T-B Collaboration in Autoimmunity, Infection, and Transplantation

Abstract

We have attempted here to provide an up-to-date review of the collaboration between helper T cells and B cells in response to protein and glycoprotein antigens. This collaboration is essential as it not only protects from many pathogens but also contributes to a litany of autoimmune and immune-mediated diseases.

OVERVIEW

Sequential collaborative interactions between helper T cells and B cells are a central aspect of adaptive immune responses initiated by protein and glycoprotein antigens. Many functions of B cells are dependent on CD4⁺ T cells. Similarly, numerous T cell–mediated processes, both physiologic and pathologic, appear to depend on the ability of activated B cells to nurture and maintain CD4⁺ T cells. Consequently, approaches designed to prevent T-B collaboration are an important part of our therapeutic armamentarium, even for diseases that appear to be driven by CD4⁺ T cells. A conceptual understanding of T-B collaboration is essential to appreciate how cellular and humoral immunity is developed and maintained and how alterations within 1 cell population propagate throughout the system. Knowledge of the mechanisms of T-B collaboration informs the pathogenesis, prevention, and treatment of numerous immune-mediated human diseases, including pathogen responses, autoimmunity, and transplant rejection.

The immune system evolved in part to mount pathogen responses. Indeed, its importance to human physiology was first appreciated in this setting. Therefore, the initial description of the biology of T-B collaboration in this review will largely be considered from the viewpoint of pathogen responses. We will separately consider the relevance of T-B collaboration in autoimmunity and transplantation.

LINKED RECOGNITION, B-CELL ACTIVATION, AND THE INDUCTION OF T-B COLLABORATION

Antigens and antigen complexes contain 2 categories of physically linked antigenic determinants (epitopes). Protrusions on the surface of an antigen serve as B-cell epitopes that are recognized by the antigen receptor on 1 or more B-cell clones. B-cell antigen receptors directly bind native conformational epitopes on antigens that can be chemically diverse; these antigens include small molecules, nucleic acids, and lipids but are most commonly carbohydrates and proteins.¹ In contrast, T-cell antigen receptors on CD4⁺ T cells recognize peptide epitopes derived from protein and glycoprotein antigens when presented on major histocompatibility complex (MHC) class II molecules.² To generate MHC class II–restricted T-cell epitopes, antigens are processed into linear stretches of approximately 15 to 25 amino acids via proteolytic degradation in late endosomes or lysosomes. These linear peptides may be capable of binding tightly to the groove of a host MHC class II molecule (Figure 1). The exposed residues of a peptide bound to an MHC class II molecule may be recognized by a T-cell receptor (TCR) on ≥1 T-cell clones.

FIGURE 1.

Distinct epitopes on the same protein are recognized by B and T cells. A, Two conformational epitopes, a “green triangle,” and a “yellow square” represent B-cell epitopes in the protein and a linear “black peptide” represents the T-cell epitope in this protein. B, The BCR specific for a “yellow square” epitope on a B cell binds specifically to and endocytoses the protein antigen. C, In the late endosomal/lysosomal compartment, the endocytosed antigen is processed to peptides and the “black peptide” binds the MHC class II groove and is transported to the cell surface of the B cell. D, An activated CD4⁺ T cell specific for the black peptide (previously activated by DCs presenting the same black peptide on an identical MHC class II molecule) now recognizes the B cell that has processed and presented the black peptide on its MHC class II molecules. BCR, B-cell receptor; DC, dendritic cell; MHC, major histocompatibility complex.

Importantly, B cells process and present T-cell epitopes contained within the specific antigen recognized by the individual B-cell clone. Given the restricted use of peptide epitopes by T cells, T-B interactions necessarily involve cells that recognize epitopes derived from a shared protein, glycoprotein, or protein complex antigen.^3,4 Therefore, T-B collaboration requires the colocalization of a B-cell clone that recognizes a conformational epitope on the surface of an antigen and a T-cell clone that recognizes a different linear peptide derived from the same antigen and bound to an MHC class II molecule on the B-cell clone.

Cognate T-B interactions are required for most antibody isotype switching, antibody affinity maturation, plasma cell development, and memory B-cell formation.⁵ These processes, and the interactions that drive them, are spatially and temporally restricted. The initial antigenic stimulation of B cells leads to partial activation with limited proliferation and differentiation. Activated B cells endocytose, process, and present antigen. They downregulate C-X-C chemokine receptor type 5 (CXCR5), a chemokine receptor that promotes localization to B-cell follicles, and upregulate C-C chemokine receptor type 7 (CCR7), the ligand for which is enriched in the T-cell zone, allowing migration to the T-B boundary.⁵ Successful engagement with a CD4⁺ T cell that recognizes a shared epitope initiates a sequence of events leading to both short-term and long-lasting humoral immunity. A broad overview of T-B collaboration is provided in Figure 2.

FIGURE 2.

An overview of T-B collaboration. A DC that has captured, processed, and presented a specific peptide on a specific MHC class II protein migrates into the T-cell zone in a draining lymph node and selects and activates a specific CD4⁺ T cell. The selected T cells proliferate, alter the expression of chemokine receptors, and migrate toward the interface between T and B cells. A B-cell clone that recognizes a B-cell epitope on the same protein antigen in the B-cell zone or follicle alters its chemokine receptor expression and migrates toward the T-B interface. At the interface, activated CD4⁺ T cells recognize B cells that present their specific peptide–MHC class II complexes and the B cells activate, proliferate, undergo class switching, and differentiate into extrafollicular plasmablasts. These activated B cells and plasmablasts in the T-B interface are called extrafollicular foci. Activated B cells may then activate previously activated CD4⁺ T cells and induce their differentiation into T follicular helper cells, which in turn contribute to the formation of GCs and the selection of higher affinity B cells that will initially form memory B cells and later give rise to long-lived plasma cells. Details in the text. DC, dendritic cell; GC, germinal center; MHC, major histocompatibility complex; Tfh, follicular helper T cell.

