Under steady-state conditions, dendritic cells (DCs) take up commensal microbes and apoptotic cells to maintain immunologic tolerance. Conversely, when DCs are exposed to an inflammatory environment, this exposure triggers DC activation, transforming them into specialized cells that process, present, and activate pathogen-derived antigen-specific T cells [1]. This process strengthens the immune response and facilitates the induction of antibody-producing plasma cells [2].

Surface molecule expression and single-cell RNA sequencing (scRNA-seq) have characterized various DC subsets in human blood and other tissues [3]. Nevertheless, blood-circulating DCs are sparse, exhibiting a semi-mature state or migrating toward their final location and differentiation [4]. Therefore, isolating them from peripheral blood may yield insufficient numbers, rendering them ineffective as stimulatory antigen-presenting cells (APCs) for in vitro assays. Fortunately, the bloodstream contains monocytes that can differentiate into DCs [3]. In vivo, monocytes serve as a continuous reserve of precursors for tissue-resident macrophages and monocyte-derived DCs (moDCs) [5]. Since the method for obtaining in vitro-produced moDC was described in 1994 by Sallusto and Lanzavecchia [6], numerous variations of this method have emerged, demonstrating successful moDC production for both research and clinical applications.

As efficient APCs, in vitro-produced moDCs have been evaluated in preclinical strategies where the immune system requires antigen-specific stimulation [e.g., tumor-specific antigens (TSA) in cancer] [7] or inactivation (e.g., alloantigens before transplantation) [8]. Given their accessibility and efficiency as stimulatory cells, moDCs have been utilized in mixed leukocyte reaction (MLR) assays, which represent an effective in vitro strategy for quantifying T cell allo- and antigen-specific responses, as well as the effects of immunomodulatory agents [9–15].

The success and efficiency of an MLR in evaluating the potential T cell response against specific alloantigens depend on selecting the appropriate APCs. Blood is the most readily available source of human mononuclear cells, including monocytes and B cells [16]. However, while these cells can effectively present alloantigens, their numbers vary among individuals and may not represent the most suitable APCs involved in graft rejection [17, 18]. DCs are specialized APCs known to be the most efficient cells at inducing T cell alloresponse; however, their numbers in peripheral blood are limited [4]. This section analyzes the characteristics of moDCs that render them the most suitable and efficient APC for MLR.

Before a transplant, a patient must undergo several tests to maximize the safety of the procedure. These evaluations include blood group and HLA typing, as well as the search for unexpected allospecific antibodies. Currently, increasing graft compatibility primarily relies on HLA matching performed through molecular methods [69]. Nevertheless, despite those theory-matching tests, the existence of donor alloantigen-specific T cells in the receptor signifies an enormous challenge in transplantation. The above is because given the T cell restriction to recognize just self-MHC molecules—even in the absence of previous events of allogenic stimulation (transfusion, transplants, or pregnancies)—each person possesses a high frequency of alloreactive naïve and, even worse, memory helper (CD4+) and cytotoxic (CD8+) T cells with the potential to become activated and instigate graft rejection [17, 70, 71]. Since the MLR serves as an in vitro assay to analyze effective T-cell responses primed for alloantigen recognition [72–74]. Therefore, in addition to the theory matching tests mentioned, MLR may be conducted to assess the functional histocompatibility of the donor-recipient pair.

Several biosafety and preclinical trials have demonstrated the feasibility of moDCs for evaluating and quantifying the alloresponse of preexisting memory T cells prior to a transplant, along with the impact of inhibitory factors, such as suppressive drugs, on T cell responses (ClinicalTrials.gov ID: NCT03726307, NCT06243289, NCT00495755, NCT00213707).

Applying the MLR to evaluate the alloresponse involves assessing the likelihood of graft rejection by coculturing recipient-derived T cells, purified from peripheral blood, as responders stimulated with donor-derived moDCs [11]. On the other hand, the procedure may involve coculturing peripheral blood-purified donor-derived T cells primed with recipient-derived moDCs to analyze the possibility of graft-versus-host disease (GvHD) [75–78] (Figure 1).

