B- and plasma cell-targeting therapies for podocytopathies: state of the art and the New Gaslini protocol

ABSTRACT

Podocytopathies, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and idiopathic nephrotic syndrome (INS), are traditionally classified by histology and corticosteroid responsiveness, but this framework does not capture their biological heterogeneity or underlying mechanisms. Over the past decade, the paradigm of podocyte injury has shifted from a T cell-centric model to a broader immunological framework in which B cells, plasma cells, and humoral immunity play a central role. The identification of circulating autoantibodies, including anti-nephrin antibodies, together with evidence of extrafollicular B-cell activation, supports a model in which humoral immune factors drive podocyte injury. Anti-CD20 therapies have improved outcomes in steroid-dependent and frequently relapsing disease, but their efficacy remains limited in steroid-resistant forms and post-transplant recurrence. This likely reflects incomplete depletion of tissue-resident B cells and persistence of long-lived plasma cells, supporting the need for strategies targeting multiple levels of the humoral immune axis. Recent studies suggest that combined targeting of CD20⁺ B cells and CD38⁺ plasma cells with monoclonal antibodies such as obinutuzumab and daratumumab may achieve deeper and more sustained disease control, including in refractory and recurrent cases. However, variability in treatment approaches across centers underscores the need for standardization. In this review, we summarize evidence supporting the role of humoral immunity in podocytopathies and discuss the strengths and limitations of B cell-directed therapies. We then propose a mechanistically driven strategy, the New Gaslini protocol, integrating sequential CD20 and CD38 targeting to provide a unified framework for managing severe and relapsing disease.

DEFINING PODOCYTOPATHIES: A BIOLOGY FRAMEWORK

Podocytopathies, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and idiopathic nephrotic syndrome (INS) in children, represent a group of glomerular diseases characterized by primary podocyte injury, leading to nephrotic-range proteinuria (>3.5 g/24 h), hypoalbuminemia, edema, and hyperlipidemia. Histologically, they share key features such as podocyte foot process effacement and, in more advanced or progressive cases, podocyte loss and segmental glomerulosclerosis, as observed in FSGS [1–5].

Despite increasing insight into disease mechanisms, classification remains largely anchored in histopathology and clinical response. In children, kidney biopsy is often deferred [6], and disease is commonly categorized according to corticosteroid responsiveness. Although practical, this approach does not reflect the underlying biological heterogeneity of these conditions [7, 8].

Current guidelines, including KDIGO [9, 10], continue to treat MCD, FSGS, and INS as distinct entities. Yet, from a clinical perspective, their disease course often overlaps. Most children initially present with steroid-sensitive (SS) nephrotic syndrome, but many later develop steroid dependence or frequent relapses, whereas ∼10%–20% are steroid resistant, a phenotype associated with a substantially higher risk of chronic kidney disease and post-transplant recurrence [11, 12].

Accordingly, corticosteroids remain the standard first-line therapy for most patients, apart from those with genetically determined disease. However, this strategy is not mechanism-based and contributes to heterogeneous treatment approaches and variable outcomes. Progress in the field has also been hampered by the rarity of these disorders, the lack of validated surrogate endpoints, and the difficulty of conducting adequately powered clinical trials, particularly for combination therapies [13].

From a mechanistic perspective, the paradigm of podocyte injury has substantially evolved over the past decade. These podocytopathies were historically considered T cell-mediated disorders [14]; however, this concept has been challenged by clinical and experimental evidence supporting a central role for B cells and humoral immunity [15–17]. The observation that B cell-depleting therapies, particularly anti-CD20 monoclonal antibodies such as rituximab, can induce sustained remission in patients with MCD and FSGS has prompted a re-evaluation of disease pathogenesis [18]. Accumulating data now indicate that B cells, plasma cells, and circulating autoantibodies contribute directly to podocyte injury. In particular, anti-nephrin autoantibodies have been detected in a large proportion of patients with MCD during active disease, where they induce nephrin phosphorylation, cytoskeletal remodeling, and slit diaphragm disruption, ultimately leading to proteinuria [19–21]. In parallel, single-cell transcriptomic studies have identified expansion of extrafollicular B-cell populations and short-lived antibody-secreting cells during disease flares, supporting a dynamic humoral immune activation [22, 23].

Complement activation further amplifies this process. Proteomic studies indicate greater complement activity in FSGS compared with MCD, suggesting a role as a secondary driver of injury [24]. In addition, natural IgM antibodies may bind glomerular epitopes and trigger classical pathway activation [25, 26], with subsequent amplification through the alternative pathway, as supported by increased complement activation products in serum and urine during active disease [27].

Although podocyte injury occurs across multiple glomerular diseases, this review focuses on primary podocytopathies presenting as MCD or FSGS and clinically manifesting as INS.