T-CELL ACTIVATION AND THE INDUCTION OF T-B COLLABORATION

At sites of inflammation, pathogen-derived molecules, such as lipopolysaccharide, flagellin, or nucleic acids, or molecules released by damaged host cells bind pattern recognition receptors on dendritic cells (DCs), leading to their activation. Activated DCs capture, process, and present protein antigens more efficiently, produce higher levels of MHC and costimulatory molecules, and express CCR7.⁶

After arriving in the T-cell zone, DCs activate rare naive CD4⁺ T cells whose TCRs recognize a relevant MHC class II–peptide complex.⁷ Activated T cells initiate diverse developmental programs and undergo clonal expansion. These primed, but not yet mature, cells begin to alter chemokine expression. Downregulation of CCR7 permits movement away from the T-cell zone. The ultimate destination and function of each cell depends on its specific developmental program. Cells developing into peripheral effector subsets (Th1, Th2, Th17, etc) upregulate sphingosine 1-posphate receptor 1 to permit egress from lymphoid tissue and additional receptors that guide them to peripheral sites.⁸ Cells developing along the follicular helper pathway upregulate CXCR5 and migrate toward the B-cell zone.⁹ Functional maturation requires reexposure to antigen at the site of inflammation or at the T-B boundary.

THE FORMATION OF AN EXTRAFOLLICULAR FOCUS

Interactions between developing cognate T and B cells lead to the formation of an extrafollicular B-cell focus. Ligation of CD40 on B cells by CD40L on T cells increases B-cell activation, promotes proliferation, and initiates class switch recombination to most human isotypes. The vast majority of isotype switching occurs at extrafollicular foci.¹⁰ Limited somatic hypermutation, and resulting affinity maturation, can occur in extrafollicular foci but does not reach the levels seen in germinal center (GC) B cells, primarily because of a lack of architectural and cellular components required for the iterative process of mutation and selection seen in GCs.¹¹

Extrafollicular foci can occur in isolation or may result in the development of GCs. The progeny of individual B-cell clones can develop into extrafollicular, GC, and memory B cells.¹² High-affinity interactions predispose B cells toward extrafollicular responses, whereas lower-affinity B cells are more likely to enter GCs.^13,14 Extrafollicular plasmablasts are short lived, whereas GCs produce long-lived plasma cells. Memory B cells arise both outside the GC (extrafollicular) and in the early stages of the GC response.¹⁵ Memory B cells thus generally exhibit a reduced degree of somatic hypermutation with an affinity for foreign antigen greater than that of naive B cells but lower than long-lived plasma cells. The diminished affinity maturation seen in memory B cells may allow for broad specificity with few restrictions on the potential direction of subsequent rounds of maturation, permitting rapid, flexible, and effective responses to antigenic variants produced by successive generations of pathogens.

Although follicular helper T (Tfh) cells are responsible for providing help to GC B cells,^16,17 the T cells involved in the initial T-B collaboration that drives the vast majority of isotype switching have not been conclusively identified. In humans and mice, CD4⁺ T cells in secondary lymphoid tissues exhibit a range of expression of Tfh markers, yet only cells with the highest expression of programed cell death protein 1 (PD1), CXCR5, and B-cell lymphoma 6 (BCL6), commonly called GC-Tfh or Tgc, migrate into GCs and provide help to GC B cells.¹⁸ Many, and often most, Tfh-like cells express lower levels of PD1 and CXCR5 and are not found within GCs. In humans, these cells are typically negative for BCL6.^19–21 They may represent precursors to GC-Tfh and are commonly called pre-Tfh, but their developmental relationship with GC-Tfh has not been formally tested. As such, we prefer to call PD1-low CXCR5-low cells extrafollicular helper T cells. The specific contributions of extrafollicular versus Tfh cells to isotype switching remains unclear. The gaps in our understanding of the phenotypic and functional heterogeneity within Tfh-like cells will be addressed in more detail in the section below on helper T cells.

THE GC RESPONSE

The histological structures described as GCs were discovered by anatomists and pathologists before any notion of the existence of immunity. Walther Flemming, the great German anatomist, is widely recognized for his description in 1882 of the doubling of the chromosomes in the nuclei of cells that occurs as a prelude to cell division, a process he called “mitosis.” Although Flemming is best remembered today for his studies on cytogenetics and mitosis, in 1885 he described structures in lymph nodes that he called “GCs,” and he speculated that these were the sites at which lymphocytes were generated. The function of lymphocytes in adaptive immunity was only established in 1957 by Gowans,²² and although indeed some lymphocytes do divide in GCs, they are not the sites of B or T lymphopoiesis. Rather, they are incubators that foster the evolution of activated B cells into high-affinity class-switched antibody-producing plasma cells.²³ In the true Darwinian sense, this process involves genetic diversification followed by natural selection and the eventual survival of only the fittest clones.

At its peak, the GC can be divided into a dark zone, in which B cells proliferate and undergo somatic hypermutation, and a light zone, in which the selection of high-affinity B cells occurs. It is the combination of somatic hypermutation and the selection of high-affinity B cells that forms the basis for the affinity maturation of antibodies. Repeated rounds of proliferation and mutation in the dark zone, followed by selection in the light zone, allow for the accumulation of mutations in both the heavy and light chain genes. These mutations are found primarily in the regions that bind B-cell epitopes because competition for limited antigen imparts a selective advantage on cells with the highest affinity.

