Mesenchymal Stromal Cells Mediate Clinically Unpromising but Favourable Immune Responses in Kidney Transplant Patients

Kaundal, Urvashi; Ramachandran, Raja; Arora, Amit; Kenwar, Deepesh B.; Sharma, Ratti Ram; Nada, Ritambhra; Minz, Mukut; Jha, Vivekanand; Rakha, Aruna

doi:https://doi.org/10.1155/2022/2154544

Stem Cells International

On this page

Abstract Introduction Methods Results Discussion Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 2154544 | https://doi.org/10.1155/2022/2154544

Mesenchymal Stromal Cells Mediate Clinically Unpromising but Favourable Immune Responses in Kidney Transplant Patients

Urvashi Kaundal,^1,2Raja Ramachandran,³Amit Arora,⁴Deepesh B. Kenwar,⁵Ratti Ram Sharma,⁶Ritambhra Nada,⁷Mukut Minz,⁸Vivekanand Jha,⁹and Aruna Rakha¹

Academic Editor: Katia Mareschi

Received01 Sept 2021

Accepted11 Jan 2022

Published15 Feb 2022

Abstract

Background. Allograft rejection postkidney transplantation (KTx) is a major clinical challenge despite increased access to a healthcare system and improvement in immunosuppressive (IS) drugs. In recent years, mesenchymal stromal cells (MSCs) have aroused considerable interest in field of transplantation due to their immunomodulatory and regenerative properties. This study was aimed at investigating safety, feasibility, and immunological effects of autologous MSCs (auto-MSCs) and allogeneic MSCs (allo-MSCs) as a complement to IS drug therapy in KTx patients. Methods. 10 patients undergoing KTx with a living-related donor were analysed along with 5 patients in the control group. Patients were given auto-MSCs or allo-MSCs at two time points, i.e., one day before transplant (D-0) and 30 days after transplant (D-30) at the rate of 1.0- MSCs per kg body weight in addition to immunosuppressants. Patients were followed up for 2 years, and 29 immunologically relevant lymphocyte subsets and 8 cytokines and important biomarkers were analysed at all time points. Results. Patients displayed no signs of discomfort or dose-related toxicities in response to MSC infusion. Flow cytometric analysis revealed an increase in B regulatory lymphocyte populations and nonconventional T regulatory cells and a decrease in T effector lymphocyte proportions in auto-MSC-infused patients. No such favourable immune responses were observed in all MSC-infused patients. Conclusion. This study provides evidence that auto-MSCs are safe and well tolerated. This is the first ever report to compare autologous and allogeneic MSC infusion in KTx patients. Importantly, our data demonstrated that MSC-induced immune responses in patients did not completely correlate with clinical outcomes. Our findings add to the current perspective of using MSCs in KTx and explore possibilities through which donor/recipient chimerism can be achieved to induce immune tolerance in KTx patients.

1. Introduction

Kidney transplantation (KTx) coupled with immunosuppressive (IS) drugs is the preferred treatment for end-stage renal disease (ESRD) [1] over the dialysis process, which is usually performed in case of nonavailability of a suitable donor or underlying medical conditions. Despite medical advances, long-term graft survival post-KTx continues to be a major challenge [2] further jeopardized by prolonged usage of IS drugs. Therefore, it would be of immense benefit to seek novel therapies that can replace/taper down the usage of immunosuppressants. The principal goal of IS therapy is immune cell inhibition [3]; however, exploiting these therapies against specific lymphocytes is difficult due to the existence of overlapping pathways used by effector and regulatory lymphocytes [4]. Therefore, it is essential to understand the effect of various cell-based therapies on lymphocyte compartments, as immunoregulatory mechanisms mediate the majority of posttransplant effects. In this regard, mesenchymal stromal cells (MSCs) have been shown to hold an immense potential to be considered an alternative or adjunct treatment for many diseased conditions for their potential of immunomodulation and regeneration through paracrine and direct effects, respectively. MSCs are well documented to affect T cells [5], but their effect has not been fully extrapolated on the T cell subset interplay. Recent studies have also highlighted the capacity of MSCs to modulate B cells [6, 7]. It would, therefore, be interesting to explore if MSCs reshape the immune balance in KTx patients and their effect on the graft outcome.

Our previously published pilot study in 4 KTx patients showed the safety and efficacy of auto-MSCs in combination with IS drug therapy postinduction [8]. The current study was designed to evaluate the effect of 2-time point MSC infusion on T and B cells in autologous and allogeneic (donor-derived) settings without any ATG induction therapy. And thereafter, the effect is analysed in the clinical setting.

