Introduction

The last two decades have seen numerous discoveries which have helped understand the biology of B cell lymphoma and lay the foundation for precision therapies. B cell lymphomas arise from the germinal center (GC), a dynamic structure that forms upon encounter of naïve B cells with a putative antigen [1], and may be secondary to i) genetic/epigenetic alterations in the GC B cells or ii) aberrant response of immune components of the microenvironment ultimately leading to lymphomagenesis [2]. Gene expression profiling (GEP) studies have divided diffuse large B cell lymphoma (DLBCL) - the most common B cell lymphoma - into two main subgroups based on the cell of origin (COO): the activated B cell (ABC) and the germinal center B cell (GCB) subtypes [3]. More recently, two additional molecular classifications have used whole exome sequencing (WES) and structural genomic abnormalities to further subdivide DLBCL into several genetically defined subgroups [4, 5]. An additional layer of complexity includes the immune cells that infiltrate the tumor. A landmark study performed on tumor biopsies from 95 untreated patients with follicular lymphoma (FL) - the second most frequent B cell lymphoma - demonstrated significant enrichment of genes associated with macrophages in patients with unfavorable outcomes whereas the expression signature was enriched for genes linked to T-cells in those with a favorable outcome [6]. Additionally, we reported the prognostic value of memory CD4+ T-cells, which play a critical role in immune surveillance, and designed a prognostic risk model (BioFLIPI) to improve the identification of high-risk patients [7]. Similarly, the prognostic relevance of TME in DLBCL has been uncovered in two recent studies which have further deconvoluted the TME in several ecosystems [8, 9]. Part of the reason for an unfavorable TME may be linked to the mutation of genes directly or indirectly involved in the control of antigen presentation, including CREBBP [10], EP300 [11], EZH2 [12], and others [13]. However, many additional mechanisms may come into play to shape the immune response against tumors [14]. Here, we dissect the function of different immune components of the TME (Table 1), the impact of their aberrant expression in B cell lymphoma and novel therapeutic avenues (Tables 2 and 3).

Table 1 Role and markers of the immune cells of the TME
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Table 2 Clinical trials including agents targeting the immune cells of the TME in B cell lymphomas
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Table 3 FDA-approved agents targeting the immune cells of the TME in B cell lymphomas
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T follicular helper cells

T follicular helper (Tfh) cells commonly reside inside the lymph nodes, tonsils, and spleen. They are defined by the expression of cell surface markers CD4, CXCR5, PD1, and ICOS, their master regulator being B cell lymphoma (BCL) 6 [15]. Tfh cells play a critical role in the formation and maintenance of GCs. Also, Tfh cells engage GC B cells to promote clonal selection and affinity maturation so that high-affinity B cells can be selected to exit the GC reaction and undergo terminal differentiation towards plasma cells or memory cells [15]. This mechanism is mediated through interaction between the co-stimulatory molecule CD40-ligand on the Tfh cells with CD40 on the B cells (Fig. 1) [15]. On the contrary, T follicular regulatory (Tfr) cells limit the output of the GC reaction counterbalancing Tfh function [1]. Of note, Tfh cells can convert to Tfr cells through FOXP3 activation in the late germinal center [16]. Several studies have shown an increased expression of Tfh CD4+PD1+ICOS+ cells [17] and/or CD4+CXCR5+Foxp3- [18] cells in diagnostic samples of malignant lymphoid disease compared to healthy controls. The same expression decreased or returned to normal at the end of effective treatment, but it increased in progressive disease [17]. It is possible that Tfh cells may contribute to lymphoma B cell survival via production of sCD40L which activates NF-kB pathway and in turn up-regulates c-FLIP and Bcl-xL [19, 20]. Increased expression of lymphoma-infiltrating Tfh cells was associated with high levels of IL-6, IL-21 [21], IL-4 [22], and CXCL13 [9] (Fig. 1). Conversely blocking these cytokines resulted in reduced infiltration of Tfh cells [21]. Additionally, the crosstalk between lymphoma B cells and Tfh cells increases the release of CCL17 and CCL22, which induces the preferential migration of regulatory T cells (Treg) and IL-4 producing CD4+ T cells, stimulating more chemokine release thus creating an immune suppressive TME that promotes tumor survival and growth [23, 24]. Another study divided Tfh cells into Tfr-like subsets (CD4+CD25+CXCR5+) and Tfh CD25- subset (CD4+CD25-CXCR5+) [25]. The difference between these two groups was associated with the higher expression of Blimp1, Foxp3, IL-10, TGF-β, and lower levels of IL-21 in Tfr-like CD25+ cells compared to Tfh CD25- cells [25]. This discovery is intriguing as it demonstrates the plasticity of the immune response and implies the possibility to leverage this characteristic as a therapeutic tool. Novel insights on the role of Tfh cells in immune evasion can usher in the opportunity for unexplored therapeutic targets [26]. In particular, identification of genetic mutations, cell markers and cytokine/chemokine signaling that impact Tfh cell function will help in improving our knowledge of the causative events that induce and/or sustain tumor development and growth. Thus, targeting these regulators may be a new approach to interrupting T cell support of lymphoma cells, which may complement other therapeutic approaches.