The concepts discussed in this review primarily apply to presumed immune-mediated podocytopathies. Accordingly, careful diagnostic evaluation, including exclusion of secondary causes and monogenic forms whenever clinically indicated, should precede consideration of intensive immunomodulatory strategies, as these conditions are unlikely to share the same pathogenic mechanisms or therapeutic response.

While the mechanisms driving individual podocytopathies may differ, recurrent disease activity and post-transplant recurrence are common clinical features, supporting the involvement of circulating pathogenic factors and shared humoral immune pathways.

Importantly, although the clinical efficacy of B-cell- and plasma cell-targeting therapies strongly supports a relevant role for adaptive immunity in at least a subset of podocytopathies, it should not be interpreted as evidence of a single B-cell-driven pathogenic mechanism. Rather, these therapies are likely to exert their effects through multiplepathways, including, among others, modulation of humoral immunity and increased regulatory T-cell function and, in the case of rituximab, direct protective effects on podocytes. The relative contribution of these mechanisms probably varies across disease subtypes and individual patients.

Here, we propose a mechanism-oriented therapeutic strategy based on combined targeting of B cells and plasma cells using anti-CD20 and anti-CD38 therapies. While this approach has shown promising results across multiple centers, current evidence remains limited to small cohorts, retrospective studies and case series, and prospective validation is still lacking. Accordingly, this proposal aims to provide a pragmatic, hypothesis-generating clinical framework to harmonize therapeutic approaches across centers, and inform the design of future prospective studies. We therefore provide a detailed protocol to facilitate harmonization and reproducibility.

This protocol was originally developed at the Gaslini Institute in Genoa, Italy and subsequently refined through collaboration with Mount Sinai in New York, NY, USA. We refer to it here as the New Gaslini Protocol, reflecting both its origins and its evolution through international collaboration.

THE HUMORAL IMMUNE ARCHITECTURE OF PODOCYTOPATHIES

From a T cell-centric paradigm to the recognition of a key role for humoral immunity

Podocytopathies have long been interpreted within a T cell-centric framework, largely based on their sensitivity to immunosuppressive therapies targeting T-cell pathways. This view began to shift with the serendipitous observation that B cell-depleting therapies can induce sustained remission, including in patients with frequently relapsing or steroid-dependent disease [28, 29]. Together, these findings have supported a broader immunological model in which humoral immunity represents an important component of disease pathogenesis, interacting with T-cell responses and other immune pathways rather than acting in isolation.

Direct evidence for this concept has come from the identification of circulating autoantibodies targeting podocyte antigens. Among these, anti-nephrin antibodies currently represent the best-characterized target [21]. Nephrin is a key structural and signaling component of the slit diaphragm, and antibody binding induces its phosphorylation, internalization, and downstream cytoskeletal remodeling, ultimately leading to foot process effacement and proteinuria [30]. Anti-nephrin antibodies have been detected in more than 70% of patients with active MCD, but appear less frequent in FSGS, with reported prevalences of 10%–20% [31]. They are more commonly observed in SS disease and are markedly less frequent in steroid-resistant (SR) forms. Their significance in FSGS, particularly in the post-transplant setting [32, 33], remains incompletely defined, and methodological limitations [34], together with the lack of standardized assays [35], continue to raise uncertainty as to whether they are directly pathogenic or instead identify a specific subtype of podocytopathy. Despite these observations, several important limitations currently restrict the clinical applicability of anti-nephrin antibodies. Available studies remain relatively small, methodologies differ substantially among laboratories, and no standardized assay has yet been adopted for routine clinical use [35, 36]. Consequently, the sensitivity, specificity, and reproducibility of anti-nephrin antibody detection remain incompletely defined. Although these antibodies represent promising biomarkers and may provide important insights into disease mechanisms, further prospective validation will be required before they can be incorporated into routine diagnostic algorithms or used to guide therapeutic decisions [23].

Other podocyte and slit diaphragm-associated targets, including Kirrel1 and podocin, have also been proposed [20], although their pathogenic relevance remains uncertain. In particular, it is difficult to explain how intracellular proteins such as podocin could act as primary antigens unless prior podocyte injury exposes normally sequestered epitopes. This would support a “second-hit” model rather than a primary autoimmune trigger. The coexistence of multiple autoantibody specificities also raises the possibility of epitope spreading, a phenomenon well described in other autoimmune diseases in which the humoral response broadens over time [37].

At the cellular level, accumulating evidence supports activation of the B-cell compartment in podocytopathies. Transcriptomic and immunophenotypic studies have shown expansion of circulating B cells with an activated phenotype during disease flares, particularly in MCD [38], whereas data in FSGS remain more limited and mainly involve the naïve and memory compartments. These findings are consistent with impaired immune tolerance, allowing autoreactive B-cell clones to persist and expand. A particularly relevant feature emerging from recent studies is the prominence of extrafollicular B-cell activation [39], which has been associated with expansion of atypical B cells and plasmablast population during disease flares [22].

