreting cells for children in groups II and III were noticeably lower compared to group I. In group III, IFN-γ SFCs only accounted for 4% of secreting cells, whereas the majority (63%) consisted of IL-22 SFCs instead (Fig. 2A). Significant differences after VZV exposure were found for the proportions of IL-22 secreting cells between group I and III (p < 0.05), for IFN-γ secreting cells between group I and III (p < 0.01) and for IL-17A secreting cells between group I and III (p < 0.01) and between group II and III (p < 0.05).

Data for each specific cytokine was also compared between the groups (Fig. 2D). No significant differences were found for IL-22. For IFN-γ, SFCs were significantly lower in group III compared to group I upon both SEA and VZV stimulation (1035 (0–12380) vs 5240 (375–9775), p < 0.05 and 2 (0–308) vs 53 (5–351), p < 0.001). In response to VZV, group III also had significantly lower ratios of IFN-γ compared to group II (2 (0–308) vs 28 (2–602), p < 0.05). For IL-10, group III had a significantly lower ratio of IL-10 SFCs compared to group I upon SEA stimulation (80 (0–1570) vs 543 (15–2730), p < 0.01), whereas no significant differences were seen following VZV stimuli (Fig. 2D). When quantifying IL-17A response, no significant differences were found between the groups upon VZV stimuli (Fig. 2D). However, upon SEA stimuli, group III had a significantly lower proportion of IL-17A SFCs compared to group I (15 (0–300) vs 165 (10–515), p < 0.01).

Finally, the volume and intensity of each spot, quantified as RSV, were used as an additional qualitative marker of cytokine secretion (Fig. 3A). Interestingly, we observed that average RSV for IFN-γ in response to VZV was markedly lower in group III; specifically, between group I and III (7810 (1407–29184) vs 462 (0–11466), p < 0.0001) as well as between group II and III (6247 (247–32453) vs 462 (0–11466), p < 0.05). Furthermore, average RSV for IL-17A was significantly lower in group III compared to that in group I (934 (0–42877) vs 8998 (0–20163), p < 0.01) upon polyclonal stimulation. No significant differences were found in RSV for IL-17A following VZV stimulation. The RSV data for IL-22 and IL-10 did not significantly differ between the study groups (Fig. 3B).

VZV infection poses challenges for pediatric oncologists, as primary infection can lead to critical illness and treatment delays. Furthermore, immunocompromised children with hematological or oncological diseases face higher VZV reactivation risks. Our previous findings highlighted IFN- g dominance in healthy children's VZV-specific memory T-cell responses¹⁶. Here, we explore polyclonal and VZV-specific responses in children with cancer or hematological diseases.

Prior studies have noted altered T-cell proportions in pediatric cancer, although to a lesser extent than for the B-cell populations (reviewed in ^{17, 18}). In the current cohort, bulk CD4⁺ and CD8⁺ were similar between the groups. However, proportions of naïve CD4⁺ and CD8⁺ cells were decreased in group III (children with lymphoid malignancies and/or post-HSCT) whilst accumulations of CD4 + and CD8 + TEM/TEFF cells as well as in the CD4 + TCM subset was noted. Age relates to accumulation of memory T cells^{1, 16}, but it has also been shown that children on cancer treatment primarily exhibit a T-cell memory phenotype^{14, 19}. Interestingly, Das et al showed that accumulation of terminal effector T cells increases with each cycle of chemotherapy²⁰. Therefore, it is possible that our results reflect variations in T-cell immunophenotype explained both by age as well as diagnosis and its entailing treatment.

We further assessed the functional status of T cells and found that the number of cytokine-secreting cells differed significantly between the groups, where children in group III showed fewer responding cells. The cytokine profile upon polyclonal SEA activation relied heavily on IFN-g, alluding to the expected Th1 response that is key to cellular immunity²¹. The cytokine profiles of SEA responding cells also aligned with those seen in a previous study in healthy children¹⁶. Interestingly, it was observed that children in group III had significantly fewer IFN-g, IL-17A and IL-10 secreting cells following polyclonal activation compared to group I, accompanied by a diminished IL-17A secretion on a single cell basis, measured as RSV. This decrease suggest a diminished T-cell response to polyclonal activation in children with haematological malignancies or children post-HSCT.