The initiation and maintenance of GC reactions requires the reciprocal induction of the transcription factor BCL6, which functions as a repressor-of-repressors in both GC B cells and cognate Tfh cells.²⁴ Expression of an enzyme called activation-induced cytidine deaminase (AID) in B cells initiates the process of somatic hypermutation, which largely targets the rearranged variable region (VDJ or VJ) exons of Ig genes.²⁵ Most somatic hypermutation occurs while B cells are dividing in the dark zone. There are 3 possible consequences of ongoing somatic hypermutation: mutations may alter the affinity for foreign antigen, they may alter affinity to self-antigen, or they may induce an overwhelming degree of DNA damage leading to apoptosis.

After multiple divisions, surviving dark zone B cells downregulate the expression of the CXCR4 chemokine receptor that originally restricted them to the dark zone, upregulate CXCR5, and drift into the light zone of the GC.²⁶ In the light zone, B cells encounter follicular DCs that exhibit antigen in immune complexes on Fc and complement receptors.¹ They also encounter Tfh cells, which only populate the light zone. B cells with the highest affinity to a foreign antigen most efficiently capture it, enhancing their ability to present linear peptides on MHC molecules and increasing the probability that they form a stable interaction with a cognate Tfh cell. This interaction depends on ligation of CD40 on B cells by CD40L on T cells and is required for survival.²⁷ Importantly, many pathogens produce epitopes with substantial overlap to human self-antigens, a phenomenon known as molecular mimicry, and mutations that reduce affinity to self-antigen potentiate T-cell collaboration and optimal affinity maturation trajectories.²⁸

Positively selected B cells that receive additional CD40 signals induce c-Myc, reexpress CXCR4, and return to the dark zone to proliferate and undergo additional somatic hypermutation.²⁹ Repeated rounds of genetic diversification and proliferation in the dark zone followed by competition for antigen and T cell help in the light zone to create a selective pressure that increases the relative frequency of cells with higher-affinity B-cell receptors. Thus, the fitness of a GC B cell is a function of its affinity for antigen, and “survival of the fittest” is the engine that drives affinity maturation.

What exactly induces the earlier emergence of memory B cells and the later emergence of the precursors of long-lived plasma cells is not known. Regardless, while memory cells exhibit only modest increases in affinity, additional rounds of selection generate plasmablasts with high-affinity B-cell receptors. These cells migrate to the bone marrow, where they differentiate further into long-lived plasma cells that secrete high-affinity antibodies for many years, often for decades.

T CELLS THAT HELP B CELLS: TFH, PRE-TFH, GC-TFH, AND PERIPHERAL HELPER T CELLS

Naive CD4⁺ T cells are pluripotent precursors that differentiate into phenotypically and functionally distinct subsets uniquely tailored to specific inflammatory contexts. The diversity of potential outcomes can be conceptually organized along 2 functional dimensions. With few exceptions, CD4⁺ T cells either promote or suppress inflammation and modulate either cellular or humoral responses. Effector T cells, including Th1, Th2, and Th17 cells, promote cellular responses at the site of inflammation. Regulatory T cells (Treg) suppress them. Tfh-like cells promote humoral responses in secondary (and possibly tertiary) lymphoid tissues, whereas follicular Treg (Tfr) cells oppose them. The heterogeneity seen among effector T cells is mirrored in Treg, Tfh, and Tfr cells, with subsets of each displaying phenotypic and functional overlap with classical effector lineages.³⁰

Differentiation begins after initial activation by professional antigen-presenting cells (APCs) in secondary lymphoid tissues. The quantity and quality of TCR, costimulatory, and cytokine signals impart specific developmental trajectories.³¹ Potent TCR activation and CD28 costimulation favor Tfh development.^32,33 The activation marker PD1 is elevated on Tfh cells, likely due in part to the strength of stimulation required to promote their differentiation.³⁴ Sequential antigen presentation and ligation of inducible T-cell costimulatory (ICOS) first by DCs and then by cognate B cells is required for sustained expression of CXCR5 and BCL6 and maturation into Tfh.^35–38 The cytokines interleukin (IL)-6 and IL-21 also promote Tfh development, whereas additional cytokines are thought to impart functional heterogeneity.³⁹

Cognate B cells are primarily located within secondary lymphoid tissues, but T-B collaboration can occur at peripheral sites of inflammation, leading to the generation of peripheral Tfh-like cells and formation of tertiary (ectopic) lymphoid structures (ELSs).^40–42 ELSs provide ongoing support for the production of class-switched antibodies in multiple inflammatory contexts, including renal transplant rejection.⁴³ They can contain mature PD1⁺ CXCR5⁺ BCL6⁺ Tfh cells and fully formed GCs or may exhibit limited spatial organization characterized by extrafollicular B-cell responses and the presence of PD1⁺ CXCR5⁻ BCL6⁻ CD4⁺ Tfh-like cells known as peripheral helper T (Tph) cells.^44,45

There is substantial heterogeneity within Tfh-like cells.⁴⁶ They can adopt partial phenotypes associated with effector subsets, and the frequency of these cells correlates with the abundance of their effector equivalent. This overlapping heterogeneity allows for coordinated and selective modulation of distinct aspects of cellular and humoral responses. Cytokines produced by CD4⁺ T cells promote cellular responses optimized for different pathogen types and also regulate humoral responses.⁴⁷ For example, IL-4 and IL-21 are important for the formation of GCs and the induction of affinity maturation. IL-4 and IL-13 promote, whereas IL-21 opposes, switching to IgE and TGF-β promotes switching to IgA.^48–52 However, the precise relationships between different cytokines and antibody isotypes remain poorly understood.

Examination of T-B conjugates in mice suggested GC-Tfh cells are the source of cytokines that guide affinity maturation and isotype switching.⁴⁸ However, more recent data indicated that the vast majority of isotype switching occurs in pre-GC or extrafollicular B cells after initial cognate interaction with T cells.¹⁰ As previously mentioned, many CD4⁺ T cells in the secondary lymphoid tissues and ELS of humans express reduced levels of PD1 and CXCR5 and are found outside of B-cell follicles.⁵³ These cells may represent developmental precursors of Tfh and central memory cells or they may be comprise cells that failed to obtain sufficient B-cell help, leading to phenotypic decay.^44,54,55 Alternatively, they may represent an overlapping but independent and functionally mature lineage that drives class switching in extrafollicular and pre-GC B cells.

