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Bone marrow haematopoietic stem and progenitor cells (HSPCs) express pattern recognition receptors such as Toll-like receptors (TLRs) to sense microbial products and activation of these innate immune receptors induces cytokine expression and redirects bone marrow haematopoiesis towards the increased production of myeloid cells. Secreted cytokines by HSPCs in response to TLR ligands can act in an autocrine or paracrine manner to regulate haematopoiesis. Moreover, tonic activation of HSPCs by microbiota-derived compounds might educate HSPCs to produce superior myeloid cells equipped with innate memory responses to combat pathogens. While haematopoietic stem cell activation through TLRs meets the increased demand for blood leucocytes to protect the host against infection, persistent exposure to inflammatory cytokines or microbial products might impair their function and even induce malignant transformation. This review highlights the potential outcomes of HSPCs in response to TLR ligands.

The therapeutic use of the immune system to specifically attack tumours has been a long-standing vision among tumour immunologists. Recently, the use of checkpoint inhibitors to turn-off immunosuppressive signals has proven to be effective in enhancing T-cell reactivity against patient-specific neoantigens, resulting from somatic mutations. Several of the identified T-cell epitopes share similarity with common bacterial and viral antigens, suggesting the involvement of pre-existing microbial cross-reactive T cells in rapid and durable tumour regression seen in some patients. This notion of T-cell cross-reactivity is further supported by the findings that intestinal bacteria can influence checkpoint-blockade therapy. Moreover, early data indicate the presence of such T cells in long-term survival breast cancer patients. This review highlights the main challenges for cancer immunotherapy and discusses the potential contribution of T-cell cross-reactivity in cancer immunotherapy and whether it can be used as a biomarker to predict the responsiveness to checkpoint inhibitors.

We recently reported that human bone marrow hematopoietic CD34(+) progenitors express functional Toll-like receptors (TLR) and can differentiate into myeloid cells just by stimulation with resiquimod (R848), a specific agonist for TLR7/8. However, the mechanisms by which R848 induces cell differentiation, the effects of other TLR agonists and the functionality of the differentiated cells are not known. Comparable to R848, loxoribine (a TLR7 agonist) and Pam(3)CSK(4) (a TLR2 agonist) induced cytokine production and cell differentiation along the myeloid lineage. R848 and loxoribine were more effective than Pam(3)CSK(4) at inducing the lineage-negative (CD11c(+) CD14(-)) dendritic cells (DC), whereas Pam(3)CSK(4) was more effective at inducing CD11c(+) CD14(+) monocytes. Both cell subsets expressed CD80/CD86 and HLA-DR molecules; however, they showed differential expression of CD1a, CD1b, CD1c, CD11b, CD206 and CD207 markers when compared with each other. Cell differentiation into DC was significantly inhibited by an anti-TNF-alpha nonoclonal antibody. The CD11c(+) CD14(-) subset was isolated and shown to be more potent in stimulating an alloreaction than the CD11c(+) CD14(+) subset. Collectively, these data highlight the differential effects of TLR agonists on human bone marow CD34(+) progenitor cells and provide a new opportunity for generating functional DC that would be useful in cancer vaccination.

Display on the surface of filamentous phages has been shown to be well suited for the enrichment of serum antibody-binding ligands. Here, we have taken the advantage of this technology to analyze the humoral immune response in patients with cancer. The cDNA repertoires from breast cancer cell lines T47D and MCF-7 were fused to the 3prime;-end of the filamentous phage M13 gene VI in all three reading frames. When the libraries were biopanned on rabbit polyclonal IgG against the human Bcl-xL protein, positive clones were selected, thus confirming the utility of the libraries. Using serum antibodies from patients with breast cancer, we specifically selected IgG-binding phage-encoded cDNA products. Sequence analysis of the selected clones identified important antigens including p53, centromere-F, int-2, pentraxin I, integrin β5, cathepsin L2 and S3 ribosomal protein. The selected phage-displayed cDNA products were recognized by a significant number of breast cancer sera as compared to sera from normal individuals. Although the human pentraxin I mRNA was reported to be exclusively localized in the nervous system, we found it also expressed by breast cancer cell lines. Four out of 30 patients with breast cancer (13 %) showed reactivity with the recombinant pentraxin expressed in Escherichia coli, while no reactivity was found in normal sera. The obtained results demonstrate that phage display could be a valuable method for the identification of antigens recognized by the humoral immune system in patients with cancer.

