The role of B-1 cells in cancer progression and anti-tumor immunity.

In recent years, in addition to the well-established role of T cells in controlling or promoting tumor growth, a new wave of research has demonstrated the active involvement of B cells in tumor immunity. B-cell subsets with distinct phenotypes and functions play various roles in tumor progression. Plasma cells and activated B cells have been linked to improved clinical outcomes in several types of cancer, whereas regulatory B cells have been associated with disease progression. However, we are only beginning to understand the role of a particular innate subset of B cells, referred to as B-1 cells, in cancer. Here, we summarize the characteristics of B-1 cells and review their ability to infiltrate tumors. We also describe the potential mechanisms through which B-1 cells suppress anti-tumor immune responses and promote tumor progression. Additionally, we highlight recent studies on the protective anti-tumor function of B-1 cells in both mouse models and humans. Understanding the functions of B-1 cells in tumor immunity could pave the way for designing more effective cancer immunotherapies.

Human B-1 cells are important contributors to the naturally-occurring IgM pool against the tumor-associated ganglioside Neu5GcGM3.

Only few studies have described the anti-tumor properties of natural antibodies (NAbs). In particular, natural IgM have been linked to cancer immunosurveillance due to its preferential binding to tumor-specific glycolipids and carbohydrate structures. Neu5GcGM3 ganglioside is a sialic acid-containing glycosphingolipid that has been considered an attractive target for cancer immunotherapy, since it is not naturally expressed in healthy human tissues and it is overexpressed in several tumors. Screening of immortalized mouse peritoneal-derived hybridomas showed that peritoneal B-1 cells contain anti-Neu5GcGM3 antibodies on its repertoire, establishing a link between B-1 cells, NAbs and anti-tumor immunity. Previously, we described the existence of naturally-occurring anti-Neu5GcGM3 antibodies with anti-tumor properties in healthy young humans. Interestingly, anti-Neu5GcGM3 antibodies level decreases with age and is almost absent in non-small cell lung cancer patients. Although anti-Neu5GcGM3 antibodies may be clinically relevant, the identity of the human B cells participating in this anti-tumor antibody response is unknown. In this work, we found an increased percentage of circulating human B-1 cells in healthy individuals with anti-Neu5GcGM3 IgM antibodies. Furthermore, anti-Neu5GcGM3 IgMs were generated predominantly by human B-1 cells and the antibodies secreted by these B-1 lymphocytes also recognized Neu5GcGM3-positive tumor cells. These data suggest a protective role for human B-1 cells against malignant transformation through the production of NAbs reactive to tumor-specific antigens such as Neu5GcGM3 ganglioside.

Human B-1 Cells and B-1 Cell Antibodies Change With Advancing Age.

Age-related deficits in the immune system have been associated with an increased incidence of infections, autoimmune diseases, and cancer. Human B cell populations change quantitatively and qualitatively in the elderly. However, the function of human B-1 cells, which play critical anti-microbial and housekeeping roles, have not been studied in the older age population. In the present work, we analyzed how the frequency, function and repertoire of human peripheral blood B-1 cells (CD19+CD20+CD27+CD38low/intCD43+) change with age. Our results show that not only the percentage of B-1 cells but also their ability to spontaneously secrete IgM decreased with age. Further, expression levels of the transcription factors XBP-1 and Blimp-1 were significantly lower, while PAX-5, characteristic of non-secreting B cells, was significantly higher, in healthy donors over 65 years (old) as compared to healthy donors between 20 and 45 years (young). To further characterize the B-1 cell population in older individuals, we performed single cell sequencing analysis of IgM heavy chains from healthy young and old donors. We found reduced repertoire diversity of IgM antibodies in B-1 cells from older donors as well as differences in usage of certain VH and DH specific genes, as compared to younger. Overall, our results show impairment of the human B-1 cell population with advancing age, which might impact the quality of life and onset of disease within the elderly population.

GM3(Neu5Gc) ganglioside: an evolution fixed neoantigen for cancer immunotherapy.

Numerous molecules have been considered as targets for cancer immunotherapy because of their levels of expression on tumor cells, their putative importance for tumor biology, and relative immunogenicity. In this review we focus on the ganglioside GM3(Neu5Gc), a glycosphingolipid present on the outer side of the plasma membrane of vertebrate cells. The reasons for selecting GM3(Neu5Gc) as a tumor-specific antigen and its use as a target for cancer immunotherapy are discussed, together with the development of antitumor therapies focused on this target by the Center of Molecular Immunology (CIM, Cuba).

Defining Natural Antibodies.

The traditional definition of natural antibodies (NAbs) states that these antibodies are present prior to the body encountering cognate antigen, providing a first line of defense against infection thereby, allowing time for a specific antibody response to be mounted. The literature has a seemingly common definition of NAbs; however, as our knowledge of antibodies and B cells is refined, re-evaluation of the common definition of Nabs may be required. Defining Nabs becomes important as the function of NAb production is used to define B cell subsets (1) and as these important molecules are shown to play numerous roles in the immune system (Figure 1). Herein, we aim to briefly summarize our current knowledge of NAbs in the context of initiating a discussion within the field of how such an important and multifaceted group of molecules should be defined.

