Zinc as a therapeutic adjunct: enhancing t-cell reconstitution in hematopoietic stem cell transplant recipients—a double-blind clinical study

Background

Effective immune reconstitution (IR) following hematopoietic stem cell transplantation (HSCT) is vital for increasing long-term patient survival and reducing the risk of posttransplant infections. A key indicator of successful immune recovery is the generation of recent thymic-enriched T cells (RTEs), which directly reflects thymic activity. Zinc (Zn), an essential trace element, has been shown to play a pivotal role in supporting immune function and thymic output. This study assessed how Zn supplementation influences RTE levels and overall immune recovery in patients undergoing HSCT.

Methods

In this double-blind, randomized, placebo-controlled clinical trial, 38 patients who underwent autologous HSCT were randomly assigned to receive either Zn supplementation (n = 19) or placebo (n = 19). The intervention group was administered three 30 mg Zn gluconate tablets daily during the first 30 days post-transplant, followed by one tablet daily from day 31 to day 90. T-cell subsets, including RTEs, were measured via flow cytometry at baseline, day 30, and day 90. Absolute lymphocyte counts (ALCs) were also monitored. Nutritional intake and serum Zn and copper levels were assessed to control for dietary confounders.

Results

On day 90 posttransplant, patients receiving Zn supplementation presented a significantly greater ALC than did those in the placebo group (p = 0.009). Furthermore, the Zn group presented a substantial increase in the percentage of RTEs at both day 30 (p = 0.008) and day 90 (p = 0.025), whereas the placebo group did not demonstrate changes. These findings suggest that Zn supplementation positively influences thymic output and contributes to early T-cell reconstitution.

Conclusion

Zn supplementation facilitates immune recovery following autologous HSCT by increasing ALC and RTE generation. These results highlight the potential utility of Zn as an adjunctive therapy to promote thymic regeneration and immune competence in transplant recipients.

Trial registration

IRCT20191211045701N1/Date: 2020–06-12. This registration confirms adherence to ethical standards and facilitates verification of study details from 2020–06–12.

Highlights

1. Zn supplementation significantly enhances T-cell recovery after HSCT.

2. RTEs increased notably in the Zn group.

3. Higher absolute lymphocyte counts were observed at 90 days post-transplant with Zn.

4. This study suggests that Zn is an effective adjunct therapy for immune reconstitution.

5. These findings support the role of Zn in improving long-term survival rates post-HSCT

Background

HSCT is a well-established therapeutic approach for various hematologic malignancies and immune disorders. Nevertheless, the immunosuppressive effects of the preparatory conditioning regimen result in a temporary state of immunodeficiency following HSCT [1]. Chemotherapy-induced thymic damage and age-associated thymic atrophy are critical factors that can delay T-cell regeneration [2–6]. T-cell reconstitution is influenced primarily by thymic function in patients undergoing HSCT, a process that may require several months or potentially years to complete [7].

RTEs through the thymus play crucial roles in enhancing the diversity of the T-cell repertoire [1]. While T cells are capable of regeneration even in the context of reduced activity within an atrophic thymus, this phenomenon frequently leads to poor outcomes following HSCT [8, 9]. Impaired thymic recovery is associated with an increased risk of opportunistic infections and unfavorable clinical outcomes in patients undergoing HSCT. Consequently, implementing strategies to enhance immune reconstitution (IR) following this procedure is paramount. Preclinical studies have explored various strategies, including the utilization of growth factors and cytokines, to enhance immune reconstitution after HSCT. However, only a limited number of these approaches have resulted in the establishment of clinical guidelines [10].

Zn, an essential trace element, is critical for various immunological mechanisms [11]. It is integral to the development, maturation, and functioning of immune cells, particularly T cells, which are pivotal in coordinating immune responses [12, 13]. Zn modulates the proinflammatory response by targeting nuclear factor kappa B (NF-kB), which is a key regulator of inflammatory responses [14, 15]. Additionally, it helps control oxidative stress and regulates inflammatory cytokines [16]. Zn deficiency can increase the inflammatory response and harm tissues [17]. Even mild Zn deficiency notably reduces thymulin activity and T-cell production [18]. Recent studies have demonstrated the beneficial effects of Zn supplementation on thymic output and its capacity to lower the risk of infection in elderly individuals [17, 19].