2. Methods

2.1. Study Objectives, Design, Safety, and Efficacy Monitoring

Recruited patients were histopathologically confirmed for ESRD. Patients who received ATG induction therapy or were suffering from any infections were excluded. Inclusion and exclusion criteria are detailed in Supplementary Table (ST 1). All protocols designed were approved by the Institutional Committee for Stem Cell Research of PGIMER (PGI-IC-SCRT-39-2013/1471), Chandigarh, and no changes were made following approval. Protocols were performed according to relevant regulations and guidelines specified in the approval letter, and informed consent was obtained from the patients.

For this open-label, parallel-group prospective study (Supplementary Figure (SF 1), a total of 30 patients, with planned KTx from a living-related donor, were assessed and 17 patients meeting the inclusion criteria were divided into 3 groups (SF 1), i.e., autologous (auto) group (; median age 24 (23, 27)), allogeneic (allo) group (; median age 31 (20.5, 37)), and control group (; median age 23.5 (25, 35)). The allocation ratio for the assignment was 1 : 1 : 1. Patients were enrolled from June 2013 till March 2015 and were followed up for 2 years. The primary and secondary objectives and endpoints have been summarized in ST 2. The patient demographics and clinical profile are described in ST 3, and the cell dosage is described in ST 4. One patient from each auto and allo group did not follow up, leaving for each group. The study design involving different time points is graphically described in Figure 1. Lymphocyte population sets, cytokines, and biomarkers characterized are tabulated in Table 1. A statistical summary of all clinical and immunological parameters measured periodically is reported in ST 5 and Table 2, respectively.

Figure 1

Treatment scheme for KTx patients recruited for the study. MSCs were isolated from bone marrow aspirate and expanded for 60 days (D-60) before transplantation. All KTx patients received immunosuppressive drugs (tacrolimus (TAC), mycophenolate mofetil (MMF), and prednisolone) 48 hours before transplantation (D-1). Allo and auto group patients received the 1^st intravenous (I.V.) MSC infusion 24 hours before transplantation (D-0) and the 2^nd I.V. MSC infusion 30 days posttransplantation (D-30). Blood samples were collected at D-0, D-30, D-90, D-180, D-365, and D-800 for determining clinical and immunological parameters. Samples were routinely processed for serum creatinine estimation.

2.2. Immunosuppressive Drug Treatment and Supportive Care

All patients received treatment with tacrolimus (TAC), mycophenolate mofetil (MMF), and prednisolone (Figure 1). TAC was started 48 hours before transplant surgery and adjusted to maintain a trough level of 10-12 ng/mL for the first month posttransplant and then between 8 and 10 ng/mL for the next 1-3 months. MMF (Cellcept®, Roche) at a dose of 1 g, twice a day, was given. Steroids at a dose of 0.5 mg/kg were given initially and tapered to 5 mg/day by the 6^th week posttransplant. Additionally, cotrimoxazole (400 mg sulfamethoxazole+160 mg trimethoprim) daily for 6 months was given. Whole blood TAC trough level (C0) was monitored till the target level was attained. Patients were monitored for changes in clinical condition or serum creatinine (Scr) levels.

The estimated glomerular filtration rate (eGFR) was determined using the Nankivell equation [9]. Biopsies were performed for allograft dysfunction or proteinuria.

2.3. Cell Preparation and Characterization

MSCs were prepared from bone marrow (BM) aspirate of patients (auto group) or the respective kidney donors (allo group) approximately 6-8 weeks (Figure 1) before transplantation in the Department of Translational and Regenerative Medicine, PGIMER, Chandigarh, as described previously [8, 10]. Briefly, 40 mL of bone marrow sample was subjected to density gradient centrifugation, and mononuclear cells were collected and resuspended in complete media (α-minimal essential media+10% pooled human platelet lysate). Cells were inoculated in T-225 flasks at a density of cells/cm² and kept in an incubator with 5% CO₂ at 37°C. MSCs were trypsinized at 80% confluency, expanded in hyperflasks till passage 2, and cryopreserved in liquid nitrogen till the time of infusion. Cryopreserved MSCs were revived and expanded in complete MEM containing 10% pHPL 7 days before infusion. On the day of infusion (D-0 or D-30), cells were trypsinized, and their count was determined using trypan blue (>95% viability) before infusion.

MSCs were also characterized phenotypically and functionally in accordance with International Society for Cell Therapy (ISCT) guidelines [11]. When observed under a light microscope, MSCs had typical spindle-shaped morphology and adhered to the surface (SF 2A). For phenotypic characterization, MSCs were stained with fluorochrome-labelled antibodies and were analysed using flow cytometry. Culture-expanded MSCs at passage 3 showed ≤2% immunoreactivity for haematopoietic lineage markers CD34, CD45, CD11b, CD19, and HLA-DR and ≥95% positivity for human-MSC specific markers CD73, CD90, and CD105 (SF 2B). Unstained MSCs were used as a negative control for analysis.