Fig. 1
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Role of T follicular helper (Tfh) cells in the normal germinal center and in lymphomagenesis

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T regulatory cells

Treg cells are CD4+ T cells expressing high CD25 (IL-2Rα) and FoxP3, and low or not CD127 (IL-7R α) [27, 28]. They suppress immune response activation and promote tolerance towards self-antigens to prevent autoimmunity [29]. However, their function can also suppress tumor immunity leading to immune escape [30]. Nevertheless, the significance of tumor-infiltrating Treg cells remains elusive due to their heterogeneity and their expression of both co-inhibitory and co-stimulatory receptors [31]. Specifically, some studies have shown that Treg FOXP3+ cells display a tumor-protective effect [32, 33] in FL [34] and DLBCL [34, 35] by suppressing T-cell proliferation and IFN-γ production [31, 36], while others found that Treg cells co-expressing activating markers such as CTLA4 [37] and TIGIT [38] result in an enhanced suppressive property and are associated with poor prognosis [39]. It is possible that the prognostic impact of Treg cells is dependent on disease context, however more clarity is still needed. Therefore, in-depth phenotypic and functional characterization of Treg cells is mandatory to identify novel targets for therapy and in turn improve patient survival. These data suggest that targeting Treg cells could be beneficial due to their antitumor immunity, however, it might also lead to unwanted immune-mediated toxicities.

In the last decade several immunomodulatory drugs (IMiDs) (e.g. lenalidomide) and targeting agents against B cell receptor (BCR) or intracellular kinases (e.g. BTK inhibitors and PI3K inhibitors) have been approved for hematologic malignancies [40]. Beside the tumor-specific effect, these molecules can also impact the immune components of the microenvironment (Fig. 2). For example, lenalidomide modulates Treg cells decreasing their suppressive function [41,42,43] and results in an enhanced anti-lymphoma activity. Similarly, PI3K inhibitors decrease the suppressive effect of Treg cells while enhancing CD8 T cell function [44,45,46]. The most recent therapeutic strategies targeting T cells include inhibition of checkpoint molecules such as PD1/PD-L1 and CTLA4 [47] or adoptive transfer of genetically engineered T cells [48]. Additional recently discovered immune checkpoint molecules that represent emerging targets for therapy are TIM3, LAG3 and TIGIT [49]. Blocking the negative T cell regulator CTLA4 reactivates immune response against the tumor in immunogenic cancers [50]. CTLA4 inhibition decreased Treg cells also in B cell lymphoma with a positive association of CD45RA-Treg ratio in responders vs non responders, however the antitumoral effects were quite modest [51]. PD1/PD-L1 inhibition prevents T cell exhaustion [52] and blocks the suppressive Treg activity [53]. Interestingly, inhibition of one checkpoint leads to compensatory increase of others. For example, blocking PD1 results in increase of LAG3 and CTLA4 [54]. On the contrary, combined inhibition of PD1 and LAG3 increased CD8 T cell cytotoxicity and decreased Treg cells [55]. Nevertheless, combination of two checkpoint blockades has shown modest activity in relapsed/refractory (R/R) B cell lymphoma [51, 56]. Similar to LAG3, TIM3 results in negative regulation of T cell response, ultimately leading to T cell exhaustion [118]. TGF-β has been shown to cause apoptosis in mouse models of B cell lymphoma [119]. Although TGF-β could promote an immunosuppressive environment, it is also a potent negative regulator of B-cell survival, proliferation, activation, and differentiation [120].

Targeting CAFs could be a challenging task due to the lack of specific cell surface markers causing difficulty to precisely target CAFs without damaging the normal tissue. However, there are a few general approaches targeting CAFs: 1) targeting the chemokine and growth factor pathways to inhibit the activation of CAFs, 2) normalization of CAFs via all-transretinoic acid or calcipotriol, 3) depletion of CAFs by transgenic technologies or immunotherapies, 4) targeting CAF-derived ECM proteins and associated signaling to induce stromal depletion, 5) cellular therapies (such as oncolytic adenoviruses, TNF-related apoptosis-inducing ligand or type I interferon) [110].