Our recent study demonstrated that Zn supplementation significantly reduced oxidative stress levels in patients undergoing autologous hematopoietic stem cell transplantation (autoHSCT) [20]. Research has investigated the potential benefits of Zn supplementation for IR after HSCT, particularly with respect to T-cell regeneration [21–26]. Preclinical studies in murine models have also shown that oral Zn supplementation enhances thymus regeneration post-HSCT [27, 28].

In light of the limitations identified in earlier studies—particularly the use of high-dose Zn and Zn sulfate formulations associated with gastrointestinal side effects, as well as smaller sample sizes relative to the current investigation—this clinical trial was designed to assess the effectiveness of Zn supplementation in promoting T-cell reconstitution in patients undergoing autoHSCT [25] (Fig. 1).

Fig. 1.

Effects of Zn supplementation on T-cell reconstitution after autologous HSCT

Materials and methods

Study design

This investigation constituted a double-blind, placebo-controlled, randomized clinical trial. Within this parallel study, patients undergoing autoHSCT were randomly allocated to either the Zn group or the placebo group. Figure 2 provides an overview of the study design. This randomized controlled trial was conducted and reported under the Consolidated Standards of Reporting Trials (CONSORT) guidelines. The completed CONSORT checklist is provided as an additional file. The research was conducted in the Bone Marrow Transplantation Department at Ayatollah Taleghani Hospital, affiliated with Shahid Beheshti University of Medical Sciences (SBMU) in Tehran, Iran. The study's protocol has been published previously [29].

Fig. 2.

Study Design [29]

Ethics consideration

This study was approved on January 14, 2020, by the Ethical Committees of Tarbiat Modares University (IR.MODARES.REC.1398.195) and Shahid Beheshti University of Medical Sciences (IR.SBUM.REC.1399.010). It has also been registered with the Iranian Registry of Clinical Trials (IRCT20191211045701N1).

All procedures involving human participants were performed according to the ethical standards of the institutional and national research committees and by the Declaration of Helsinki and its subsequent amendments or comparable ethical guidelines. Before enrollment, all participants provided written informed consent. The study population was rigorously defined, and the participants were fully informed of the potential side effects of Zn supplementation. Upon providing consent, each participant was assigned a unique numerical identifier, and baseline data were collected via a standardized questionnaire administered by the study interviewer. The participants retained the right to withdraw from the study without penalty. No financial compensation was provided for participation, and all study-related interventions, including pharmaceutical treatments and laboratory investigations, were administered without additional cost to participants.

Patients

The study participants were selected from patients with a history of multiple myeloma (MM) who were candidates for autoHSCT and had been referred to the Bone Marrow Transplantation Department of Taleghani Hospital in Tehran. Eligible patients were enrolled on the basis of the inclusion and exclusion criteria. Patients were instructed to promptly contact the researchers or their assistants in cases of any adverse events, such as nausea, vomiting, diarrhea, rash, or other abnormal symptoms [30].

Inclusion and exclusion criteria

The inclusion criteria were as follows: age between 40 and 60 years, ability to swallow tablets, history of MM, complete response to treatment, and eligibility for autoHSCT without comorbidities.

The 40–60 year age range was selected to target both the peak incidence demographic for MM [31] and the period of established thymic involution when the T-cell regeneration capacity becomes clinically compromised [32], thereby optimizing the assessment of the immunomodulatory effects of Zn.

The exclusion criteria included a history of allergic reactions to oral Zn supplementation, serum Zn levels above 200 µg/dL, and the use of oral Zn supplements within three months prior to the intervention.

Blinding and randomization

In this meticulously conducted double-blind study, the researchers and the participants were unaware of the Zn and placebo groups. The eligible patients were evenly distributed into two groups via a block randomization process. The research assistant carefully packaged the Zn supplement and placebo into A and B packs and then delivered them to the principal researcher according to the randomization.

Intervention

The dosage of Zn was derived from a previous clinical trial that employed high-dose oral Zn supplementation to enhance T-cell reconstitution following HSCT [33].

"Zn gluconate" 30 mg tablets were acquired from Dineh Iran Industries Complex located in Tehran, Iran. To ensure consistency in color, shape, and weight, placebo tablets were formulated by the SBMU School of Pharmacy. The placebo consisted of microcrystalline cellulose (Avicel) and included propylene glycol, hydroxypropyl methyl cellulose, titanium dioxide, alcohol, and edible coloring agents.