B-cell differentiation

The humoral immune response in podocytopathies involves distinct B-cell subsets at different stages of differentiation, each with specific functional roles and possible therapeutic implications. Beyond antibody production, B cells may also contribute to podocyte injury through cytokine-mediated cross-talk with T cells and podocytes, including IL-4 and IL-13 release and induction of CD80 expression [15]. Likewise, B-cell depletion may influence disease activity not only by reducing autoantibody production, but also through broader immunomodulatory effects, including altered antigen presentation, cytokine production, and cross-talk with T-cell subsets.

Naïve B cells originate in the bone marrow and undergo central and peripheral tolerance checkpoints designed to eliminate or silence autoreactive clones. Despite these mechanisms, a substantial proportion of circulating B cells retain some degree of autoreactivity. Under physiological conditions, these cells remain quiescent, but in the presence of appropriate immune signals they may become activated [40].

Upon antigen encounter, B cells can follow two main differentiation pathways. The germinal center pathway leads to the generation of class-switched memory B cells and long-lived plasma cells. These plasma cells, typically residing in the bone marrow, produce high-affinity antibodies over prolonged periods and are largely resistant to conventional immunosuppressive therapies, including glucocorticoids and anti-CD20 agents [41].

In parallel, extrafollicular pathways generate short-lived plasmablasts and memory B cells with less extensive affinity maturation. These responses are rapid and flexible, providing an immediate source of antibody production, but are also more dependent on ongoing immune stimulation. Cytokines such as BAFF and APRIL play a central role in sustaining these responses by promoting B-cell survival and differentiation [42].

In podocytopathies, both compartments appear to be involved. Expansion of class-switched memory B cells has been associated with disease relapse and with reconstitution following anti-CD20 therapy, suggesting that this compartment may harbor autoreactive clones capable of reinitiating disease. At the same time, extrafollicular responses, characterized by increased plasmablasts and atypical B cells, likely contribute to acute disease activity and antibody production [38].

Long-lived plasma cells represent a particularly relevant compartment from a therapeutic perspective. These cells reside in specialized survival niches, do not express CD20, and are therefore not targeted by conventional B-cell depletion strategies. Their persistence may explain incomplete responses to anti-CD20 therapies and ongoing antibody production despite apparent B-cell depletion [43].

This layered organization of the B-cell compartment provides a framework to understand differential responses to therapy and supports the rationale for targeting multiple levels of the humoral immune axis [44]. Supporting this concept, high-dimensional immune profiling in children with steroid-dependent nephrotic syndrome treated with anti-CD20 therapy showed that relapse was associated with the selective expansion of class-switched memory B-cell subsets, despite similar total B-cell counts between relapsing and non-relapsing patients. Notably, five distinct class-switched B-cell populations were significantly increased in relapsing individuals, whereas other immune compartments, including T-cell subsets, remained unchanged [38]. These findings indicate that qualitative features of B-cell reconstitution, rather than absolute cell counts, are critical determinants of disease recurrence and may represent relevant biomarkers to guide treatment strategies.

ANTI-CD20 THERAPY: DEPTH AND DURATION OF B CELL DEPLETION AND TREATMENT LIMITATIONS

Rituximab, a chimeric anti-CD20 monoclonal antibody, depletes naïve and memory B cells through complement- and cell-mediated cytotoxicity, while sparing long-lived plasma cells. The immunological effects of anti-CD20 therapy likely extend beyond B-cell depletion and include modulation of T-cell homeostasis, particularly regulatory T-cell function, as well as other immune pathways, although the relative contribution of these mechanisms remains incompletely understood. (PMID: 26147676). Initially introduced based on serendipitous clinical observations [29, 45], its efficacy in podocytopathies has been confirmed in randomized trials and cohort studies, particularly in steroid-dependent and frequently relapsing disease [28, 46–49]. Although its clinical efficacy has provided important insights into disease biology, therapeutic response alone should not be interpreted as proof of a purely B-cell-mediated pathogenesis, as rituximab also exerts broader immunomodulatory effects. In this setting, rituximab prolongs relapse-free survival, reduces steroid exposure, and achieves remission rates of ∼60%–80% at 12 months. Repeated courses and maintenance strategies may further extend disease control, especially in SS patients [50–55].