A random nanopeptide phage library was used to screen a pool of immunoglobulin fractions obtained from rheumatoid arthritis (RA) patients. After three rounds of panning, random individual phages were selected by their capacity to react with individual sera from RA patients. By sequencing the inserts corresponding to the peptides displayed on the surface of the phages, we found that phages displaying particular peptides were overrepresented in the selected libraries. The peptides displayed by these phages were: pep1 = Ala-Asp-Gly-Gly-Ala-Gln-Gly-Thr-Ala; pep2 = Pro-Gly-Pro-Ser-Arg-Ala-His-Phe-Leu; pep3 = Leu-Ser-Ser-Arg-Glu-Pro-Gln-Ala-Arg; pep4 = Arg-Leu-Thr-Arg-Glu-Leu-Tyr-Ala-Gln and pep5 = Tyr-Thr-Gln-Lys-His-Gln-Ala. The percentage of sera positive for pep1 was higher in RA patients as compared to the normal adults (p 0.0004) and the reacting antibody was mainly of IgG isotype. The specificity of binding to the phage displaying pep1 was confirmed by competition experiments using both isolated phages and a synthetic peptide. Interestingly, a mutated phage displaying only Ala-Asp-Gln-Gly-Thr-Ala had no significant reactivity with the sera, indicating that the amino acids (Gly-Gly-Ala) of pep1 are the vital for the binding. Taken together this study demonstrates that it is possible to select specific ligands from a random phage library using sera from RA patients. In addition, this approach could be useful for identifying peptide antigens that might be part of causitive agents in autoimmune diseases.

In rheumatoid arthritis and other inflammatory diseases we and others have found that gamma delta T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate gamma delta T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the gamma delta but not the alpha beta T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the gamma delta T cells and a corresponding reduction of the fraction of alpha beta T cells. Limiting dilution analysis revealed that approximately 1/60 of the gamma delta T cells responded to IL-2 in contrast to only 1/250 of the alpha beta T cells. Comparison of the expression of the IL-2 receptor (IL-2R) alpha and beta chains showed that there was a similar expression of the alpha chain on gamma delta and alpha beta T cells whereas the relative density of the beta chain was more than twice as high on gamma delta T cells. Both the IL-2-induced proliferation of gamma delta T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2R beta monoclonal antibody (mAb) but not by an anti-IL-2R alpha mAb. Expression of CD69 on gamma delta T cells was dependent neither on the presence of B cells, monocytes, nor alpha beta T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of gamma delta T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on gamma delta T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated alpha beta T cells.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.

We have investigated both the humoral and the cellular immune responses of patients with juvenile rheumatoid arthritis (JRA) and rheumatoid arthritis (RA) to mycobacterial antigens. The JRA group was not Bacillus Calmette Guerin (BCG) vaccinated whilst the majority of the RA group was. As determined by immunoblotting, 79% of sera from patients with JRA reacted mainly with a 18.6-kDa protein (P18.6), whilst 70% of sera from patients with RA reacted mainly with a 30-kDa protein (P30) of BCG, M. tuberculosis and M. kansasii. In contrast, only a moderate proportion of the control sera (25% of adult and 20% of children) showed reactivity to P30, and none of the samples had significant reactivity with the P18.6 antigen. Furthermore, T-cell proliferation to the P18.6 and P30 antigens was detected in the majority of JRA and RA patients, and was nearly always higher in synovial fluid (SF) than in the peripheral blood (PB). We also investigated the usage of V beta family genes in P18.6 and P30 antigen-specific T-cell lines established from the SF of one patient with active RA. We showed that V beta 2, -4, -5, -6, -7, -14, -17, -18 and V beta 19 were over-represented compared with other known V beta families. We also noted that the proportion of V beta 14 was higher in freshly isolated SF mononuclear cells compared with the blood in this patient and in 2 out of 4 other RA patients examined. Other V beta families such as V beta 6, V beta 8, V beta 16, V beta 18 and V beta 19 were also over-represented in the SF compared with the blood in some patients. Taken together our results provide more information concerning the role of mycobacterial antigens in RA and suggest that there may be an in vivo clonal expansion of T lymphocytes in the synovium.

It has recently been shown that TLR are capable of regulating the differentiation of haematopoietic progenitor cells. Specifically, R848 (resquimod, a specific TLR7/8 agonist) is capable of stimulating the differentiation of human bone marrow CD34+ progenitors into myeloid DC. Sioud and Floisand (pp. 2834–2846) investigate the differentiation induced by other TLR agonists (loxoribine (TLR7) and Pam3CSK4 (TLR2)), as well as further investigating the effects of R848. The cover image (a composite, artificially coloured figure) is based on May-Grunwald-Giesma staining of the CD11c– CD14– (green section of the image), CD11c+ CD14+ (yellow section) and CD11c+ CD14– (red section) cell subsets induced following stimulation of CD34+ progenitors with R848 for seven days. Importantly, around 30% of cells of the CD11c+ CD14– subset display DC morphology (multiple fine dendrites) and, furthermore, this CD11c+ CD14– subset is shown to be capable of activating allogeneic T cells.