Assessments

Questionnaires

All participants completed a comprehensive demographic questionnaire before entering the study.

To ensure that there were no differences in food intake between the intervention and placebo groups, the participants completed two 3-day food intake registration questionnaires. All the participants received training on how to complete these food records. Each patient completed two "3-day food intake registration" questionnaires before and after HSCT during the intervention at home, resulting in six questionnaires per patient by the end of the study.

On the basis of the six completed food records, we determined the average caloric intake by analyzing the macronutrients and micronutrients of both groups. Nutrient calculations were performed via Nutritionist 4 software (First Data Bank, San Bruno, CA, USA) during the intervention [33].

Owing to the interference between Zn and copper absorption, these two micronutrients were measured in the clinical laboratory via the colorimetric technique before and one month after the intervention.

Flow cytometry

Blood samples were collected in 2 ml EDTA-coated tubes at three time points: upon admission and on the 30th and 90th days following HSCT. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation (Ficoll-Hypaque, etc.) and counted in a Neubauer chamber. After rinsing with PBS, 10⁶ mononuclear cells were meticulously stained with a panel of fluorochrome-conjugated anti-human monoclonal antibodies, including CD4, CD31, CD45RA, and CD45RO, for 20 min at 4 °C and then analyzed by flow cytometry (Attune et al.).

The results for the two populations of RTEs are expressed as percentages of CD4 + CD45RA + CD31 + T cells within the CD4 + T-cell population and subsequently as absolute cell counts per microliter of blood. CD45RA + CD4 + naïve T cells and CD45RO + CD4 + memory T cells were quantified in the same sample [34].

TREC assessment

Despite the challenges posed by the embargo, we conducted the signal joint T-cell receptor excision circles (sjTREC) assessment in Iran. Alternatively, we collected dried blood spots to determine the TREC/KREC copy number and sent them to Immuno IVD, a company based in Switzerland. Unfortunately, most samples did not yield results, so the report needed to be completed.

Follow-up

Relapse, cytomegalovirus (CMV) reactivation, and Epstein–Barr virus (EBV) occurrence were monitored within 365 days post-HSCT. Bone marrow aspirates and serum/urine protein electrophoresis were used to detect relapse in MM patients during follow-up.

Figure 2, published in the protocol study [29], summarize [29]s the trial's intervention and assessment schedule.

Statistical analysis

The repeated-measures ANOVA model was used to assess within-subject variability and the difference between RTEs and the absolute lymphocyte count (ALC) between the treatment groups. Data normality was evaluated via the Kolmogorov‒Smirnov test, and transformations were applied when necessary to satisfy model assumptions. Temporal differences between treatment groups were further examined through post hoc pairwise comparisons with Tukey's adjustment for multiple testing.

Zn and Cu levels after HSCT were analyzed via an ANCOVA model to control for baseline levels. Paired t tests were used to evaluate changes in Zn and Cu levels before and after transplantation.

The Cox proportional hazards model was applied to investigate the time to relapse following HSCT, estimating hazard ratios while accounting for censored data. Proportionality assumptions were assessed via Schoenfeld residuals, and model diagnostics were performed to ensure validity and robustness.

Despite the fact that randomization plays a crucial role in the formation of comparable groups, we evaluated the potential confounding effects within both the repeated measures model and the Cox model.

All the statistical analyses and visualizations were performed via R software (version 4.4.1).

Results

From January 2019 to December 2021, 49 patients between 40 and 60 years of age who underwent autoHSCT signed the consent form and were included in the study. After excluding 11 patients who discontinued follow-up (due to travel constraints or withdrawn consent), complete data from 38 participants were followed for at least three months and analyzed.

The information about the patients, including age, sex, chemotherapy regimen, treatment response, relapse history, and latest disease status before transplantation, was derived from medical records and is reported in Table 1.

Table 1.