Tumor-infiltrating natural killer cells

Natural killer (NK) cells are innate cytotoxic lymphocytes of the immune system, contributing to the prevention of infection and tumor growth [121]. NK cells can be divided into two subtypes: CD56dim CD16+ NK cells (a mature cytotoxic population) and CD56bright CD16- NK cells (an immature and mostly immunomodulatory population) [121]. For both populations the most important cell surface inhibitory receptors are i) the members’ killer cell immunoglobulin-like receptor (KIR) family and ii) the CD94/NKG2A heterodimer [122, 123]. In physiologic conditions, normal cells are spared by the NK cells due to the recognition of MHC Class I engaged with KIRs. By contrast, lack of “self-recognition” signals to the NK cells to attack abnormal cells, such as tumor cells which present downregulated antigen presentation molecules as immune evasion strategy (Fig. 6) [124].

Fig. 6
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Role of natural killer (NK) cells in physiologic and pathologic conditions

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The role of NK cells in tumor immunosurveillance is well established [125, 126]. Importantly, NK cells seem to prevent development of tumors including B cell lymphoma [127, 128]. Recent evidence has shown that tumor infiltrating NK cells unleashed cytotoxic T cells, ultimately resulting in tumor eradication [129]. In line with the role of NK cells in suppressing malignancies, several studies have demonstrated a survival advantage of tumor infiltration by NK cells [129,130,131,132,133]. Even though a direct correlation may be less clear due to the frequent co-expression of T cells, these studies support a critical role of NK cells in promoting antitumor immune response. Tumor immune escape includes mechanisms that prevent NK cell activation or recruitment. For examples, suppressive cytokines (e.g. TGF- β) [134, 135] and prostaglandin [136, 137] clearly suppress NK cell activation. TGF- β also induces differentiation of Treg cells, which in turn suppress NK cells [138, 139]. Additional escape mechanisms include engagement of inhibitory receptors. Besides expressing NK-cell inhibitory receptors, NK cells also express other immune checkpoint molecules (e.g., PD1, TIM3, TIGIT, SIRP α) [140,141,142,143,144]. For example, increased expression of PD1 on NK cells was observed in several tumors [145,146,147,148], including HL and DLBCL [148]. By contrast, the inhibitory ligand PDL1 was found on tumor cells and macrophages, thus favoring the PD1/PDL1 interaction which limits the anti-tumor effect of NK cells. Recent studies have shown that PD1 blockades disrupt the suppressive PD1/PDL1 axis, reactivating NK cells with clinical implication [148]. Blockade of other immune checkpoint molecules has also shown encouraging potential for NK cell-based immunotherapy [124]. TIGIT was associated with NK cell exhaustion. On the contrary, TIGIT blockade antibodies restored anti-tumor activity [149]. Monalizumab, a humanized antibody against NKG2A, unleashes NK and T cells, thus promoting an enhanced tumor immunity [150]. STING agonists, such as cyclic dinucleotides, enhance NK cell fitness and anti-tumor effect [130, 151]. Another approach to amplify NK cell function against tumor is using “NK cell engagers”: bi- or tri-specific antibodies that bind NK and tumor cells [152, 153]. Furthermore, FDA has recently approved the first NK cell-based immunotherapy, NK-92, for clinical testing [154, 155]. Of note, NK cells provide a safer chimeric antigen receptor (CAR)-engineering platform compared to T cells [156]. Additionally, since they lack most of the KIRs, CAR-NK cells are less likely to become exhausted [157]. Several ongoing efforts have attempted to further potentiate and prolong NK-CAR potency by combining checkpoint inhibitor, cytokines and co-stimulatory signaling [157]. However, this promising off-the-shelf approach needs additional improvements to maximize its therapeutic efficacy.