Functional characterization of MSCs was done at passage 4 based on their differentiation into adipocytes, osteocytes, and chondrocytes (SF 2C). For adipogenic differentiation, cells were plated in a 6-well plate at a density of cells/cm² and maintained in an adipogenic medium comprising α-MEM, isomethylbutylxanthine, insulin, dexamethasone, and indomethacin. Similarly, for osteogenic differentiation, cells were plated at a density of cells/cm² and with α-MEM supplemented with dexamethasone, ascorbic acid, and glycerophosphate. To evaluate chondrogenic potential, a chondrocyte differentiating kit (HiMedia) was used per the manufacturer’s protocol.

The culture medium was changed every 3 to 4 days. On the 21st day, staining was performed using Oil Red O to estimate the neutral lipid accumulation in fat vacuoles of differentiated adipocytes. Likewise, the staining for differentiated osteocytes was performed using Alizarin Red S, which detects the alkaline phosphatase activity, and chondrogenic differentiation was demonstrated by staining with Alcian Blue, which detects the expression of aggrecans in chondrocytes.

Karyotyping was also performed for the culture-expanded MSCs (passage 3) to confirm chromosomal stability (SF 2D). By actively dividing cells from 70%, confluent culture flasks were treated with KaryoMAX® (Gibco) to inhibit the proliferation of cells at the metaphase stage. After the mitotic arrest, the cells were harvested using trypsin/EDTA and immersed in KCl solution at 37°C for hypotonic treatment. The treated cells were centrifuged, followed by fixation using Carnoy’s fixative. Cells were resuspended in a fresh fixative solution at room temperature for slide preparation. The cell suspension was dropped on the slide and kept on a hot plate for 2-3 min at 38-40°C. Once dried, the slides were kept at room temperature overnight and afterwards were immersed in cold trypsin solution, and staining was performed using Giemsa. The trypsin and Giemsa bands (GTG) were analysed microscopically (100x). Metaphases were captured through a CCD camera and analysed using the GenASIs Bandview software (Applied Spectral Imaging). A minimum of 20 banded metaphases was captured for all samples.

MSC culture medium was used to detect bacterial and fungal contaminants or the incidence of mycoplasma pathogen (SF 2E). The BACTEC blood culture system was used to rule out aerobic and anaerobic bacterial contaminations, and agar plates were used to detect fungal contaminations. For mycoplasma testing, nested PCR using a mycoplasma detection set (TaKaRa) was performed. Cells were infused once the sterility was confirmed.

2.4. Cell Administration

Auto and allo group patients received two intravenous MSC infusions at D-0 and D-30, and for each dose, approximately 1- cells/kg body weight were given (ST 4). Patients were premedicated with paracetamol and chlorpheniramine as a precautionary measure to prevent any reactions postinfusion. The patient’s vitals were monitored for 4-6 hours postinfusion.

2.5. Clinical Evaluation

Routine clinical parameters (ST 5) were measured at days (D) 0, 30, 90, 180, 365, and 800, along with serum creatinine and eGFR.

2.6. Immunological Evaluation

Immunophenotyping was performed on isolated peripheral blood mononuclear cells, and cytokine assays were performed on serum samples collected at days (D) 0, 30, 90, 180, 365, and 800. Lymphocyte subpopulations were analysed using fluorochrome-labelled monoclonal antibodies on a FACSAria flow cytometer (ST 6). Th1/Th2/Th17 cytokines and TGF-β1 were quantified using commercially available kits (ST 6). Gating strategies for phenotyping are provided in Supplementary Figures SF 3 and 4.

2.7. Statistical Analysis

Analysis was undertaken by using in-house R scripts [12]. Wherever applicable, values were first adjusted to the respective parent population. Adjusted values were further normalized (min-max) before statistical analysis. Linear mixed models using the R package lme4, followed by ANOVA, were used to access significantly contributing factors. The R method Wilcox test - Wilcox.test(x, y, paired = FALSE) was used to carry out the Wilcoxon rank sum test in order to test the null hypothesis that the distributions of two variables under investigation differ by a location shift of . Plots of the resultant values were created using the R method boxplot().

3. Results

3.1. MSCs are Well Tolerated with No Clinical Impact on Graft Survival

Patients displayed no signs of discomfort, allergies, or infections during or post-MSC infusion. In the auto group, 40% patients had rejection episodes immediately after transplantation (Pa5 and Pa6), and in the allo group as well, 40% patients had rejection episodes (P3—TCMR at 3.5 months, P6—immediate rejection posttransplantation) (ST 3), but afterwards stable graft function was achieved for all. On the contrary, no rejection episodes were observed in the control group (ST 3). All the routine clinical parameters analysed showed no significant changes over the period of follow-up and were in the normal range (ST 5). However, levels of Scr and eGFR are normalized within all groups posttransplantation (Figure 2).