In contrast, efficacy in SR disease is limited [3, 56]. Early case series suggested potential benefit in selected patients; however, randomized trials failed to demonstrate superiority over standard therapy [57, 58]. In the best-characterized trial, the addition of rituximab to calcineurin inhibitors and steroids did not improve remission rates compared to conventional treatment alone [56]. Subsequent retrospective studies reported partial responses in a subset of patients, but overall remission rates were markedly lower than in steroid-dependent disease. These findings likely reflect the biological heterogeneity of SR forms rather than insufficient treatment intensity. This difference in response between SS and SR disease suggests that distinct pathogenic mechanisms may underlie these conditions, with a more prominent role for CD20-positive B cells in relapsing SS forms, and a more heterogeneous and less clearly B cell-dependent biology in SR disease [59].

The increasing use of anti-CD20 therapies, together with retrospective cohort analyses, has provided a better understanding of their limitations and of the B-cell compartments most effectively targeted [60].

The incomplete depth of B-cell depletion represents a major limitation of anti-CD20 therapy. Rituximab efficiently depletes circulating B cells but achieves less complete depletion within secondary lymphoid tissues. As a result, autoreactive memory B cells may persist within tissue niches and contribute to disease relapse after peripheral reconstitution [61]. The association between relapse and the re-emergence of class-switched memory B cells further supports this concept [38, 62]. In line, repopulation of memory B-cell subsets after anti-CD20 therapy has been associated with disease relapse, supporting their role as both biomarkers and potential drivers of disease activity [62].

In addition, long-lived plasma cells, which do not express CD20, remain unaffected and may sustain antibody production. Immunogenicity, related to its chimeric structure, may reduce efficacy over time [63, 64]. Safety concerns include hypogammaglobulinemia with repeated dosing, infection risk, and less commonly neutropenia and hepatitis B reactivation [65, 66].

The optimal dosing regimen of rituximab remains uncertain. Initial protocols were derived from hematologic practice and consisted of four weekly infusions of 375 mg/m². Subsequently, alternative strategies, including two-dose and single-dose regimens, have been adopted [67]. Available evidence does not clearly support higher cumulative dosing, and real-world data suggest that concomitant maintenance immunosuppression may have a greater impact on relapse prevention than dose escalation alone. Variability in dosing strategies across studies reflects the absence of standardized approaches.

The limitations of rituximab have prompted the development of alternative anti-CD20 strategies. Other anti-CD20 monoclonal antibodies have been investigated to overcome some of these limitations [59] [46]. As a result, its role in podocytopathies remains limited [68].

More recently, attention has shifted toward next-generation anti-CD20 agents. Obinutuzumab, a humanized type II anti-CD20 antibody, has distinct pharmacodynamic properties compared to rituximab. It induces enhanced antibody-dependent cellular cytotoxicity and direct B-cell death, with reduced reliance on complement activation [68]. These features result in deeper and more sustained B-cell depletion, including within lymphoid tissues, where rituximab activity is less pronounced. In addition, obinutuzumab appears less immunogenic, potentially reducing the risk of anti-drug antibody formation [69].

Early clinical experience supports its potential role in difficult-to-treat cases [70]. Clinical experience with obinutuzumab in podocytopathies is still limited but suggests efficacy in both rituximab-refractory and SR disease. Small series reported complete or partial remission in patients with early relapse after rituximab, as well as in difficult-to-treat cases [71–73]. Activity has also been described in steroid-dependent and frequently relapsing forms, with some reports indicating longer relapse-free survival compared to rituximab [74]. These findings support the concept that deeper and more sustained B-cell depletion may translate into improved clinical outcomes. However, evidence remains preliminary and requires further validation. Safety data remain limited, but the available evidence suggests a profile broadly comparable to other anti-CD20 agents, with infusion reactions and hypogammaglobulinemia as the most commonly reported adverse events. However, recent observations, including our experience, indicate that clinically relevant hematologic toxicity may occur, even in the absence of concomitant immunosuppressive therapy [71, 75, 76]. Severe neutropenia and persistent anemia have been reported following obinutuzumab administration in patients with glomerular diseases, raising concerns about its safety profile, particularly in pediatric and young adult populations. Long-term safety, especially with repeated administration, requires further evaluation.

ANTI-CD38 THERAPY

The limited efficacy of anti-CD20 therapies in complex or refractory podocytopathies has fueled increasing interest in strategies targeting long-lived plasma cells, which are not affected by CD20-directed agents and represent a persistent source of autoreactive immunoglobulins. These cells express high levels of CD38, providing a rationale for therapeutic targeting with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab.