In mice, PD1-low, CXCR5-low, CD4⁺ T cells phenotypically similar to human extrafollicular helpers can be found at the T-B border. These cells express BCL6 and are required for extrafollicular B-cell class switching to IgG1 and IgG2c in some models.¹⁹ This suggests that extrafollicular helper T cells may collaborate with B cells to drive class switching. In support of this, signaling lymphocytic activation molecule-associated protein (SAP) is required for GC formation and long-term humoral responses but appears dispensable for class-switched extrafollicular responses.^56,57 In humans, PD1-low, CXCR5-low, BCL6⁺ tonsil CD4⁺ cells can induce proliferation and differentiation of naive B cells into class-switched antibody-producing cells in vitro but are unable to provide help to GC B cells.⁵⁸

However, most human extrafollicular helper T cells do not express BCL6.^20,21 Interestingly, extrafollicular class switching events may not necessarily require BCL6. Although deletion of BCL6 in murine CD4⁺ T cells leads to complete loss of Tfh cells and GCs, its effect on class switching is less pronounced, and the magnitude of this effect varies by isotype and by inflammatory context. The dependence of IgG1 and IgE appears to be absolute in rodents, but deletion of BCL6 leads to inconsistent, modest, or absent reductions in T cell–dependent isotype switching to IgG2b, IgG2c, IgG3, and IgA.^{16,56,57,59–64} There is evidence that BCL6-negative T cells can drive class switching in humans. CXCR5⁺ BCL6⁻ extrafollicular CD4⁺ T cells can provide help to B cells in vitro.⁵³ Tph cells have been observed adjacent to B cells and are capable of driving IgG plasma cell differentiation in vitro.⁴⁴ Patients with severe coronavirus disease 2019 (COVID-19) exhibited abundant class-switched antibodies despite losing BCL6⁺ Tfh cells.^20,65,66 Interestingly, IgG1 was the most abundant class-switched isotype in these patients, and IgG1 antibodies harbored the fewest number of somatic mutations, indicating that class switching to IgG1 may not require BCL6 in humans.

As with B cells, the progeny of individual naive CD4⁺ T-cell clones can develop into multiple lineages, and the affinity of a TCR biases these fate outcomes.^32,33 High affinity for antigen predisposes cells to develop into Tfh versus effector T cells. The role of affinity in driving extrafollicular helper differentiation remains unexplored. Does increased affinity promote extrafollicular versus follicular responses, as in B cells, or is the established relationship between affinity and Tfh development paramount? The former is supported by observations that cognate interactions are long-lasting at the T-B border and shorter-lived in the follicle and GC.⁶⁷ Additional studies are needed to identify the cells responsible for driving extrafollicular help to B cells, both central and peripheral, their developmental relationship with other cell lineages, the role of specific antigenic determinants in guiding their differentiation, and their contribution to health and disease. Immunophenotypes T helper cell subsets of relevance are listed in Table 1.

TABLE 1.

T-cell subsets important in T-B collaboration

T-cell subsets	Key markers	Location	Key cytokines
Pre-Tfh	CD4+ ICOS+ PD1 + CXCR5+	SLO	?
GC-Tfh	CD4+ ICOS+ PD1+ CXCR5++ BCL6+	SLO, GC	IL-4, IL-21, CXCL13
Tph	CD4+ICOS+ PD1+ CXCR5− BCL6−	Inflamed tissue	CXCL13
cTfh	CD4+ ICOS+/− PD1+/− CXCR5−/− BCL6−	Inflamed tissue	IL-21
Tfr	CD4+ICOS+ PD1+ CXCR5+ BCL6+ Foxp3+	SLO	IL-10, TGF-β

cTfh, circulating Tfh cell; GC, germinal center; ICOS, inducible T cell costimulatory; IL, interleukin; PD1, programed cell death protein 1; SLO, secondary lymphoid organ; Tfh, follicular helper T cell; Tfr, follicular regulatory T cell; Tph, peripheral helper T cell.

THE REGULATION OF GC RESPONSES: ANTIGEN CLEARANCE, TREG, AND TFR

Tolerance must be maintained during GC reactions. Additionally, eventually, GC reactions must recede and, ultimately, dissolve. These processes are mediated in part by the suppressive effects of Tfr cells.^68,69 Self-reactive natural Treg cells (nTreg) are generated in the thymus from developing T-cell precursors and maintain tolerance to self-antigen. Peripheral Treg (pTreg) are generated in secondary lymphoid tissues from naive precursors in response to both self and foreign epitopes but function primarily to maintain tolerance to foreign antigens of the commensal microbiota.⁷⁰ Tfr exhibit phenotypic and functional overlap with Treg and Tfh cells and exert suppressive effects on GC reactions. They are predominantly derived from thymic natural Treg (nTreg) and can also develop from naive precursors, possibly via transdifferentiation of pTreg or Tfh cells.^71–73

Regulatory populations expand in response to inflammation. nTreg prevent loss of T-cell tolerance to self-antigen. Tfr cells derived from nTreg constrain GC reactions, preventing the development of autoreactive GC B cells and the production of autoantibodies. In contrast, Tfr derived from naive precursors, whether pTreg or Tfh, appear to oppose GC reactions to foreign antigens.^71–73 Interestingly, this opposition is required for optimal antibody affinity. As the supply of antigen diminishes, and the positive feedback loops driving inflammation are reduced, the suppressive effects of Tfr cells come to predominate, leading to resolution of GCs.