(a)

(b)

3.2. MSCs Alter the Frequency of T and B Lymphocytes

Flow cytometric analysis pointed that the auto group had a reduction in CD4 T cells at the end of follow-up while CD8 T cells remained unaffected (Figures 3(a) and 3(b)). We further compared metabolically inactive T_NAI cells to identify the impact of MSC infusion on the cell differentiation process.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 3

Lymphocyte subsets in peripheral blood of kidney transplant patients. Multicolour FACS analysis for the normalized proportion of (a) CD4 T cells and (b) CD8 T cells, (c) CD4 T_NAI:CD4 T_EFF cells, (d) CD8 T_NAI:CD8 T_EFF cells, (e) CD4 T_NAI:CD4 T_MEM cells, (f) CD8 T_NAI:CD8 T_MEM cells, (g) T_REGS, and (h) DN T cells at D-0, D-30, D-90, D-180, D-365, and D-800 for different groups (auto (), control (), allo (), and healthy control ()). Box plots depict median of respective lymphocyte subsets. Significant differences are indicated as value < 0.05.

Analysis of T_NAI against T_EFF cells revealed a higher proportion of T_NAI cells within the auto group at D-800 for both CD4 (Figure 3(c)) and CD8 T cells (Figure 3(d)). Additionally, analysis of T_NAI:T_MEM cells showed elevation at D-800 within the auto group for both CD4 (Figure 3(e)) and CD8 (Figure 3(f)) T cell subsets. This points towards expanded T_NAI cells as compared to T_EFF and T_MEM cells in the auto group. This trend was more pronounced for the effector memory (T_MEM-EM) subset than for the central memory (T_MEM-CM) subset (Table 2). We found a drop in T_REGS in the auto and control groups and a decreasing trend in the allo group (Figure 3(g)). An increase in double-negative (DN) T cells at multiple time points was observed within the auto group, which turned out to be significantly higher at D-800 than that within the control group or healthy controls (Figure 3(h)). No significant differences in other T cell subsets were evident in either the allo or control group (Table 2).

In our previous study, CD19 B cells decreased in auto-MSC-treated patients posttransplantation [6]. To evaluate the specific impact of this change, we further characterized CD19 B cell subsets using bm-bm5 and CD27/IgD classification. No relevant difference was evident in either of the subsets within the auto or control group. Intriguingly, within the allo group, bm2, bm2, and bm3+4 cells displayed a decrease at D-800 (Table 2). These results indicate differences in long-term effects of auto- and allo-MSCs on the B cell profile of KTx patients.

Since B_REGS contribute to transplant tolerance, we studied immature B (B_IM) cells along with other two B_REG subsets, with phenotypes similar to classical B_reg and B₁₀ populations, i.e., CD19⁺CD5⁺CD1d^hi (B_regs) and CD19⁺CD27⁺CD24^hi (B₁₀ cells). Comparative analysis of B₁₀ and B_IM subsets against CD4/CD8 T_EFF populations indicated a significant increase within the auto group at varied time points (Figures 4(a)–4(d)).

(a)

(b)

(c)

(d)

Figure 4

Comparison of regulatory B and effector T cell subset distribution in peripheral blood of kidney transplant patients. Multicolour FACS analysis for the normalized proportion of (a) B₁₀:CD4 T_EFF cells, (b) B_IM: CD4 T_EFF cells, (c) B₁₀:CD8 T_EFF cells, and (d) B_IM:CD8 T_EFF cells at D-0, D-30, D-90, D-180, D-365, and D-800 for different groups (auto (), control (), allo (), and healthy control ()). Box plots depict the median of the respective cell subset ratios. Significant differences are indicated as value < 0.1 and value < 0.05.

On the contrary, in the allo group, B_IM:T_EFF cells reduced within the group and variedly decreased when compared to the HC (Table 2). No significant changes were observed in B cell populations within the control group.

3.3. MSCs Modulate Cytokine Levels

A significant increase in TGF-β1 levels was evident within the auto group until D-800 (Table 2), which overlapped with a decrease in CD4 T_EFF cells and an increase in DN T and B_REG subsets. The allo group showed an intermittent increase in TGF-β1 post-D-30 (Table 2). IL-2 MFI in the auto group at D-365 was lower than that in the allo group (Table 2). None of the other cytokines had any significant changes in the auto or allo group (Table 2). No significant differences in cytokine levels were observed in the control group.