The VZV-specific T-cell memory cytokine profile showed a significant lower proportion of IFN-g in groups II and III, with a bias towards IL-22 and IL-17A secretion²². This shift in cytokine profile is explained primarily by a low number of IFN-g secreting cells, rather than an increase in the number of IL-22 or IL-17A secreting cells. IFN-g is the key cytokine secreted by Th1 cells, and a cytokine of significant importance during HZ²³. Our data suggest that these cells are particularly affected in children belonging to group III, possibly due to disease itself or HSCT treatment affecting the lymphoid compartment. Another possible explanation could be differences in drugs used in the applied chemotherapy protocols for different malignancies. Work by Mackall in the 1990s demonstrated, for example, that the drug cyclophosphamide more effectively depleted lymphocytes compared to other drugs²⁴. There are also previous reports suggesting that 6-mercaptopurine, often used in the treatment of ALL, reduces the number of IFN-g⁺ T cells in peripheral blood^{25, 26}. Conversely, Haining et al showed that T-cell proliferation in response to VZV whole virus lysate and tetanus toxoid remained intact in children treated for ALL and was indeed greater than the proliferation in age-matched controls due to memory T-cell accumulation¹⁴. In contrast, our study demonstrates that children with lymphoid malignancies may instead have a qualitative difference at the cytokine responses level, by means of altered IFN-g secretion which could affect the immune response to VZV reactivation. In that context, it is also interesting that individuals with anti-IFN-g autoantibodies often experience HZ²⁷.

This study's limitations stem from a diverse cohort in age, diagnosis, and treatment, and limited sample size for inclusion of VZV + patients in every analysis. Expanding the cohort with stratification by age, diagnosis, treatment, and sampling time could enhance our understanding of T-cell IFN-γ responses to VZV. Emerging immunotherapies may influence results; two patients received CAR-T cell treatment (Table 1).

In a broader context, the question demanding a more comprehensive answer pertains to how cancer and its treatments impact the overall functional status of T-cell memory. While our understanding of the late effects of cancer treatment in children is gradually expanding, certain gaps persist. Although we acknowledge that chemotherapy exerts a lasting effect on T cells¹⁹, a thorough understanding of how chemotherapy, corticosteroids, HSCT, and the underlying diagnoses collectively influence T-cell memory in the long term remains elusive¹⁷. Our findings may also have implications beyond infection immunity. Given that some novel therapies hinge on the functionality of autologous T cells, it is plausible that dysfunctional T cells¹⁹ and/or an altered ratio between effector and regulatory T cells²⁸ could affect the effectiveness of immunotherapeutic treatments. Understanding these dynamics becomes particularly relevant as we navigate the complexities of immunotherapeutic approaches and seek to enhance anti-cancer response while reducing treatment toxicity, including infection sensitivity, in well-characterized study cohorts. Additional qualitative studies are imperative to unravel the intricate interplay of disease, treatment, and their collective impact on T-cell function in the broader context of childhood health.

ACKNOWLEDGEMENT

This work was supported by grants from the Swedish Childhood Cancer Foundation (AN, AHA), Swedish Research Council (VR 2020-02244) (AN). JP and AHA were supported by Lions Cancer Foundation, Uppsala University Hospital, Uppsala.

AUTHOR CONTRIBUTION

SSH, AHA, JP and AN conceptualised and designed the study and were responsible for supervision and funding; ES and AHA were responsible for clinical assessment of patients and their inclusion in the cohort; ET, AHÖ, AHA and SSH were responsible for sample biobanking and performance of experiments; ET, SSH, AN and PJ were responsible for data compilation and analysis; ET and SSH performed statistical analysis; ET, SSH, and AN interpreted the data and wrote the manuscript. All authors read and approved the final version of the manuscript.

COMPETING INTEREST

Peter Jahnmatz was, at the time of the study, employed by Mabtech AB, Sweden. Several of the reagents and the FluoroSpot reader system used in this study are produced by Mabtech. Mabtech had no influence on the content of this study. At the time of study conceptualization and performance of experiments, data compilation and analysis SHS was employed by Karolinska Institutet. As of April 2023, SHS is employed by Amgen, Sweden. Amgen has not had any influence on this study.

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Competing interest reported. Peter Jahnmatz was, at the time of the study, employed by Mabtech AB, Sweden. Several of the reagents and the FluoroSpot reader system used in this study are produced by Mabtech. Mabtech had no influence on the content of this study. At the time of study conceptualization and performance of experiments, data compilation and analysis SHS was employed by Karolinska Institutet. As of April 2023, SHS is employed by Amgen, Sweden. Amgen has not had any influence on this study.

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Children treated against lymphoid malignancies display diminished IFN-gamma producing T cells after polyclonal and Varicella zoster virus peptide activation

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Cohort description

Handling of PBMCs and cryopreservation

Assessment of VZV IgG in plasma through enzyme-linked immunosorbent assay (ELISA)

Immunophenotyping of T-cell subsets by Flow Cytometry

FluoroSpot for simultaneous detection of IL-22, IFN-γ, IL-10 and IL-17A secreting cells

Statistics

RESULTS

Demographic and clinical cohort characteristics

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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