MLR can be performed using peripheral blood mononuclear cells (PBMC) purified by differential centrifugation with Ficoll from the whole blood of the donor and recipient [79]. However, under these conditions, APC function is performed by monocytes and B cells, whose numbers may vary among patients. These cells do not represent the most suitable APCs involved in graft rejection and for MLR with effective results. A high density of costimulatory and MHC class II molecules is crucial to triggering an efficient MLR. Although monocytes and B cells possess these molecules, their expression levels are low, which provides them with weak allostimulatory capabilities [16].

Depending on the specific transplanted tissue, responder cells for an MLR may be obtained from different sources, albeit with certain limitations. For example, conducting a one-way MLR with bone marrow-derived responder cells indicates that, within this tissue, most cells are not T cells, and many others demonstrate persistent basal proliferation [80]. On the other hand, purifying lymphocytes from the thymus, lymph nodes, or spleen as responder cells is appropriate for MLR in animal models [81, 82]. However, it is not feasible in human assays, and stimulator cells must be obtained from PBMC or induced in vitro [10–12, 83].

The MLR can be two-way when both T cell populations, from the donor and recipient, proliferate [84] or one-way, when only one of those lymphocyte populations can respond [85]. Although the first provides a general view of the donor-recipient’s histocompatibility, it does not allow for the evaluation of each response. Therefore, the latter is preferable because it enables the evaluation of the likelihood of graft rejection by quantifying the proliferation of only recipient-derived T cells as responders or the possibility of GvHD by analyzing donor-derived T cells. This is achieved by abolishing the proliferative ability of stimulator cells through irradiation [86] or treating them with mitomycin C [85].

Some recipients of stem cells or kidney allografts—because of their inherent condition—are often immunocompromised, and their peripheral blood T cells may be affected. Therefore, in this case, to evaluate whether these cells have the potential to proliferate, the inclusion of HLA-DR mismatched controls should be analyzed. Then, the response in the MLR between donor and recipient is compared with the reaction toward the health controls [83].

MLR application in immunosuppressive drug testing involves using moDCs in preclinical studies to evaluate the in vitro effect of immunosuppressive drugs on alloreactivity, such as calcineurin and mTOR inhibitors, as well as co-stimulatory blockade agents on both DC stimulatory abilities and T cell activation, proliferation, and cytokine profiles [51, 87–91]. On the other hand, in addition to methods such as flow cytometry for analyzing T cell activation, monitoring donor-specific antibodies, and using specific radiotracers to visualize T cell trafficking, among others [92], moDC can be used in immune monitoring in clinical settings to track specific T cell responses to alloantigens during the post-transplant phase. This procedure assesses the frequency and intensity of T-cell alloresponse, providing insights into the risk of rejection or tolerance [93].

moDCs are widely used to assess the immune responses triggered by cancer immunotherapies, such as allogeneic tumor cell-based vaccines or adoptive tumor-specific T-cell transfer. In these clinical interventions, moDCs are utilized in vivo to present TSA to T cells and subsequently in vitro through an MLR to monitor the resulting immune activation toward TSA, allo, and autoantigens [9, 10, 94–100].

The MLR can evaluate the strength of tumor-specific T-cell responses. This is accomplished by mixing T cells from the patient with moDCs previously loaded with TSA or tumor-derived lysates. The resulting cellular events—proliferation, activation, and cytokine profiling—provide information about tumor immunogenicity and the immune system’s ability to recognize and trigger a tumor-specific immune response or even the risk of autoimmunity [100].

Adoptive cell therapy (ACT) involves the infusion of autologous, tumor-specific T-cells that have been expanded ex vivo or genetically engineered to target cancer cells. In this immunotherapy, MLR is used to measure whether these transferred cells recognize, proliferate, and kill tumor cells [101]. However, in allogeneic ACT, MLR may also predict the risk of unwanted immune alloresponse, such as donor T cell rejection by the recipient’s immune system or GvHD mediated by donor T cells that damage recipient cells. This could compromise the therapy’s success, which depends on the compatibility and persistence of donor T cells that must recognize and destroy host tumor cells [102].