4. Discussion

Immunosuppressants are given to KTx recipients to hamper immune cell-mediated rejection, thereby promoting successful engraftment of the donor kidney. Despite improvements in IS drug management, KTx patients not only suffer from life-threatening complications but are also predisposed to opportunistic infections. There is an utmost need to develop an approach that will aid donor-specific immune-hyporesponsiveness, thereby reducing the patient’s dependence on immunosuppressants. The immune milieu is ever changing, and graft acceptance in transplant settings is determined by how well the immune system adapts to challenges that an engraft imposes. Nevertheless, simultaneous assessment of the cellular and humoral arm of the immune system is paramount in the transplant setting for predicting graft quality.

MSCs have been major contenders for their potential towards therapeutic, regenerative, and immunomodulatory activities. This study evaluates safety and efficacy of auto-MSCs and allo-MSCs in patients who underwent KTx. The first infusion was given at D-0 to establish a protolerogenic microenvironment that might promote graft acceptance and avoid acute deterioration of graft function, and the second infusion at D-30 was given to combat inflammatory environment postsurgery and to prolong protolerogenic effect mediated by MSCs.

Our study signifies that MSC infusion is feasible with favourable immune response in renal transplant patients, but there is no short-term clinical benefit of such an intervention in a normal risk renal transplant. We show that auto-MSC infusion upregulates naive T (T_NAI) subsets, and B regulatory (B_REGS) and double-negative (DN) T cells may contribute to a decrease in circulating effector T (T_EFF) cells.

It is known that donor-specific tolerance is considered Holy Grail for transplant immunology, and studies suggest that T_MEM cells can directly stimulate T_EFF cells and prove to be deleterious to the graft [13]. There has been increased incidence of rejections related to increased circulating memory T cells [14]. Therefore, low T_EFF/T_MEM cell proportions relative to T_NAI cells post-auto-MSC infusion that were observed in our study could be of potential therapeutic value.

T_REGS have been reported to maintain donor-specific nonresponsiveness in KTx patients [15]; however, we found a drop in T_REGS in all groups irrespective of MSC infusion, which challenges the present view of MSC-induced T_REGS expansion. However, the number of T_REGS might not even correlate with the functional ability of MSCs to suppress T cell functions [16]. Downregulation of T_REGS can be attributed to the use of calcineurin inhibitors as a part of IS therapy [17], which is known to block IL-2 production, required for T_REGS expansion. Lesser-known nonconventional T_REGS subsets such as double-negative T (DN T) cells are also known to have immunosuppressive properties [18]. DN T cells lack FoxP3 expression and therefore are resistant to calcium release-activated calcium channel inhibition [19] which supports the increase in these cells in our study. Also, studies so far have suggested the importance of B_REGS in preclinical transplant models and patients [20, 21]. B_REGS have a direct impact by inhibiting effector T cells in addition to their role in antibody production. Their profiling may help identify patients with immunotolerance thereby minimizing immunosuppressive regimens. The increasing trend of B_REGS in relation to effector T cells in our setup indicates well-guarded B cell tolerance checkpoints post-auto-MSC infusion; however, the functional status of these cells has not been determined in our study.

On the contrary, allo-MSC infusion led to no significant change in T cell subsets but decreased regulatory B cell subsets. Although various studies advocate the use of allo-MSCs (Table 3), our data suggest that prior to considering the application of MSCs of allo origin in kidney transplant patients, further studies are needed to analyse their effects on the immune cell phenotype and function.

We identified TGF-β as the primary immunomodulatory cytokine in our study. Increase in this anti-inflammatory cytokine might indicate a shift from Th1 to Th2 response in auto group patients.

MSCs have been major contenders for their potential towards immunomodulatory properties [22, 23].

Numerous studies have reported the safety and efficacy of MSCs (Table 3); however, there are differences in the source, dosage, route of MSC administration, time points, IS regimen, and follow-up periods. Moreover, differences in efficacy endpoints of these studies make it further challenging to infer the therapeutic efficacy of MSCs.

The novelty of our study lies in the comparison of the immune profile of two groups administered with 2 time point doses of autologous and allogeneic MSCs, and the major findings point towards a controlled immune environment (Figure 5) for the graft, especially in the auto group with lesser impact on clinical parameters used for determining the graft survivability. While few of the studies, as pointed in Table 3, have pointed towards basic immune repertoire, some have pointed towards clinical safety and feasibility of auto- or allo-MSCs. Our study is unique in comparative analysis of 29 T and B cell subsets with cytokine profiling in two groups with an uncertain impact on clinical outcome, emphasizing conducting more regulated trials utilizing MSCs in solid organ transplantation.

Our study is limited by small sample size and lack of functional assessment data. However, our findings would contribute substantially toward understanding the long-term immunomodulatory effects of MSCs, considering the inadequacy of available MSC efficacy data. Although we believe that favourable immune response is taking the front seat post-auto-MSC infusion, clinical relevance can only be stated upon in large sample size and more follow-up years.