Detection of Naturally Occurring Human Antibodies Against Gangliosides by ELISA.

Gangliosides are sialic acid-containing glycolipids that have been considered attractive targets for cancer immunotherapy, based on the qualitative and quantitative changes they suffer during malignant transformation and due to their importance for tumor biology. Natural antibodies against gangliosides have been detected not only in cancer patients but also in healthy donors. The presence of these antibodies can be used as diagnostic or prognostic factor. However, these responses are difficult to detect because anti-ganglioside antibodies are usually of IgM isotype and low affinity. Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay based on the specific binding of antibodies to antigens bound to a solid phase. These antigens can be glycolipids like gangliosides. An enzyme linked to the last reactant allows the detection of specific binding through the development of color after the addition of a suitable substrate. ELISA combines the specificity of antibodies with the sensitivity of enzyme reactions. The ELISA method described herein can be used to detect antibody responses against gangliosides not only related to cancer but also to autoimmune diseases and infections, both in healthy donors, and patients, untreated or receiving specific immunotherapy.

Distinctions among Circulating Antibody-Secreting Cell Populations, Including B-1 Cells, in Human Adult Peripheral Blood.

Human Ab-secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20(+)CD27(+)CD38(lo/int)CD43(+)) cell and conventional plasmablast (PB) (CD20-CD27(hi)CD38(hi)) cell populations, in this study, we identified a novel B cell population termed 20(+)38(hi) B cells (CD20(+)CD27(hi)CD38(hi)) that spontaneously secretes Ab. At steady-state, 20(+)38(hi) B cells are distinct from PBs on the basis of CD20 expression, amount of Ab production, frequency of mutation, and diversity of BCR repertoire. However, cytokine treatment of 20(+)38(hi) B cells induces loss of CD20 and acquisition of CD138, suggesting that 20(+)38(hi) B cells are precursors to PBs or pre-PBs. We then evaluated similarities and differences among CD20(+)CD27(+)CD38(lo/int)CD43(+) B-1 cells, CD20(+)CD27(hi)CD38(hi) 20(+)38(hi) B cells, CD20(-)CD27(hi)CD38(hi) PBs, and CD20(+)CD27(+)CD38(lo/int)CD43(-) memory B cells. We found that B-1 cells differ from 20(+)38(hi) B cells and PBs in a number of ways, including Ag expression, morphological appearance, transcriptional profiling, Ab skewing, Ab repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20(+)38(hi) B cells or PBs, but differ in that memory B cells do not express Ab secretion-related genes. We found that B-1 cell Abs use Vh4-34, which is often associated with autoreactivity, 3- to 6-fold more often than other B cell populations. Along with selective production of IgM anti-phosphoryl choline, these data suggest that human B-1 cells might be preferentially selected for autoreactivity/natural specificity. In summary, our results indicate that human healthy adult peripheral blood at steady-state consists of three distinct ASC populations.

Anti-NeuGcGM3 reactivity: a possible role of natural antibodies and B-1 cells in tumor immunosurveillance.

While not naturally expressed in normal human tissues, N-glycolylated (NeuGc) gangliosides are overexpressed in several tumors and have immunosuppressive capacity, which contributes to cancer progression. Naturally occurring antibodies against NeuGcGM3 exist in healthy donors that specifically recognize and kill tumor cells expressing the antigen by complement-dependent and -independent mechanisms, the latter resembling an oncotic necrosis-type of cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with these natural antibodies decrease with age. Our work has shown that anti-NeuGcGM3 antibodies are not detected in the sera of non-small cell lung cancer (NSCLC) patients, compared to age- and sex-matched healthy donors, which have anti-NeuGcGM3. Interestingly, the level of serum total IgM, but not IgG, was significantly lower in cancer patients than in healthy donors. Screening of immortalized mouse splenic and peritoneal-derived hybridomas showed that peritoneal B-1 cells secrete anti-NeuGcGM3 with tumor cytotoxic capacity. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors developing cancer and affect the capacity of cancer patients to effectively fight against tumor cells.

Human antibodies reactive to NeuGcGM3 ganglioside have cytotoxic antitumor properties.

N-glycolylated gangliosides are not naturally expressed in healthy human tissues but are overexpressed in several tumors. We demonstrate the existence of antibodies that bind (N-glycolylneuraminyl)-lactosylceramide (NeuGcGM3) and are detectable in the sera of 65 from the 100 donors (65%) tested by ELISA. From those 65 NeuGcGM3 antibody-positive donors, 35 had antibodies that were able to recognize and kill NeuGcGM3-expressing tumor cells by a complement-mediated mechanism. After complement inactivation, 11 of the 35 positive sera showed a direct cytotoxic effect on the tumor cells. This complement-independent cytotoxicity was dependent on the presence of antigen on the membrane and resembles an oncotic necrosis cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera as well as the percentage of healthy donors with this immunity decreased with the age of the donor. In contrast to age and gender-matched healthy donors, we could only detect low reactivity against NeuGcGM3 in the sera of six out of 53 non-small cell lung cancer patients. These results suggest the existence of antibodies against NeuGcGM3 with antitumor immune surveillance functions, reinforcing the importance of N-glycolylated gangliosides as antitumor targets.