THE LOSS OF GCS IN SEVERE INTRACELLULAR INFECTIONS

Disruption of the delicate interplay of T and B cells can have devastating consequences. For example, severe COVID-19 infections appear linked to a failure in TFH cells and GC development.^20,65 Although thoracic lymph node from age-matched controls contained abundant GCs, we observed a dramatic loss of BCL6⁺ T_FH cells and GC B cells in patients with severe COVID-19.²⁰ However, T-B conjugates and AID⁺ B cells were frequent and consistent with extrafollicular T-B collaboration. In support of this, vigorous extrafollicular B-cell responses have been observed in patients with severe COVID-19.⁶⁸

This phenomenon has been observed in other severe intracellular infections and may be related to dysregulated production of type 1 inflammatory cytokines. Loss of GCs was reported in severe acute respiratory syndrome.⁷⁴ GCs failed to develop in response to the murine rickettsia Ehrlichia muris. This phenomenon was reversed by tumor necrosis factor (TNF)-α blockade and by deletion of the gene encoding TNF-α.⁷⁵ The loss of T_FH cells and GCs observed in a murine malaria model was reversed by blockade of TNF-α or interferon (IFN)-β. Deletion of TBX21, which encodes the transcription factor T-box expression in T cells and is required for the differentiation of IFN-γ producing TH1 cells, also prevented GC loss.76 In a Salmonella infection model, IL-12 was responsible for both a block in Tfh cell differentiation and a loss of GCs.⁷⁷ IL-12 contributes to T_H1 cell differentiation. Both IL-12 and T_H1 cells increase production of TNF-α in the lymph node. Finally, mice immunized with lymphocytic choriomeningitis peptide and then subsequently infected with lymphocytic choriomeningitis clone 13 developed a severe viral infection involving the lungs and other organs, pronounced lymphopenia, and the loss of GCs in lymph nodes, a syndrome eerily similar to that seen in severe COVID-19. Very high levels of circulating IL-12 were documented in these mice.⁷⁸

Interestingly, examination of thoracic lymph nodes from patients with severe COVID-19 revealed high levels of TNF-α and increased numbers of T_H1 cells.²⁰ Based on these findings, we suspect that high levels of TNF-α and IL-12 may promote T_H1 differentiation and block T_FH development, leading to a loss of GCs. Thus, the sequential induction of IL-12, IFN-γ and TNF-α could be linked to the phenotype seen in severe COVID-19, a phenomenon that may be relevant to other human diseases, and murine infectious models may afford an opportunity to understand the mechanisms underlying it.

T-B COLLABORATION IN AUTOIMMUNE AND CHRONIC INFLAMMATORY DISEASES

Most common autoimmune diseases are complex multigene disorders. Some genetic variants linked to autoimmunity result in a breakdown in B- or T-cell tolerance. Others enhance activation of adaptive or innate immunity or alter cellular processes that indirectly impinge on immune function. Environmental challenges can exacerbate underlying genetic susceptibilities by inducing autoreactive responses because of molecular mimicry.

Very few autoimmune diseases are entirely dependent on only T cells or only B cells. In many diseases, including systemic lupus erythematosus, pemphigus vulgaris, and myasthenia gravis to name but a few, autoantibodies are pathophysiologically relevant (as opposed to representing an epiphenomenon within a disease). Almost all disease-causing autoantibodies are IgG antibodies.⁷⁹ Therefore, T-B collaboration is of obvious relevance and is known to occur at both central and peripheral sites.⁸⁰

In lupus, extrafollicular IgD⁻ CD27⁻ CD11c⁺ CXCR5⁻ DN2 B cells have been suggested to be the key precursors of autoantibody-secreting cells.⁸¹ As a result of this finding and other studies in animal models, it is thought that many autoantibodies are generated at extrafollicular sites.⁸² A widely held and related view, not substantiated by very strong evidence to date, posits that this may be because it is easier to break tolerance at extrafollicular sites than in GCs. It may be theoretically argued that although Treg function at both follicular and extrafollicular sites, it is only in the GC that successive rounds of selection iteratively reduce affinity to self-antigen and increase affinity to foreign antigen. In contrast, somatic hypermutation in extrafollicular sites is not accompanied by selection and thus may produce a greater propensity for autoreactivity.

The role of T-B collaboration in autoimmunity extends beyond autoantibody production. Depletion of B cells using monoclonal antibodies to CD20 has been shown to be therapeutically beneficial in a number of autoimmune and inflammatory disease contexts, although these diseases are often considered to be largely T-cell driven.^83–85 Activated B cells may play an important role in presenting antigen to CD4⁺ T cells, or provide these T cells with essential cytokines or costimulation, in these disorders. In support of this view, it has been argued on the basis of studies in rodents that in some circumstances, CD4⁺ T-cell memory may be sustained by B cells.⁸⁶ Other possible mechanisms need to be considered. For instance, B-cell depletion is a very effective therapy in multiple sclerosis. Although there may be many possible contributions of B cells to the pathogenesis of multiple sclerosis, recent evidence shows that the induction of T cell–dependent B-cell responses to an antigen from Epstein-Barr virus may induce cross-reactive B-cell responses that drive the disease process.^87,88

B CELLS AND ANTI-HLA ANTIBODIES IN TRANSPLANT REJECTION

Solid organ transplant serves as a lifesaving therapy for end-stage organ dysfunction. However, long-term allograft survival is limited despite potent, broad-based immune suppression. Chronic antibody-mediated rejection (AMR) is a major barrier to improving long-term graft survival. Donor-specific anti-HLA antibodies (DSAs) are one of the diagnostic criteria of AMR and play a key role in its pathophysiology.⁸⁹ Seminal studies by Paul Terasaki’s group established the importance of anti-HLA antibodies in AMR.⁹⁰ It is thought that DSAs bind to antigen, primarily HLA molecules, on endothelial cells in glomerular and peritubular capillaries, leading to complement activation and endothelial injury.⁹¹ Studies show that 8% to 10% of kidney transplant recipients develop de novo DSA within 1 y of transplant.⁹² Acquisition of anti-HLA antibodies within 1 y posttransplant is associated with reduced allograft survival.⁹³ Inhibition of DSA production prevents graft rejection in nonhuman primate kidney transplant models.^94,95