Additionally, including moDCs loaded with different TSAs in various MLR assays allows for the identification of the most immunogenic TSA that elicits strong T-cell responses as potential candidates for developing targeted immunotherapies, such as personalized cancer vaccines or ACT [98–100]. Likewise, the likely alterations of T-cell activation mediated by the tumor microenvironment (TME) can be evaluated by adding tumor-derived factors (e.g., soluble components or immune cells from the tumor) to the MLR, providing insights into how immunotherapies could counteract these effects [98, 100]. Finally, the MLR can be developed for personalized immunotherapy approaches tailored to the specific immune reactivity of the tumor, utilizing autologous moDCs pulsed with their tumor-derived antigens [94].

ADs are characterized by the pathological activation of preexisting low-affinity T cells that recognize self-antigens. Depending on their maturation state, DCs have been involved as both instigators and protectors of AD [103]. Therefore, as the best APC, moDCs-based MLR provides a suitable tool to investigate the presence and contributions of autoreactive T cells for the diagnosis, treatment, and monitoring of these diseases (e.g., rheumatoid arthritis, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, etc.).

Self-antigen-loaded moDCs (e.g., myelin in multiple sclerosis, GAD65 in type 1 diabetes, β2GPI in antiphospholipid syndrome, or joint proteins in rheumatoid arthritis) are utilized in MLR to quantify the presence of autoreactive T cells and analyze their activation state and cytokine profile (e.g., IFN-γ, TNF-α, IL-6, IL-17) in autoimmune patients, as well as assess the functionality of Tregs in maintaining immune tolerance [59, 103–105].

moDCs-based MLR enables the distinction of each specific T cell polarization contribution (e.g., Th1, Th17, or Treg cells) to autoimmune pathology. MLR can also be employed to examine the Th1/Th17 dominance. In many ADs, IL-17 (Th17 cells) or IFN-γ (Th1 cells) are critical cytokines driving chronic inflammation and tissue damage [106].

MLR can also be applied to analyze the role of individual pro- and anti-inflammatory cytokines, environmental factors (e.g., toxins), pathogen-derived antigens, immunosuppressive drugs (e.g., corticosteroids, methotrexate), biologic agents (e.g., TNF inhibitors), drugs targeting specific immune checkpoints (such as CTLA-4 or PD-1), tolerance-inducing vaccines, or peptide-based therapies in the beginning, progression, and counteraction of the autoimmune process [107] and simultaneously investigate the signaling pathways activated in T cells, such as the NF-kB, JAK/STAT, and MAPK pathways, which are frequently dysregulated in autoimmune conditions [108]. Therefore, since the immune response can differ among AD patients, the MLR can be used to predict how a specific AD patient might respond to a particular treatment in order to create tailored therapeutic strategies for that individual [109, 110].

The MLR applications in vaccine development encompass evaluating antigen immunogenicity, biosafety, and personalized vaccine design. This allows for the simultaneous assessment of T cells and cytokine profiles to optimize vaccine formulations [94].

moDC-based MLR can be used to analyze T cell activation and proliferation, as well as polarization [CD4+ helper T cells (Th1, Th2, Th17), CD8+ cytotoxic T cells, Tregs], and cytokine secretion (e.g., IFN-γ, IL-2, TNF-α) against specific candidate antigens for a vaccine. Given this, the immune balance will determine whether this antigen may induce an immune response with cell memory and antibody production, while avoiding excessive immune tolerance or even autoimmunity [111].

Likewise, in combination with vaccine antigens, MLR may be applied to optimize vaccine formulations, test different adjuvants, evaluate various vaccine delivery systems such as liposomes, nanoparticles, or viral vectors, assess cross-reactivity (i.e., whether T cells might respond to similar antigens), identify the most immunodominant epitopes within a vaccine antigen, and measure the recall response of memory T cells to subsequent doses [112–114].