Initial clinical evidence was provided by Dossier et al., who treated 14 children with steroid-dependent or frequently relapsing podocytopathies that had relapsed after rituximab therapy [77]. The combination of obinutuzumab and daratumumab resulted in prolonged remission compared with historical rituximab-based approaches, although the specific contribution of daratumumab could not be clearly isolated in this cohort. Subsequent studies extended this approach to more severe phenotypes. In a cohort of 23 patients with multidrug-resistant or multidrug-dependent disease, a single course of rituximab combined with daratumumab induced remission of proteinuria in the majority of resistant cases and allowed complete withdrawal of background immunosuppression in dependent forms. Clinical response was associated with depletion of CD19⁺ B cells, CD38⁺ plasma cells, and reduction of circulating IgM levels [78]. Treatment was generally well tolerated, with only mild infusion-related reactions reported.

These findings provide preliminary support for the hypothesis that combined targeting of B cells and plasma cells may achieve deeper immunological control than anti-CD20 therapy alone, particularly in patients with refractory disease. However, available evidence is derived from small observational studies and does not yet allow definitive conclusions regarding comparative efficacy. Available immunophenotyping studies further suggest that clinical response may be associated not only with depletion of total CD19⁺ B cells, but also with changes in specific immune subsets, including CD19⁺CD38⁺ B cells, plasma cells, and circulating immunoglobulin levels [79]. These observations highlight the potential value of longitudinal immune monitoring to better characterize treatment response, immune reconstitution, and mechanisms of relapse. However, standardized flow-cytometry panels and monitoring strategies have not yet been established and represent an important area for future investigation.

A similar therapeutic approach has been explored in post-transplant FSGS recurrence. Recurrence frequently occurs early after transplant and remains difficult to manage, with plasma exchange (PLEX) and rituximab representing the most commonly used interventions despite variable efficacy and significant treatment burden [80]. Therefore, we and others [81–83] started testing combination strategies targeting both CD20⁺ B cells and CD38⁺ plasma cells in patients unresponsive to conventional approaches. Small case series have reported that regimens including rituximab or obinutuzumab in combination with daratumumab can induce sustained remission and allow PLEX discontinuation. We described five patients with early post-transplant recurrence resistant to both PLEX and rituximab achieving complete remission following combined rituximab and daratumumab therapy, with a parallel reduction in circulating plasma cells [80]. Additional reports using obinutuzumab plus daratumumab have described rapid remission and sustained control of proteinuria, further supporting the role of plasma cell depletion in suppressing pathogenic circulating factors [81, 83].

SAFETY CONSIDERATIONS AND BENEFIT-RISK ASSESSMENT

While the rationale for combined CD20/CD38 targeting is biologically compelling, its potential benefits must be balanced against the risks associated with deeper and more prolonged immune depletion. This consideration is particularly relevant in pediatric populations, where treatment-related immune alterations may persist over long periods and where long-term safety data remain limited.

Mechanistically, the simultaneous eradication of CD20+ B cells and CD38+ plasma cells disrupts both immediate and long-lived humoral responses. Data from systemic reviews and clinical cohorts of anti-CD38 use in refractory autoimmune and glomerular diseases indicate that secondary hypogammaglobulinemia occurs in ∼33% of patients, frequently leading to impaired vaccine-induced antibody titers and a delayed recovery of polyclonal humoral immunity [84–87].

Although the safety profile of individual anti-CD20 and anti-CD38 agents has been described in several studies, less is known about the long-term consequences of their combined use.

Given the limited duration of follow-up in currently available studies, the long-term consequences of repeated combined B-cell and plasma-cell depletion remain incompletely defined. Although no clear signal of increased malignancy risk has emerged so far, available cohorts are too small and follow-up periods too short to draw definitive conclusions.

THE NEW GASLINI PROTOCOL: A COMBINED CD20/CD38 STRATEGY IN SEVERE PODOCYTOPATHIES

Building on the emerging use of combined B-cell and plasma cell targeting, we developed a stepwise, response-guided treatment algorithm for podocytopathies, based on clinical disease behavior rather than histological classification. The proposed algorithm is based on the currently available clinical and biological evidence; however, several components remain hypothesis-generating and have not yet been validated in prospective randomized studies. Therefore, the protocol should be viewed as a framework for therapeutic escalation in selected refractory patients rather than as a replacement for current KDIGO recommendations.

The New Gaslini protocol follows a stepwise approach starting from the distinction between SS and SR disease. At present, available evidence does not support the replacement of steroids with anti-CD20 therapy as initial treatment. Although recent reports have explored rituximab as a first-line therapy in children with new-onset INS and in adults, mostly with MCD, these data are limited to small, single-center observational studies. In these cohorts, rituximab induced remission in ∼60%–70% of patients [46]; however, the absence of randomized trials comparing rituximab versus standard steroid therapy, together with the well-established efficacy and rapid response of corticosteroids, does not support a change in first-line treatment strategies. Therefore, corticosteroids remain the reference initial therapy, while anti-CD20 agents are positioned as steroid-sparing or second-line options (Fig. 3). There are very well-defined protocols for steroid treatment and patients should follow them strictly before they can be defined as steroid resistant [9, 88, 89].