Alloreactive B cells can be derived from naive and memory B cells. Historically, IgM DSAs were considered harmless but may be associated with worse allograft survival.⁹⁶ Nevertheless, most clinically relevant alloreactive antibodies are IgG. Their pathogenicity varies by isotype; data suggest that complement-fixing IgG subclasses are associated with acute AMR, whereas responses dominated by IgG4, which cannot bind FC receptors, are associated with subclinical AMR.^97,98 IgG1 is the most common DSA isotype, but most responses are mixed and IgG3, which has the highest affinity for Fc gamma receptor IIIA (FCγRIIIA), correlates best with AMR frequency and severity.^99,100

The role of extrafollicular versus GC responses in DSA production and AMR remains unclear. In mice, although deletion of SAP in host CD4⁺ T cells failed to abrogate early, low-affinity antibody production in a cardiac transplant model, late alloantibody responses were abolished and grafts survived indefinitely.¹⁰¹ Secretion of GC-dependent class-switched, high-affinity antibodies was required for rejection. A separate study found that graft-versus-host allorecognition by tissue-resident donor CD4⁺ T cells was required to initiate alloimmune humoral responses.¹⁰² This process was not dependent on SAP, indicating that induction of DSA may depend on extrafollicular T-B interactions. Maximal DSA levels were achieved in the absence of SAP in host CD4⁺ T cells, but epitope spreading, vasculopathy, and graft rejection were dependent on germinal reactions. Finally, GC B-cell numbers correlated with DSA levels and kidney allograft rejection.¹⁰³

Both extrafollicular and GC alloreactive B cells are seen in human transplant recipients. Although DSA levels broadly correlate with AMR, many patients with high DSA titers do not exhibit graft inflammation or vasculopathy.^104,105 Notably, epitope spreading appears to correlate with coronary artery vasculopathy in human cardiac transplant recipients.¹⁰⁶ Somatic hypermutation is an important driver of epitope spreading and, once tolerance is broken, GCs can recruit additional autoreactive clones.^107,108 Furthermore, pretransplant IgG4, which is indicative of a preexisting, advanced GC-dependent humoral response, predicts posttransplant IgG3 production and acute AMR.¹⁰⁹ However, extrafollicular responses are a major source of autoreactive antibodies in multiple diseases.^82,110,111 Given these findings, it is possible that DSA production and epitope spreading may begin with extrafollicular T-B interactions but that GC dependent, high-affinity antibodies preferentially drive AMR. Differential reliance on extrafollicular versus follicular T-cell help may help explain why DSA alone is insufficient to predict rejection.

Antigen presentation by B cells is critical for DSA and AMR. Studies suggest that it may drive other aspects of allo responses as well. Chronic cardiac allograft vasculopathy was absent in mice lacking B cells but unchanged in mice deficient for antibodies but not B cells.¹¹² Although effector cell numbers were unaffected, IFN-γ-producing memory CD4⁺ and CD8⁺ T-cell numbers were dramatically reduced in the absence of B cells in a mouse skin transplant model.¹¹³ Interestingly, B cells may be important for more than just antigen presentation and antibody production. In a mouse model of cardiac transplant tolerization, alloantigen-specific B cells were identified in transplant-tolerant recipients that supported T_FH development but were unable themselves to become GC B cells.¹¹⁴ These tolerized B cells inhibited DSA production by naive B cells. Thus, B cell–intrinsic properties may impact alloimmune responses in unexpected ways.

T_FH, CT_FH, AND T_FR IN TRANSPLANT REJECTION

T cells are important mediators of both cellular and humoral alloreactions. Loss of CD8⁺ T cells by anti-CD8 mAb impairs cellular responses and delays, but does not prevent, rejection in a mouse model of heart transplantation.¹¹⁵ Depletion of CD4⁺ T cells, or costimulatory blockade (eg, CD154 cytotoxic T lymphocyte–associated antigen-4 immunoglobulin fusion protein [CTLA4-Ig]), suppresses both cellular and humoral immunity and can achieve long-term tolerance.^116–118 Blockade of CD40 or CD40L prevents DSA production and dramatically extends graft survival in nonhuman primate kidney transplant models, suggesting T-cell help to B cells is a key driver of transplant rejection.^94,95

T-cell responses begin with alloantigen presentation by professional APC in secondary lymphoid tissues. Direct and indirect allorecognitions have long been understood to be major pathways of alloantigen presentation. In direct allorecognition, recipient T cells recognize intact donor MHC molecules expressed on the donor cells, whereas in the indirect pathway, recipient T cells recognize donor-derived peptides presented in the context of recipient MHC expressed on the recipient APCs. The semidirect pathway (cross-dressing) was more recently described as an additional and critical mechanism of alloantigen recognition. In the semidirect pathway, intact donor peptide–MHC complex is transferred from donor DCs to recipient DCs and presented to recipient T cells by the recipient DCs. Fadi Lakkis’ group showed that cross-dressed DCs stably engaged TCR-transgenic effector CD8⁺ T cells in the allograft immediately after transplantation and for up to 8 wk in mouse models of islet and kidney transplantation.¹¹⁹ Mouse models suggest that the indirect pathway is the primary contributor to AMR, although donor-derived T cells may play some role in initiating DSA production via “inverted direct allorecognition.”^{102,120–122}