Patient characteristics

Variable	Total		Intervention				P Value
			Zinc (N = 19)		Placebo (N = 19)
Categorical Variables
	frequency	percent	frequency	percent	frequency	percent
Gender
Male	20	52.6	11	57.9	9	47.4	0.7401
Female	18	47.4	8	42.1	10	52.6
Relapse
Yes	11	28.9	7	35	4	22.2	0.6101
No	27	71.1	13	65	14	77.8
Chemotherapy Regimen
VCD	15	39.5	8	42.1	7	36.8	0.212
VRD	19	50.0	7	36.8	12	63.2
VD	1	2.6	1	5.3	0	0
VTD	2	5.3	2	10.5	0	0
Missing	1	2.6	1	5.3	0	0
Chemotherapy Response
CR	28	73.7	14	73.7	14	73.7	0.999
VGPR	7	18.4	4	21.1	3	15.8
PR	2	5.3	1	5.3	1	5.3
Missing	1	2.6	0	0	1	5.3
Disease status
Stage1	14	36.8	7	35.0	7	38.9	0.132
Stage2	8	21.1	2	10.0	6	33.3
Stage3	16	42.1	11	55.0	5	27.8
Continuous Variables
	mean	sd	mean	sd	mean	sd
Age at diagnosis	49.62	4.63	49.61	5.59	49.64	3.57	0.987
Age at HSCT	51.48	4.69	51.57	5.46	51.38	3.93	0.902

P values were used to assess independence for categorical variables via the chi-square test or Fisher's exact test, and differences for continuous variables were evaluated via the independent t test

VCD Bortezomib, cyclophosphamide and dexamethasone, VRD Bortezomib, lenalidomide and dexamethasone, VD Bortezomib and dexamethasone, VTD Bortezomib, Thalidomide and Dexamethasone, CR Complete remission, VGPR Very good partial remission, PR Partial Remission

The average age of patients at the time of transplantation was approximately 51.5 years in both groups, with no significant difference (p = 0.902), confirming a balanced age distribution between the groups (Table 1).

As shown in Table 2, before the intervention, the average serum Zn levels in the Zn and placebo groups were 101.35 ± 14 µg/dL and 101.71 ± 22.02 µg/dL, respectively, with no statistical evidence of a difference (p = 0.98). The average serum Cu levels in the Zn and placebo groups were 128.35 ± 16.46 µg/dL and 152.33 ± 29.16 µg/dL, respectively, with no significant difference (p = 0.467).

Table 2.

Comparison of Zn (µg/dl) and copper (µg/dl) serum levels between the two groups before and after the intervention

Variable	Treatmnet		P value
	Zn	Placebo
Zn before	6.506 ± 0.351	7.532 ± 0.251	0.027
Zn after	6.999 ± 0.290	7.648 ± 0.261	0.11
P value	0.275	0.778
Cu before	1.016 ± 0.068	1.169 ± 0.053	0.018
Cu after	0.990 ± 0.038	1.033 ± 0.072	0.851
Pvalue	0.88	0.005

On the basis of the three-day food registration questionnaire analysis, the average Zn intake from food before the intervention was 6.5 mg in the Zn group and 7.53 mg in the placebo group. After the intervention, the average Zn intake was 6.99 mg in the Zn group and 7.64 mg in the placebo group (Fig. 3). The results indicated that the amount of Zn received from food was greater in the placebo group (p = 0.01). The ANCOVA model revealed no confounding effect on the Zn levels before the intervention (p = 0.868). Additionally, there was no substantial evidence of the intervention's effect (p = 0.129).

Fig. 3.

Comparison of dietary Zn intake, measured by a three-day food record questionnaire, between the two groups before and after the intervention, along with the individual line plots

The results of the repeated measures analysis revealed that overall, the evidence for a time effect (within-subject effect) on the logarithm of ALC values was not statistically significant (p = 0.073). However, the Zn group demonstrated a significant overall difference in the logarithm of ALC values over time (p = 0.019). The effect was particularly pronounced on day 90 + (p = 0.009) (Fig. 4a, Table 3). Notably, the growth of the log ALC became apparent on day 90 +, where a significant difference was observed compared with that on day 30 + (p = 0.018) (Fig. 4b). The results also indicated that the overall time effect on RTEs was weak (p = 0.292). However, the Zn group demonstrated a significant overall difference (p = 0.016). In particular, we observed a significant difference in days 30 + (p = 0.008) and 90 + (p = 0.025), with no significant difference in the number of admission days (p = 0.219) (Fig. 4c, Table 3). Moreover, we evaluated the confounding effects of age, sex, chemotherapy response, BMI, WBC count, hemoglobin, platelet count, and LDH before HSCT. Our analysis revealed no statistically significant results (p > 0.1).