Figure 3:

The New Gaslini protocol: a response-guided treatment algorithm for native podocytopathies. Schematic representation of a stepwise, response-guided therapeutic algorithm for podocytopathies based on clinical disease behavior. Initial treatment follows KDIGO-based steroid therapy to define steroid sensitivity. Patients are subsequently stratified into SS and SR forms. In SS patients progressing to steroid-dependent or frequently relapsing disease, anti-CD20 therapy is introduced as a steroid-sparing strategy. In younger children, MMF may be considered prior to anti-CD20 therapy. After anti-CD20 antibody initiation, or MMF in younger than 10 yr, patients can undergo progressive steroid tapering and withdrawal. In SR disease, calcineurin inhibitors (CNI) are initiated as first-line treatment. In CNI-sensitive patients, anti-CD20 therapy is introduced after stable remission (3–6 months) to allow CNI withdrawal. In CNI-resistant patients, MMF should be added, followed by reassessment to define multidrug-dependent or multidrug-resistant disease. In both multidrug-dependent and multidrug-resistant settings, escalation to combined CD20/CD38 targeting should be considered. The protocol includes anti-CD20 therapy (rituximab or obinutuzumab) followed by anti-CD38 therapy (daratumumab), with progressive withdrawal of background immunosuppression. Treatment response is assessed after 2–4 weeks based on proteinuria. In the absence of complete remission, daratumumab may be repeated, up to a maximum of four infusions.

Figure 1:

Schematic representation of B-cell development in physiological conditions and its dysregulation in podocytopathies. In physiological conditions (upper panel), B cells originate in the bone marrow and progress through sequential maturation stages from precursors to immature B cells, followed by central tolerance checkpoints, including negative selection. Transitional B cells migrate to peripheral lymphoid organs, where BAFF-dependent signals support maturation into naïve B cells. Upon antigen (Ag) encounter, B cells differentiate through two main pathways: the germinal center reaction, which, with the help of follicular helper T cells, leads to affinity maturation and the generation of class-switched memory B cells and long-lived plasma cells; and extrafollicular responses, which are rapidly induced outside germinal centers and generate short-lived plasmablasts and plasma cells under the influence of innate immune signals, BAFF, and APRIL. In podocytopathies (lower panel), based on current pathogenic models, defects in immune tolerance, HLA-II–associated genetic susceptibility, and impaired immunoregulation favor the persistence and activation of autoreactive B and T cells. These cells may recognize podocyte antigens and activate both germinal center and extrafollicular pathways, leading to expansion of class-switched memory B cells and plasma cell subsets, with sustained autoantibody production. However, the relative contribution of germinal center versus extrafollicular activation remains incompletely defined and may vary across disease subtypes and age groups. Environmental agents, such as viral infections, may further amplify these processes through type I interferon signaling and increased BAFF/APRIL production by innate immune cells.

Figure 2:

Therapeutic targeting across the B-cell and plasma cell continuum in podocytopathies

Schematic representation of the main B-cell differentiation stages and corresponding therapeutic targets. Naïve B cells, activated B cells, and memory B cells express CD20 and are therefore susceptible to anti-CD20 monoclonal antibodies (rituximab, obinutuzumab). Upon antigen (Ag) stimulation, activated B cells differentiate through germinal center and extrafollicular pathways. Extrafollicular responses generate plasmablasts and short-lived plasma cells, whereas germinal center reactions give rise to class-switched memory B cells and long-lived plasma cells. Plasma cell survival and differentiation are supported by BAFF and APRIL signaling through receptors including BAFF-R, TACI, and BCMA. Anti-CD38 monoclonal antibodies (daratumumab) target plasmablasts and plasma cells, including long-lived antibody-secreting cells that are not affected by anti-CD20 therapies. BAFF/APRIL inhibitors and BCMA-targeted therapies further modulate B-cell maturation and plasma cell survival.

In SS patients who evolve into steroid-dependent or frequently relapsing forms, we propose anti-CD20 therapy as a steroid-sparing strategy. While rituximab has historically been the most widely used agent, emerging data and mechanistic considerations support a potential advantage of obinutuzumab, including deeper and more sustained B-cell depletion and lower immunogenicity. Although direct comparative randomized studies between rituximab and obinutuzumab are lacking, the biological properties of obinutuzumab, together with the currently available clinical experience and the positive results of a recent randomized trial comparing obinutuzumab with mycophenolate mofetil (MMF) [90], support its emerging role as a steroid-sparing therapy. At present, however, there is insufficient evidence to recommend one anti-CD20 agent over the other. When anti-CD20 therapy is indicated, a single infusion of either rituximab (375 mg/m², maximum 1 g) or obinutuzumab (375 mg/m², maximum 1 g) may be considered according to the clinical context, previous treatment history, and local availability. Future comparative studies should define whether the greater biological activity of obinutuzumab translates into superior long-term clinical outcomes while maintaining an acceptable safety profile. In younger children, typically those under 10 years of age, MMF may be considered instead as a primary steroid-sparing agent prior to biologic therapy, given the faster B-cell reconstitution observed in this age group, which may limit the duration of response to anti-CD20 treatment [62]. This approach is also consistent with the current KDIGO 2025 recommendations, which recognize MMF as an established steroid-sparing option in childhood steroid-dependent and frequently relapsing forms [88].