Regardless of the source of initial antigenic stimulation, development of IgG DSA requires T-B collaboration.^{94,95,101–103,123} In secondary lymphoid tissues, T cells primed by APCs presenting alloantigens proliferate and differentiate. Some cells migrate to the graft and modulate cellular inflammation, whereas others move to the T-B boundary and provide help to B cells. Additionally, T and B cells sometimes form organized tertiary lymphoid structures in graft tissue. The role of these structures in transplant rejection is not fully understood.¹²⁴ B cells can be found in close proximity to Tfh-like T cells in kidney allograft biopsies not only in patients with mixed cellular rejection and AMR but also in patients with isolated cellular rejection.^43,125,126 Intragraft tertiary lymphoid structures were reported to be associated with worse long-term allograft function, but tissue-resident class-switched innate-like B cells appear to bind renal self-antigens and are not enriched for reactivity against alloantigen.^127,128 Furthermore, although B cells have been shown to expand and undergo limited somatic hypermutation in situ, the relative abundance of silent mutations in complementarity determining regions and the absence of follicular DCs suggested that these cells were not part of functional GCs.¹⁰⁵

Tfh cells are thought to be critical for T-B collaboration during allo responses. Tfh and GCs develop in mouse models of skin, heart, and kidney transplantation. Using Tfh-deleter mice (CD4-cre; CXCR5-IRES-loxp-STOP-loxp-DTR), Sage et al showed that IgG DSA and AMR were virtually eliminated in the absence of CXCR5⁺ CD4⁺ T cells in a model of kidney transplantation.¹²⁹ Graft survival was dramatically enhanced, but cell-mediated allograft responses were increased and ultimately drove rejection, indicating that abrogation of T-B collaboration is not sufficient to prevent disease. These data may suggest that DSA production in kidney transplantation is dependent on Tfh, but CXCR5⁺ CD4⁺ T cells also drive extrafollicular responses.^19,53,58,130 Although GCs appear to preferentially drive AMR, extrafollicular T-B interactions are sufficient for DSA production and may be required to initiate follicular responses. The precise role of specific subsets of Tfh-like cells, their influence on GC versus extrafollicular B-cell responses, and their role in graft failure remain incompletely defined.

In transplant patients, circulating Tfh (cTfh) cells, defined as CXCR5⁺ or ICOS⁺, are studied as a surrogate marker for humoral alloimmune responses. cTfh cells are a distinct population of T cells found in peripheral blood that exhibits functional overlap with Tfh cells; they secrete IL-21 and can induce naive B cells to proliferate and differentiate into antibody-producing cells in vitro.^131,132 The proportion of cTfh cells is higher in kidney transplant recipients with AMR and lower in patients who achieve operational tolerance or who are at low risk of rejection.^133,134 La Muraglia et al showed that cTfh cells are an early biomarker of humoral alloreactivity and precede development of DSAs after transplantation, using a mouse skin transplant model.¹³⁵ However, cTfh cells are heterogenous, do not express BCL6, and their ontogeny is poorly understood. The manner and degree to which these cells reflect the nuances of T-B collaboration is unclear.

The role of Tfr cells in transplant is also poorly understood. Tfr-deleter mice (Foxp3-YFP-cre; CXCR5-IRES-loxp-STOP-loxp-DTR) revealed that Tfr modestly inhibited DSA production in response to allogeneic splenocyte challenge but did not alter DSA levels or graft survival in a model of kidney transplant.¹²⁹ The relevance of Tfr to human transplant biology is unknown.

THERAPEUTIC TARGETING OF T-B INTERACTION

Several therapies have been tested to treat or prevent allo-specific antibody production: anti-CD20 antibodies to deplete B cells (eg, Rituximab), proteasome inhibitors to deplete plasma cells (eg, Bortezomib), plasmapheresis to remove circulating antibodies, enzymatic digestion by IgG endopeptidase, and anti-IL-6 receptor monoclonal antibodies to modulate B-cell differentiation.^136–139 However, their efficacy is limited and, given their lack of specificity, they carry substantial risks of infections.

More recent efforts have focused on the ability of T cells to help B cells. Nonhuman primate transplant models indicate that blockade of CD40/CD40L interactions prevents AMR.^118,140 Early antibodies targeting CD40L led to thromboembolic complications and were discontinued. Newer agents engineered to avoid platelet activation, and monoclonals targeting CD40, are under investigation. CD28 blockade also leads to a sharp reduction in Tfh in a mouse model of skin transplantation, and the Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial and Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial-EXTended criteria donors trials found that fewer patients receiving belatacept, a CTLA4-Ig that interferes with CD28 costimulation, developed DSAs compared with those receiving cyclosporine.^141–143 Similarly, although proteasome inhibitor monotherapy frequently leads to DSA rebound, combined pretransplant costimulation blockade and proteasome inhibition reduced Tfh numbers and DSA levels, delayed DSA rebound, and prolonged graft survival in nonhuman primate–sensitized kidney and skin transplant models and in highly sensitized human cardiac transplant candidates.^144–146 Combined costimulation blockade and proteasome inhibition were also able to reverse acute AMR in a sensitized mouse skin transplant model and in human kidney transplant recipients, and prolonged costimulation blockade prevented DSA rebound.¹⁴⁷ These data indicate that T-B collaboration is required for the initiation, maintenance, and rebound of DSA-mediated AMR. The therapeutics targeting T-B collaboration are summarized in Table 2. However, CD28 costimulation is required for all T-cell inflammatory responses, and prospective cohort studies have consistently shown that belatacept and antimetabolite (eg, mycophenolic mofetil and azathioprine) therapy is associated with impaired seroconversion after COVID-19 vaccination.^148–150 Caution is warranted when wielding such blunt tools.

TABLE 2.