The primary outcome (ST 2) of the study is that the infusion of auto-MSCs is safe and well tolerated in KTx patients. As far as the graft outcome is concerned, all KTx patients showed stable graft function eventually after rejection episodes in few patients. Variations in immunological responses were evident, regardless of the same origin, isolation, expansion conditions, and dosage of MSCs. The exact reason behind these differences remains unclear; however, these could have been elicited by donor-dependent variability or host microenvironment. As a secondary outcome (ST 2), the results collectively stress upon a unique trend of change in lymphocyte subsets that will help us to conduct more targeted clinical trials to improve long-term graft survival eventually. MSCs of autologous origin may be the safer choice in terms of avoiding unwanted immune responses while MSCs of allogenic origin might elicit specific cellular and humoral immune responses against donor antigens.

In spite of a seemingly favourable immune profile, the clinical ineffectiveness is evident in this study. Therefore, our findings add to the current perspective of using MSCs in KTx and explore possibilities through which donor/recipient chimerism can be achieved to induce immune tolerance in KTx patients.

Data Availability

Supporting data were available as supplementary figures.

Disclosure

The current address of Urvashi Kaundal is NIH, Bethesda, USA.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Raja Ramachandran and Amit Arora contributed equally to this work.

Acknowledgments

We are very thankful to Prof. Remuzzi and Dr. Casiraghi from Mario Negri Institute, Bergamo, Italy, for facilitating short-term training for AR on immunological techniques relating to the project. The study was registered on ClinicalTrials.gov under NCT02409940.

Supplementary Materials

Supplementary Tables (ST 1 to ST 7). ST 1: patient eligibility criteria. ST 2: primary and secondary study objectives and endpoints. ST 3: patient demographics and clinical profile of renal transplant patients. ST 4: cell characteristics postmanufacturing. ST 5: statistical summary of the clinical parameters in study groups. ST 6: resource table. ST 7: HLA typing of the renal recipient and donor. Supplementary Figures (SF 1 to SF 4). SF 1: CONSORT flow diagram: trial reporting for screening, enrollment, allocation, follow-up, and analysis for MSC infusion in autologous (auto) and allogeneic (allo) groups along with the control group. SF 2: MSC characterization of bone marrow-derived mesenchymal stromal cells. (A) Representative light microscopy picture of spindle-shaped adherent MSCs (P-2) (magnification: 20x). (B) MSC gating according to the FSC and SSC profile. Flow cytometric analysis indicated that BM-MSCs are negative for CD34, CD45, CD11b, CD19, and HLA-DR (negative cocktail) and positive for MSC surface markers CD73, CD90, and CD105. Dark grey-coloured plots represent specific antibody staining, and light grey plots represent negative control. (C) Representative images depicting in vitro differentiation assays revealing formation of lipid droplets stained with Oil Red O (20x, formation of chondrocytes stained with Alcian Blue (40x), formation of osteocytes stained with Alizarin Red S (20X). (D) Representative normal complete karyogram -46, XX, of culture-expanded MSCs at passage 3. Karyotypic analysis was done for all samples of expanded MSCs that were used for infusions. (E) Sterility testing was performed for all samples used for infusion. Anaerobic bacterial, aerobic bacterial, mycoplasma, and fungal contamination was ruled out before the infusion. SF 3: representative figures depicting the gating strategy for the identification of human T cell subsets. (A) Lymphocyte gating according to the FSC and SSC profile. Lymphocytes were then gated to determine the proportion of (B) CD3⁺ cells, (C) CD4⁺ T cells, CD8⁺ T cells, and CD4^-CD8^- T (double-negative (DN) T) cells. For detecting effector and memory cell subsets, first (D) CD4⁺CD45RO⁺CD45RA^- and CD4⁺CD45RO^-CD45RA⁺ cells and (E) CD8⁺CD45RO⁺CD45RA^- and CD8⁺CD45RO^-CD45RA⁺ cells were gated, and then, on the basis of CD62L staining, the cells were identified as (F) CD4 central memory (CD4 T_MEM-CM) and CD4 effector memory cells (CD4 T_MEM-EM), (G) CD4 naive (CD4 T_NAI) and CD4 effector (CD4 T_EFF) cells, (H) CD8 central memory (CD8 T_MEM-CM) and CD8 effector memory cells (CD8 T_MEM-EM), and (I) CD8 naive (CD8 T_NAI) and CD8 effector cells (CD8 T_EFF). (J) Lymphocyte gating to determine the proportion of (K) CD3⁺CD4⁺ T cells. Further from CD3⁺CD4⁺ T cells, (L) CD25^hi cells were gated for the (M) T_REGS (FoxP3⁺ CD127^lo) population. FCS-A: forward scatter area; SSC-A: side scatter area. SF 4: representative figures depicting the gating strategy for the identification of human B cell subsets. (A) Lymphocyte gating according to the FSC and SSC profile. Lymphocytes were then gated to determine the proportion of (B) CD19⁺ B cells, which on the basis of CD38 and IgD double staining were further classified as (C) virgin naive (bm1) IgD⁺CD38⁻, activated naive (bm2) IgD⁺CD38⁺, pregerminal (bm2) IgD⁺CD38^hi, germinal center (bm3⁺ bm4) IgD⁻CD38^hi, early memory (ebm5) IgD⁻CD38⁺, and late memory (bm5) IgD⁻CD38⁻ cells. On the basis of IgD-CD27 double staining, CD19⁺ B cells were also identified as (D) switched B cells—IgD⁻CD27⁺; unswitched B cells—IgD⁺CD27⁺; naive (B_NAI)—IgD⁺CD27⁻; and double negative (DN B) IgD⁻CD27⁻ cells. Regulatory B cell subsets were determined from the CD19⁺ B cells by gating (E) CD5⁺ cells and (F) CD1d^hi cells for the B_REG subset, (G) CD27⁺ cells and (H) CD24^hi for B₁₀ cells, and (I) CD24^hi cells and (J) CD38^hi cells for immature B (B_IM) cells. FCS-A: forward scatter area; SSC-A: side scatter area. (Supplementary Materials)