Subcutaneous administration of anti-CD20 therapy, particularly rituximab, has shown comparable efficacy and safety to intravenous infusion [91], with potential advantages in terms of cost and feasibility; however, data on subcutaneous obinutuzumab in nephrological settings are currently lacking and will be required to support its implementation.

In SR disease, we propose calcineurin inhibitors as first-line therapy, given their high rate of response. Patients are subsequently stratified according to response. In CNI-sensitive patients, treatment is maintained until stable remission is achieved. To prevent long-term CNI-related toxicity, we propose the introduction of anti-CD20 therapy after 3–6 months of stable remission, followed by gradual CNI tapering and eventual withdrawal over the subsequent months. Clinical evidence suggests that CNI-sensitive patients have a high likelihood of responding to anti-CD20 therapy and achieving sustained remission [92], with outcomes comparable to those observed in steroid-dependent or frequently relapsing disease, in contrast to CNI-resistant forms. However, the optimal timing and pace of CNI tapering remain to be established and should be individualized according to treatment response.

Therefore, in CNI-resistant cases, we propose add-on therapy with MMF.

This strategy is supported by emerging clinical experience suggesting that the combination of calcineurin inhibitors and MMF may improve outcomes in selected patients with resistant disease while maintaining an acceptable safety profile [93, 94].

After 3–6 months of combined therapy, patients should be re-evaluated. Those achieving complete remission are classified as multidrug dependent if ongoing therapy is required to maintain disease control, whereas those with partial or no response are classified as multidrug resistant.

In both multidrug-dependent and multidrug-resistant settings, we propose escalation to combined CD20/CD38 targeting. The New Gaslini protocol consists of a single infusion of anti-CD20 therapy, preferentially with a single infusion of obinutuzumab (375 mg/m², maximum 1 gr), if available, followed by a single infusion of daratumumab (16 mg/kg, maximum 1200 mg). Oral immunosuppressive therapy, including calcineurin inhibitors and MMF, is progressively withdrawn within a short time frame following combined treatment. After daratumumab infusion, we propose to assess treatment response based on proteinuria after ∼2–4 weeks. In the absence of complete remission, daratumumab may be repeated, up to a maximum of four infusions.

Recent immunophenotyping data further support this approach, showing that clinical response to combined CD20/CD38 targeting is associated with near-complete depletion of CD19⁺CD38⁺ B cells and a significant reduction in circulating IgM levels, whereas non-responders retain higher levels of these cells [79]. These findings suggest a potential pathogenic role of CD38⁺ B-cell subsets and IgM-mediated mechanisms, and indicate that monitoring these compartments may help guide response to therapy.

Based on our experience, we do not propose maintenance therapy once remission is achieved. Instead, patients should undergo close clinical follow-up, and retreatment with the same combined CD20/CD38 strategy is initiated only in the event of disease recurrence, which is currently defined by the onset of proteinuria. At present, available evidence does not support fixed-interval retreatment, and prospective studies will be required to determine whether biomarker-guided or scheduled maintenance strategies may further improve long-term outcomes.

Therefore, treatment decisions and monitoring remain primarily based on clinical parameters, particularly proteinuria. Although emerging biomarkers, including circulating anti-nephrin antibodies [31] and immune cell subsets [38], hold promise, their role in guiding treatment remains to be defined. For anti-nephrin antibodies, available evidence is largely retrospective and their utility appears limited to steroid sensitive forms [95]. In addition, significant methodological variability across assays, lack of standardized thresholds, and limited reproducibility currently prevent their use as reliable biomarkers in clinical practice [34, 35]. Standardization of detection methods and prospective validation in clinical trials will be required before these markers can be integrated into therapeutic algorithms.

Given the increased risk of treatment-related over-immunosuppression, appropriate infectious screening and monitoring are integral components of the proposed protocol. All patients should undergo hepatitis B virus screening before treatment with anti-CD20 monoclonal antibody monoclonal antibody, according to current recommendations. In patients receiving combined CD20/CD38 therapy, we recommend trimethoprim-sulfamethoxazole prophylaxis three times weekly for 6 months, together with serial monitoring of complete blood count and serum IgG levels at 1, 3, and 6 months to facilitate the early detection of leukopenia, neutropenia, and hypogammaglobulinemia. Immunoglobulin replacement should be considered when clinically appropriate, according to patient characteristics and local practice. Additional antiviral prophylaxis should be considered according to individual infectious risk and local guidelines.