Fig. 4.

Boxplots of log(ALC) and RTE values over the study period stratified by intervention, accompanied by line plots illustrating individual trends

Table 3.

Results of multiple comparisons between treatment groups for log(ALC) and RTEs across the study period

		Response
		Log (ALC)			RTE
Time	Treatment	Estimated Mean	95% confidence interval	p	Estimated Mean	95% confidence interval	p
Pretransplant	Zinc	0.403	0.249, 0.556	0.120	23.4	20.7, 26.0	0.219
	Placebo	0.228	0.065, 0.389		21.0	18.1, 23.8
day + 30	Zinc	0.277	0.124, 0.431	0.448	24.8	22.1, 27.5	0.008
	Placebo	0.193	0.031, 0.355		19.4	16.6, 22.2
day + 90	Zinc	0.540	0.386, 0.693	0.009	25.6	22.9, 28.2	0.025
	Placebo	0.236	0.074, 0.398		21.1	18.3, 23.9

All patients were followed for at least six months until relapse. Only one female participant experienced a relapse in this study, which occurred within the zinc treatment group. In contrast, all other relapses, totaling ten, were observed among male participants: six relapses occurred in the Zn group, and four occurred in the placebo group. The survival analysis suggested a possible trend toward a greater risk of recurrence in males than in females (hazard ratio = 8.346; p = 0.043). Given the confounding effect of sex, our analysis revealed no significant difference in relapse risk between the zinc group and the placebo group (hazard ratio = 1.647; p = 0.433) (Fig. 5).

Fig. 5.

The probability of relapse after transplantation in the Zn and placebo groups

Discussion

This study evaluated the effects of Zn supplementation on enhancing immune reconstitution (IR) after HSCT. Delayed T-cell recovery can increase the risk of infection in patients [30, 35–38].

In this study, patients received Zn supplementation at a dosage of 90 mg per day for the first 30 days, followed by 30 mg per day for the next 90 days. The results revealed improved T-cell recovery, with no dietary influence noted (P = 0.868).

Zn plays a significant role in thymic function and lymphocyte proliferation, supporting its use as a safe and cost-effective strategy to accelerate immune reconstitution, which is crucial for reducing posttransplant complications [6, 39–42].

These findings highlight the potential of Zn in HSCT protocols; however, further studies with larger sample sizes are needed to confirm the long-term benefits of Zn on survival and infection rates.

Our results indicated that patients receiving oral Zn supplements exhibited a significant increase in ALC 90 days post-transplantation compared with the placebo group (P = 0.008). No significant differences were observed at the initial time points, suggesting that Zn supplementation may effectively enhance immune recovery. Furthermore, continued Zn consumption over specified periods led to significant increases in the ALC between days 30 and 90 posttransplantation (P = 0.003), a trend not observed in the placebo group. These findings underscore the importance of monitoring the ALC as a critical indicator of immune recovery and long-term outcomes following HSCT and provide reassurance about the effectiveness of our research [43]. ALC predicts long-term and recurrence-free survival [44–46]. Three pivotal studies established the absolute lymphocyte count (ALC) as a significant prognostic marker following hematopoietic stem cell transplantation (HSCT). In a pediatric cohort undergoing allogeneic HSCT (n = 111), an ALC of ≥ 500 cells/μL by day 100 was associated with improved survival [36]. Kim et al. reported that a low ALC is an independent risk factor for increased nonrelapse mortality in adults (n = 1,109), regardless of the severity of GVHD [45]. In patients with multiple myeloma (n = 769), an ALC of ≥ 1400 cells/μL measured at days 0, 15, and 90 correlated with increased overall survival, further validating ALC as an independent predictor of immune function and survival [47]. These findings reinforce the clinical significance of ALC across different transplant scenarios.