References

M. Abecassis, “Kidney transplantation as primary therapy for end-stage renal disease: a National Kidney Foundation/Kidney Disease Outcomes Quality Initiative (NKF/KDOQITM) conference,” Clinical Journal of the American Society of Nephrology, vol. 3, no. 2, pp. 471–480, 2008.
View at: Publisher Site | Google Scholar
J. M. Neuberger, “Practical recommendations for long-term management of modifiable risks in kidney and liver transplant Recipients,” Group. Transplantation, vol. 101, no. 4S, pp. S1–S56, 2017.
View at: Publisher Site | Google Scholar
C. Hartono, T. Muthukumar, and M. Suthanthiran, “Immunosuppressive drug therapy,” Cold Spring Harbor Perspectives in Medicine, vol. 3, no. 9, article a015487, 2013.
View at: Publisher Site | Google Scholar
B. M. Hall, “T cells: soldiers and spies--the surveillance and control of effector T cells by regulatory T cells,” Clinical Journal of the American Society of Nephrology, vol. 10, no. 11, pp. 2050–2064, 2015.
View at: Publisher Site | Google Scholar
M. E. Castro-Manrreza and J. J. Montesinos, “Immunoregulation by mesenchymal stem cells: biological aspects and clinical applications,” Journal of Immunology Research, vol. 2015, Article ID 394917, 20 pages, 2015.
View at: Publisher Site | Google Scholar
U. Kaundal, “B cells: a new player in mesenchymal stromal cell-mediated immune modulation in renal transplant patients,” Kidney International, vol. 96, no. 1, pp. 249-250, 2019.
View at: Publisher Site | Google Scholar
F. Casiraghi, “Kidney transplant tolerance associated with remote autologous mesenchymal stromal cell administration,” Stem Cells Translational Medicine, vol. 9, no. 4, pp. 427–432, 2020.
View at: Publisher Site | Google Scholar
C. Mudrabettu, “Safety and efficacy of autologous mesenchymal stromal cells transplantation in patients undergoing living donor kidney transplantation: a pilot study,” Nephrology (Carlton), vol. 20, no. 1, pp. 25–33, 2015.
View at: Publisher Site | Google Scholar
B. J. Nankivell, “Predicting glomerular filtration rate after kidney transplantation,” Transplantation, vol. 59, no. 12, pp. 1683–1689, 1995.
View at: Publisher Site | Google Scholar
C. Capelli, “Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts,” Bone Marrow Transplantation, vol. 40, no. 8, pp. 785–791, 2007.
View at: Publisher Site | Google Scholar
M. Dominici, “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement,” Cytotherapy, vol. 8, no. 4, pp. 315–317, 2006.
View at: Publisher Site | Google Scholar
Team, R C, R: a language and environment for statistical computing, 2013.
G. Benichou, B. Gonzalez, J. Marino, K. Ayasoufi, and A. Valujskikh, “Role of memory T cells in allograft rejection and tolerance,” Frontiers in Immunology, vol. 8, 2017.
View at: Publisher Site | Google Scholar
D. S. Segundo, G. Fernández-Fresnedo, M. Gago et al., “Changes in the number of circulating TCM and TEM subsets in renal transplantation: relationship with acute rejection and induction therapy,” Kidney international supplements, vol. 1, no. 2, pp. 31–35, 2011.
View at: Publisher Site | Google Scholar
T. K. Hendrikx, “Generation of donor-specific regulatory T-cell function in kidney transplant patients,” Transplantation, vol. 87, no. 3, pp. 376–383, 2009.
View at: Publisher Site | Google Scholar
M. Krampera, “Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide,” Blood, vol. 101, 2003.
View at: Publisher Site | Google Scholar
D. S. Segundo, “Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients,” Transplantation, vol. 82, no. 4, pp. 550–557, 2006.
View at: Publisher Site | Google Scholar
Z. McIver, B. Serio, A. Dunbar et al., “Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation,” British Journal of Haematology, vol. 141, no. 2, pp. 170–178, 2008.