This protocol aims to standardize therapeutic escalation across different clinical phenotypes, providing a unified framework for the use of combined B-cell and plasma cell targeting. Ongoing prospective evaluation will define its efficacy and safety across podocytopathy subgroups.

THE NEW GASLINI PROTOCOL IN RECURRENT PODOCYTOPATY

Podocytopathies frequently recur after kidney transplantation, often within hours to days, supporting the presence of pathogenic circulating factors. PLEX and anti-CD20 therapy remain the most widely used first-line treatments; however, their efficacy is inconsistent [96, 97], and a substantial proportion of patients either fail to respond or become dependent on repeated PLEX sessions, with significant treatment burden [98].

Recent clinical experience has shifted toward strategies targeting both B cells and plasma cells. Small case series indicate that combined CD20/CD38 targeting can induce rapid and sustained remission in refractory cases, including those unresponsive to rituximab and PLEX [83]. In our experience, this approach leads to complete remission in the majority of patients, further supporting a mechanistic role for the humoral immune axis in disease recurrence [80, 82].

Based on these observations, we propose an early, stepwise approach to post-transplant recurrence. At disease recurrence, defined by new-onset proteinuria and/or histological evidence, treatment should be initiated promptly with PLEX and anti-CD20 therapy, preferably with a single obinutuzumab infusion (375 mg/m², maximum 1 gr). In our clinical practice, PLEX is performed using 1.5 plasma-volume exchanges per session, with treatment frequency individualized according to disease severity and clinical response.

Given the limitations of targeting B cells alone, and the severity of post-transplant recurrence, early introduction of anti-CD38 therapy is recommended within a risk-benefit framework, irrespective of the initial response to anti-CD20 therapy. To optimize drug exposure, biologic therapy should be administered after completion of PLEX cycles, with subsequent PLEX withheld for at least 1 week.

Treatment response should be assessed within 2–4 weeks based on proteinuria. In responders, PLEX can be progressively tapered and discontinued. In patients with persistent or PLEX-dependent disease, escalation to CD38 targeting is recommended, with additional daratumumab infusions as needed (Fig. 4).

Figure 4:

The New Gaslini protocol in post-transplant podocytopathy recurrence. Schematic representation of a response-guided therapeutic approach for post-transplant recurrence of podocytopathies. Recurrence should be highly suspected in case of early onset of proteinuria and/or delayed graft function with no clear explanation. This should prompt a graft biopsy that can provide histological evidence of podocyte injury, including foot process effacement. Initial management consists of PLEX combined with anti-CD20 therapy (rituximab or obinutuzumab), administered after PLEX to optimize drug exposure. Anti-CD38 therapy (daratumumab) is introduced early, irrespective of the initial response to anti-CD20 treatment, to target residual pathogenic cells. To minimize drug clearance, subsequent PLEX sessions should be delayed following biologic infusion. Treatment response is based on proteinuria. In patients achieving complete or partial remission, PLEX is progressively tapered and discontinued. In non-responders, daratumumab is repeated (up to four infusions), while PLEX schedule may be continued and adapted according to clinical response.

CONCLUSION

Podocytopathies are increasingly recognized as disorders driven, at least in part, by dysregulation of the humoral immune axis, including antibodies and other B cell- and plasma cell-derived factors. This framework helps explain both responsiveness to B cell-depleting therapies and the persistence or recurrence of disease despite CD20 targeting alone.

Combined targeting of B cells and plasma cells may therefore enable deeper and more durable disease control, particularly in refractory and recurrent cases. However, current evidence remains largely hypothesis-generating and further prospective studies are needed before definitive conclusions can be drawn regarding its place in clinical practice. The New Gaslini protocol provides a structured, mechanism-based approach to therapeutic escalation, but requires prospective validation to define its efficacy, safety, and optimal application across patient subgroups.

AUTHORS' CONTRIBUTIONS

A.A. wrote the first draft, designed the New Gaslini Protocol, and refined it with P.C. All authors participated in the literature search, data analysis and interpretation, and were involved in critically revising the manuscript.

CONFLICT OF INTEREST STATEMENT

None declared.

FUNDING

A.A. was supported with public funds granted by the European Union—Next Generation EU—NRRP M6C2–Investment 2.1 Enhancement and strengthening of biomedical research in the NHS.

DATA AVAILABILITY STATEMENT

No new data were generated or analyzed in support of this research.