RTEs, which are characterized by CD31 + CD4 + T naïve cells, maintain relatively stable numbers even in the face of age-related declines in thymic function. This preservation is vital, as RTEs contribute a diverse array of T-cell receptors (TCRs) that are essential for fighting infections and tumors. Following HSCT, rapid recovery from RTEs may help mitigate two significant complications: infections and disease relapse [48]. Zn plays a pivotal role in this process by supporting lymphocyte proliferation, thymic epithelial cell regeneration, and thymic output [49, 50]. These findings highlight the critical immunoprotective functions of RTEs and the potential of Zn to enhance immune reconstitution following HSCT, particularly in maintaining TCR diversity and preventing posttransplant complications.

Our results provide strong evidence that oral Zn supplementation significantly increases the incidence of RTEs following transplantation. Longitudinal analysis at three time points revealed similar baseline RTE percentages between the two groups (P = 0.086). The placebo group exhibited no significant fluctuations in RTE percentages between days 30 and 90. In contrast, patients receiving Zn treatment presented significantly greater RTE percentages at both day 30 (P = 0.008) and day 90 (P = 0.025) than did the control group.

While the absolute RTE counts remained stable over time in both groups (P = 0.292), a combined analysis confirmed a significant association between Zn administration and elevated RT percentages across all time points (P = 0.016). Intragroup comparisons demonstrated a consistent upward trend in mean RTE values among Zn recipients throughout the study period, whereas placebo patients maintained stable levels. Although these trends did not achieve individual statistical significance—likely owing to sample size limitations and associated variance—the persistent differences between the groups strongly indicate the beneficial effect of Zn on maintaining RTE levels.

These findings lead to two important conclusions: first, Zn supplementation appears to sustain RTE proportions at significantly higher levels than those observed in the placebo control group, despite comparable baseline characteristics; second, the intervention may prevent the natural decline in RTEs typically seen after transplantation. The demonstrated dose‒response relationship, although it requires validation in larger cohorts, provides mechanistic support for the role of Zn in promoting thymic-dependent immune reconstitution through enhanced preservation of RTEs.

Previous clinical research involving 18 HSCT patients demonstrated that high-dose Zn supplementation (150 mg/day for 100 days) effectively increased TCD4 + cell counts and sjTREC copy numbers [25]. In our current protocol, we used Zn gluconate, which is chosen for its favorable safety profile, which includes low levels of cadmium contamination and minimal renal or gastrointestinal toxicity [51]. We modified the dosing regimen to 90 mg/day for the first month, followed by 30 mg/day for the subsequent two months. This adjusted protocol was designed to optimize the immunomodulatory effects while ensuring safety and efficacy. These findings are further supported by preclinical work from Iovino et al., whose murine allogeneic HSCT models consistently demonstrated the ability of Zn to increase thymic regeneration and output [27, 52, 53].

Relapse following HSCT remains a major therapeutic challenge in MM, representing the primary cause of treatment failure. Early postautoHSCT relapse is associated with an inferior prognosis. The key determinants of relapse include time to recurrence (early relapse portends worse outcomes), disease characteristics (high-risk cytogenetics, tumor burden, and pretransplant response), and transplant type [54]. Thus, we investigated the impact of Zn supplementation on early relapse incidence in MM patients post-HSCT.

Although the relapse rate in patients receiving Zn treatment was slightly higher, the results were not statistically significant. This lack of significant evidence can be attributed to the small sample size, making the findings unreliable. Additionally, the difference may be related to cytogenetic factors, which we did not evaluate. Other factors, such as patient genetics, the country where chemotherapy drugs are produced, the timing of a patient's presentation after relapse, and various other considerations, may influence the occurrence of relapse after transplantation. However, owing to the limitations of this study, these factors were not included in our analysis.

Additionally, regarding the incidence of CMV and EBV infections, no acute viral reactivation or new infections were observed in patients from either group. The simultaneous initiation of sampling for this study with the COVID-19 pandemic and the use of masks may be one reason for patients' lack of viral infections such as CMV and EBV.