Innate lymphoid cells

Innate lymphoid cells (ILCs) belong to the adaptive immune system and have a similar phenotype and function of T cells but differ from them for the lack of antigen receptors and clonal selection and expansion after stimulation [158]. ILCs are relatively rare (≤ 1% lymphocytes in mucosal tissues) [159] and can be distinguished in three main subsets: 1) type 1 ILCs include ILC1s and conventional NK cells [160, 161], express Tbx21, produce IFN-γ, and contribute to anti-viral and Th1 immunity [162]; 2) type 2 ILCs express Gata3, ROR α, TCF1 and Notch [163, 164], produce Th2 cell-associated cytokines (IL-4, IL-5, IL-9 and IL-13), and contribute to respond to Helminths infections and allergic diseases [165]; 3) type 3 ILCs express ROR γ t, present a different expression of T-bet [161, 166,167,168], produce IL-17A and IL-22, and participate in the homeostasis and mucosal defense and preservation of memory CD4 T cells [164, 169]. Notably, ILCs have a remarkable plasticity that allows them to acquire features of another ILCs subtype as required by changes in the TME. For examples, NK cells can switch to ILC1-like cells upon increase of TGF- β [135]. The existence of a continuous conversion from NK cells to ILC1s and vice versa is also plausible [170, 171]. Similarly, IL-12 has been shown to induce differentiation of ILC2s into ILC1 [172, 173] and ILC3s into ILC1s [173, 174]. ILCs also regulate tumor surveillance through a dynamic crosstalk with different immune components of the TME. Among ILCs, NK cells are the most active population as previously described. ILC2s can suppress immune response against tumor through IL-13-mediated enhancement of MDSCs expansion [174], alternatively they favor anti-tumor immunity through IL-5-mediated cooperation with DCs [175, 176]. ILC2s may potentiate the suppressive function of Treg through release of the growth factor AREG [177], or limit T cell activation through production of Arg1 [178]. ILC3s favor chronic inflammation, which in turn may promote tumor initiation [179, 180]. A group of ILC3s produce IL-17 and IL-22 [181, 182], which have been associated with poor prognosis in cancer patients [183, 184]. Collectively, these studies support the interplay between ILCs and the immune cells of the TME, which influence both innate and adaptive immune response against tumor. Future studies may be directed to investigating strategy blocking ILCs-myeloid or ILCs-Treg axes as a promising therapeutic strategy.

Lymphomas of the immune-privileged sites

The lymphomas of the immune-privileged sites include those arising from the central nervous system (PCNSL) and testes (PTL) [185]. Unlike other lymphomas, PCNSL and PTL are invisible to the immune system and have a suppression of anti-tumor T-cell response. Typically, they are localized diseases at presentation, even though they may be disseminated within the compartment (CNS-CNS, testis-testis) and between the compartments (CNS to testis) but rarely systemically, and have a poor prognosis [186, 187]. Constitutive activation of NF-kB via BCR (e.g. CD79B mutation) and toll-like receptor (e.g. MYD88 L265P mutation) is the canonical oncogenic pathway [188,189,190]. They share genetic features with classical ABC-DLBCL as well as with the recently defined molecular clusters MCD and C5 [4, 5]. However, the precise relationship between these classes remains to be elucidated. They present a high prevalence of genetic mutations causing loss of MHC class I and II expression [189, 191, 192]. Additionally, structural alterations at 9p24.1, which is the PD-L1 and PD-L2 locus, increase the abundance of transcriptional and translational expression of PD-L1 and PD-L2, further reinforcing immune evasion [189]. The predominant immune components of TME in these diseases are CD8+ cytotoxic T cells with a direct correlation between their number and outcome. Macrophages are also frequently identified, being an increased M1/M2 ratio associated with a better survival. Of note, PD1 and TIM3 appear to be concomitantly upregulated in CD8+ cytotoxic T cells and M2 macrophages with prognostic implications [193, 194]. However, further investigation is required to uncover the immune landscape of these diseases. The specific features of the lymphomas of the immune-privileged sites impact on treatment option. Especially, NF-kB/BTK inhibition has shown promise, with ibrutinib-based therapy being at the forefront of clinical investigation [195,196,197]. Additionally, checkpoint inhibition (e.g. nivolumab/pembrolizumab) has had an emerging role in the therapeutic armory [198].

Conclusion

The crosstalk between malignant B cells and immune cells in the lymphoma TME is highly complicated and might be affected by often interconnected intrinsic and/or extrinsic mechanisms which ultimately can lead to immune escape. This notion suggests the need to adopt a more comprehensive therapeutic strategy that does not limit its focus to tumor cells but that considers a global approach including the TME. Targeting the TME has long been considered a promising strategy, but much more work is needed to identify novel prognostic and predictive targets. Stratification of the patients for precision medicine as well as monitoring of immune response remain unmet clinical needs. Several advancements have been made towards this direction, such as the recent development of liquid biopsy that monitors circulating tumor DNA and immune components [199] or immune-imaging tools [200,201,202] to assess the efficacy of immunotherapy. The horizon of B cell lymphoma allows for a glimpse of a therapeutic strategy that considers the tumor in its whole, and maybe such an approach might be able to overcome the current clinical hurdles and rescue the still high therapeutic failures.