Therapies targeting T-B collaboration

Categories	Target	Reagent	Target	Clinical trial and indications	Target disease	NCT number
Costimulatory blockade	CD28	FR104/VEL-101	Pegylated anti-CD28 Fab’ fragment	Phase I/II; phase I	Solid organ transplant	NCT05238493; NCT04837092
		Lulizumab	Pegylated anti-CD28 specific domain antibody	Phase IIa	Solid organ transplant, SLE	NCT04903054; NCT04066114; NCT-2265744
	CD80/86	Abatacept	CTLA4-Ig	FDA approved for rheumatoid arthritis and prophylaxis of acute GVHD	Rheumatoid arthritis, acute GVHD
		Belatacept	CTLA4-Ig	FDA approved for kidney transplant patients	Solid organ transplant
	CD40L	Ruplizumab (hu5C8)a	Anti-CD40L mAb	Discontinued because of thromboembolic events	Solid organ transplant
		Toralizumab (IDEC-131)a	Anti-CD40L mAb	Discontinued because of thromboembolic events	Solid organ transplant
		Toralizumab (IDEC-131)a	Anti-CD40L mAb	Discontinued because of thromboembolic events	Solid organ transplant
		Dapirolizumab	Anti-CD40L mAb	Phase I	SLE	NCT01764594
	CD40	Iscalimab	Anti-CD40 mAb	Halted because of nonsuperiority compared with CNI	Solid organ transplant	NCT03663335
		Bleselumab	Anti-CD40 mAb	Phase I	Solid organ transplant	NCT01780844; NCT01279538
	Syk tylosine kinase	Fostamatinib	Tylosine kinase inhibitor	Phase II	Solid organ transplant	NCT03991780
	BAFF	Atacicept	TACI-Ig	Phase II	SLE	NCT01972568
Plasma cell–targeted therapy	Proteasome	Bortezomib	Proteasome inhibitor	FDA approved for multiple myeloma	Multiple myeloma
		Carfilzomib	Proteasome inhibitor	FDA approved for multiple myeloma	Multiple myeloma
	CD38	Daratumumab	Anti-CD38 mAb	FDA approved for multiple myeloma	Multiple myeloma, solid organ transplant
		Felzartamab	Anti-CD38 mAb	Phase III	Antibody-mediated rejection	NCT05021484
	BCMA; CD3	REGN5458/9	Anti-BCMA+ anti-CD3 bispecific mAb	Phase I/II	Multiple myeloma	NCT05137054
	CD19	Inebilizumab	Humanized anti-CD19 mAb	Phase III	Neuromyelitis optic spectrum disorder	NCT02200770
	CD20	Rituximab	Chimeric anti-CD20 mAb	FDA approved for non-Hodgkin’s lymphoma, CLL, rheumatoid arthritis, granulomatous with polyangiitis, microscopic polyangiitis, pemphigus vulgaris
		Ofatumumab	Humanized anti-CD20 mAb (IgG1k)	FDA approved for CLL, multiple sclerosis
		Obinutuzumab	Humanized anti-CD20 mAb (defucosylated, IgG1)	FDA approved for CLL
Cytokine	IL-2	Aldesleukin	IL-2 (low dose)	Phase I/II	HCV vasculitis, GVHD, Type 1 diabetes, SLE, Solid organ transplant	NCT00574652; NCT00529035; NCT00525889; NCT04077684; NCT02417870
		Efavaleukin alfa	Mutant IL-2 Fc	Phase I/II	GVHD, SLE, Rheumatoid arthritis	NCT03422627; NCT03451422; NCT03410056
	IL-21	NNC0114-0006	Anti-IL-21 mAb	Phase II	Type 1 diabetes, Rheumatoid arthritis, Crohn’s disease	NCT02443155; NCT01208506
		BOS161721	Anti-IL-21 mAb	Phase I/II	SLE	NCT03371251
		ATR-107	Anti-IL-21R mAb	Phase I	PK/PD study in healthy subjects	NCT01162889
	IL-6	Clazakizumab	Anti-IL-6 mAb (humanized IgG1)	Phase III	Solid organ transplant	NCT03744910
		Tocilizumab	Anti-IL-6Ra mAb	Phase III	Solid organ transplant	NCT04561986
	IL-7	RN168	Anti-IL-7Ra mAb	Phase I	Type 1 diabetes	NCT02038764
		OSE-127	Anti-IL-7Ra mAb	Phase II	Ulcerative colitis	NCT04882007

^aDiscontinued because of side-effect profile.

CLL, chronic lymphocytic leukemia; CNI, calcineurin inhibitor; CTLA4, cytotoxic T lymphocyte–associated protein 4; FDA, Food and Drug Administration; GVHD, graft-versus-host disease; HCV, hepatitis C virus; IL, interleukin; mAb, monoclonal antibody; PK/PD, pharmacokinetics/ pharmacodynamics; SLE, systemic lupus erythematosus; TACI, transmembrane activator and CAML interactor.

CONCLUDING REMARKS

T-B collaboration initiates a complicated network of sequential interactions, both direct and indirect, that positively reinforce alloimmune antibody responses. Disruption or modulation of this collaboration may aid efforts to treat or prevent AMR. Selective manipulation of isotype switching could improve outcomes, but the mechanisms driving isotype rearrangements are poorly understood and given their roles in other aspects of immune responses, efforts targeting the relevant cytokines may lack specificity. The ability to prevent allo-specific antibody production without disrupting normal humoral response would revolutionize organ transplantation. However, a more sophisticated understanding of humoral responses to alloantigen is needed before this goal can be achieved. The relative contribution of GC versus extrafollicular antibody production to AMR is ill-defined and may be context dependent. Furthermore, antigen-specific interventions are required to prevent alloantibody production without compromising beneficial T-B collaboration or to augment desired T-B collaboration without driving rejection. B-cell allo-epitopes are well described, but little is known about the T-cell epitope landscape. What types of mismatches generate T-cell epitopes? Which ones are responsible for AMR? What types of T-B collaboration do they lead to? T-cell epitopes are difficult to identify, but linked recognition may offer a roadmap forward.