Since the MLR involves human moDC-T cell interactions, it represents a better humanized preclinical model than animal models. This assay may provide more accurate predictions of how a vaccine would perform in humans. The MLR can be adapted to assess immune responses under different genetic backgrounds or in individuals with immunocompromised conditions (e.g., elderly subjects or patients with cancer), chronic diseases (e.g., diabetes, obesity), co-infections (HIV), or even specific T cell responses in children. These variations ensure that vaccine formulations can be adjusted for different immune system profiles, enhancing safety and efficacy across all populations [114–116].

A better approach to assessing a preexisting T-cell response should consider that several DC subsets, with specific functions and tissue distribution near the inflammatory process, may contribute differentially to antigen presentation [18]. However, due to their accessibility and efficiency, only in vitro-produced moDCs have been extensively used as the best APCs (stimulatory cells) in MLR assays [9–12, 83].

The most common source for obtaining monocytes is peripheral blood or leukapheresis products—centrifugation gradients (Ficoll) separate PBMC. Monocytes are purified by magnetic cell sorting (MACS), which achieves high purity rates above 95%. Monocytes are differentiated in vitro by culturing them with GM-CSF and IL-4 for 4 to 7 days, generating immature moDCs (imoDCs). Then, maturation is induced by adding stimuli such as LPS, TNF, Poly I: C, IL-6, and PGE₂, either alone or in different combinations, for 2 more days to obtain mature moDCs (mmoDCs). The status of the obtained cells is assessed by measuring the expression balance of differentiation/maturation and inhibitory markers such as CD1a, CD83, CD86, CD40, Tim-3, PD-L1, and HLA-DR using flow cytometry.

Responder cells can be represented by whole PBMC or partially purified naïve or CD3+ T cells. However, the alloresponse can be analyzed in greater detail by purifying CD4+, CD8+, and CD45RA-memory T cells using MACS.

The moDC: T cell cocultures are performed in a 1:10 ratio for 4 to 7 days. The balance between cell proliferation and death can be measured using simple methods such as cell counting and viability assessments through light microscopy and trypan blue staining in the Neubauer chamber, although these may yield indefinite and unquantifiable results [117]. Cell proliferation can be quantified by integrating radioactive tritium [³H]-labeled thymidine [118] into the MLR culture. Still, this method does not evaluate cell death or activation and requires a scintillation counter. Therefore, flow cytometry represents an advanced technology for cell analysis and data acquisition, enabling the simultaneous analysis and quantification of cell proliferation [e.g., CFSE-dilution method, 5-bromo-2’-deoxyuridine (BrdU), Ki-67 expression, WST-8/CCK8 tetrazolium assay] related to CD4- or CD8-specific responses, cytokine expression (e.g., IFN-γ, IL-2), cell death (e.g., annexin V, propidium iodide, 7AAD), activation (e.g., CD25, CD69), and the induction of Tregs (e.g., Foxp3, CTLA-4) [13, 59, 104] and coculture supernatants can be utilized to quantify cytokines and chemokines related to the alloresponse intensity and T cell migratory abilities, such as IFN-γ, IL-12, CCL2, CCL3, CXCL9, and CXCL10 (Figure 1 and Table 1).

In the MLR, T cells recognize alloantigens processed and presented on allogeneic MHC molecules by allogeneic APCs. This cell interaction (immunological synapse) induces that, among the entire T cell repertoire, only alloantigen-specific naïve and memory T cells become activated and proliferate. The highest measurable cellular proliferation occurs around 5–8 days [9, 10, 12, 83].

Given their feasibility for in vitro generation and high allostimulatory capability, moDCs are the most efficient stimulatory cells in a mixed lymphocyte reaction. These properties support their application in evaluating preexisting memory T-cell alloresponse to analyze the likelihood of transplant success and the effects of immunomodulatory factors, immune monitoring, and tolerogenic strategies. Furthermore, the use of moDC in MLRs has provided critical insights into alloimmunity processes relevant to cancer immunotherapy, ADs, and vaccine development, facilitating the optimization of immunotherapeutic interventions.