This investigation offers novel insights into zinc-mediated immune reconstitution; however, several inherent constraints associated with complex transplant research exist. The age restriction (40–60 years), although biologically justified by thymic decline and the epidemiology of multiple myeloma (MM), limits the generalizability of the findings to other age groups. While our CD31-based gating method (CD4 + CD45RA + CD31 +) effectively excludes most Temra cells, any minimal contamination from CD31 + Temra subsets could be addressed in future studies through the use of additional markers such as CCR7, CD27, and CD38. Key challenges included (1) a limited amount of cytogenetic data (available for only 16% of participants), which constrains the assessment of relapse risk; (2) a modest sample size (n = 38) that diminishes the power for subgroup analyses (with relapse power estimated at 42%); (3) the technical inability to conduct TREC analysis; and (4) pandemic-related confounders impacting the monitoring of infections. Although all patients completed the protocol-defined 3-month follow-up, the short duration limited the ability to assess long-term survival, and operational hurdles (including patient attrition in high-risk autologous hematopoietic stem cell transplant) underscore the importance of multicenter validation with extended follow-up and comprehensive molecular profiling. Despite these limitations, the study maintained rigorous ethical standards and covariate adjustment, ensuring its integrity throughout the 3-year investigation.

Conclusion

This clinical study evaluated the impact of Zn supplementation on improving IR in patients after autoHSCT. According to the results of this study, no severe Zn deficiency was observed in patients; however, since even mild Zn deficiency can affect the immune system, the consumption of oral Zn supplements may contribute to enhancing IR, particularly cellular immunity. Daily Zn supplementation increases ALCs; thus, Zn may accelerate the reconstitution of cellular immunity in patients undergoing autoHSCT. Additionally, Zn intake has been associated with increased production of RTEs from the thymus, effectively improving thymic function. Therefore, Zn directly impacts the number of RTEs and accelerates their production, ultimately reducing the risk of infections, especially viral infections, and relapses in these patients.

While our findings show promise for Zn supplementation as a feasible approach to enhance thymic recovery post-HCT, further validation in larger trials is warranted.

Although these preliminary results are promising, they need validation through studies that include comprehensive baseline molecular characterization.

However, this trial has several limitations, including slow patient recruitment and the need for long-term follow-up. Furthermore, the sampling process may be time-consuming due to the use of multiple eligibility criteria, such as a single-center study, participants' self-reported dietary intake, and a need for more cooperation from some participants in completing the intervention, which may necessitate replacing them with other patients.

Evaluating the functional recovery of T cells may provide insights into the positive effects of Zn supplementation, sparking new avenues of research. A follow-up period extending beyond six months and up to five years is recommended to obtain more accurate data regarding the impact of Zn on recurrence and infection rates. Furthermore, it would be beneficial to measure and analyze other immune cell populations, particularly B lymphocytes, which oral Zn may also influence. Finally, assessing the effects of oral Zn supplementation in patients undergoing allogeneic transplantation could be valuable, as previous studies suggest that it may help prevent complications related to graft-versus-host disease, opening new possibilities for patient care.

Abbreviations

SBMU

Shahid Beheshti University of Medical Sciences

HSCT

Hematopoietic Stem Cell Transplantation

GVHD

Graft Versus Host Disease

MM

Multiple Myeloma

PBMC

Peripheral Blood Mononuclear Cell

ALC

Absolute Lymphocyte Count

RTEs

Recent Thymic Emigrant T cells

sjTRECs

Signal joint T-cell receptor excision circles

CMV

Cytomegalovirus

EBV

Epstein–Barr virus

NRM

Nonrelapse mortality

EBMT

European Society for Blood and Marrow Transplantation protocols

NF-kb

Nuclear Factor Kappa B

Authors’ contributions

M.N. Conceptualization, study design, and writing – original draft. A.Z.H. Lead author, review, and editing. S.P. Clinical data management, patient monitoring, review, and editing. D.K. Data curation, statistical analysis, review, and editing. K.J. Data collection, laboratory test, review, and editing. Y.S. Laboratory supervision, review, and editing. A.H. Oversight of clinical aspects and critical revision of the manuscript, review, and editing. M.S. Patient selection, clinical supervision, review, and editing. H.Z. Nutrition clinical data management, review, and editing.

Funding

This research was funded by the Hematopoietic Stem Cell Research Center (HSCRC), an independent institution that operates independently from governmental influence.

Data availability

The datasets generated and analyzed during the current study are not publicly available owing to patient confidentiality and institutional restrictions but are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Tarbiat Modares University (IR.MODARES.REC.1398.195) and Shahid Beheshti University of Medical Sciences (IR.SBUM.REC.1399.010). The trial was registered under IRCT20191211045701N1.

All procedures involving human participants followed the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Written informed consent was obtained from all participants before enrollment.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.