View at: Publisher Site | Google Scholar
S. Jin, J. Chin, C. Kitson et al., “Natural regulatory T cells are resistant to calcium release-activated calcium (CRAC/ORAI) channel inhibition,” International Immunology, vol. 25, no. 9, pp. 497–506, 2013.
View at: Publisher Site | Google Scholar
C. M. Wortel and S. Heidt, “Regulatory B cells: phenotype, function and role in transplantation,” Transplant Immunology, vol. 41, pp. 1–9, 2017.
View at: Publisher Site | Google Scholar
Y. Peng, “Donor-derived mesenchymal stem cells combined with low-dose tacrolimus prevent acute rejection after renal transplantation: a clinical pilot study,” Transplantation, vol. 95, no. 1, pp. 161–168, 2013.
View at: Publisher Site | Google Scholar
U. Kaundal, U. Bagai, and A. Rakha, “Immunomodulatory plasticity of mesenchymal stem cells: a potential key to successful solid organ transplantation,” Journal of Translational Medicine, vol. 16, no. 1, p. 31, 2018.
View at: Publisher Site | Google Scholar
M. E. J. Reinders, C. van Kooten, T. J. Rabelink, and J. W. de Fijter, “Mesenchymal stromal cell therapy for solid organ transplantation,” Transplantation, vol. 102, no. 1, pp. 35–43, 2018.
View at: Publisher Site | Google Scholar
N. Perico, F. Casiraghi, M. Introna et al., “Autologous mesenchymal stromal cells and kidney transplantation: a pilot study of safety and clinical feasibility,” Clinical Journal of the American Society of Nephrology, vol. 6, no. 2, pp. 412–422, 2011.
View at: Publisher Site | Google Scholar
J. Tan, W. Wu, X. Xu et al., “Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial,” JAMA, vol. 307, no. 11, pp. 1169–1177, 2012.
View at: Publisher Site | Google Scholar
M. E. J. Reinders, “Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study,” Stem Cells Translational Medicine, vol. 2, no. 2, pp. 107–111, 2013.
View at: Publisher Site | Google Scholar
N. Perico, F. Casiraghi, E. Gotti et al., “Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation,” Transplant International, vol. 26, no. 9, pp. 867–878, 2013.
View at: Publisher Site | Google Scholar
A. V. Vanikar, H. L. Trivedi, A. Kumar et al., “Co-infusion of donor adipose tissue-derived mesenchymal and hematopoietic stem cells helps safe minimization of immunosuppression in renal transplantation - single center experience,” Renal Failure, vol. 36, no. 9, pp. 1376–1384, 2014.
View at: Publisher Site | Google Scholar
G.-h. Pan, Z. Chen, L. Xu et al., “Low-dose tacrolimus combined with donor-derived mesenchymal stem cells after renal transplantation: a prospective, non-randomized study,” Oncotarget, vol. 7, no. 11, pp. 12089–12101, 2016.
View at: Publisher Site | Google Scholar
N. Perico, “Long-term clinical and immunological profile of kidney transplant patients given mesenchymal stromal cell immunotherapy,” Frontiers in Immunology, vol. 9, pp. 1359–1359, 2018.
View at: Publisher Site | Google Scholar
Q. Sun, Z. Huang, F. Han et al., “Allogeneic mesenchymal stem cells as induction therapy are safe and feasible in renal allografts: pilot results of a multicenter randomized controlled trial,” Journal of Translational Medicine, vol. 16, no. 1, p. 52, 2018.
View at: Publisher Site | Google Scholar
P. Erpicum, “Infusion of third-party mesenchymal stromal cells after kidney transplantation: a phase I-II, open-label, clinical study,” Kidney International, vol. 95, no. 3, pp. 693–707, 2019.
View at: Publisher Site | Google Scholar
G. J. Dreyer, “Human leukocyte antigen selected allogeneic mesenchymal stromal cell therapy in renal transplantation: the Neptune study, a phase I single-center study,” American Journal of Transplantation, vol. 20, no. 10, pp. 2905–2915, 2020.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2022 Urvashi Kaundal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies