A combination of BR101801 and venetoclax enhances antitumor effect in DLBCL cells via c-Myc/Bcl-2/Mcl-1 triple targeting.

Double hit diffuse large B-cell lymphoma (DLBCL) with rearrangement and overexpression of both c-Myc and Bcl-2 responds poorly to standard R-CHOP therapy. In a recent phase I study, Venetoclax (ABT-199) targeting Bcl-2 also exhibited disappointing response rates in patients with relapsed/refractory DLBCL, suggesting that targeting only Bcl-2 is not sufficient for achieving successful efficacy due to the concurrent oncogenic function of c-Myc expression and drug resistance following an increase in Mcl-1. Therefore, co-targeting c-Myc and Mcl-1 could be a key combinatorial strategy to enhance the efficacy of Venetoclax. In this study, BR101801 a novel drug for DLBCL, effectively inhibited DLBCL cell growth/proliferation, induced cell cycle arrest, and markedly inhibited G0/G1 arrest. The apoptotic effect of BR101801 was also observed by increased Cytochrome C, cleaved PARP, and Annexin V-positive cell populations. This anti-cancer effect of BR101801 was confirmed in animal models, where it effectively inhibited tumor growth by reducing the expression of both c-Myc and Mcl-1. Furthermore, BR101801 exhibited a significant synergistic antitumor effect even in late xenograft models when combined with Venetoclax. Our data strongly suggest that c-Myc/Bcl-2/Mcl-1 triple targeting through a combination of BR101801 and Venetoclax could be a potential clinical option for double-hit DLBCL.

Green tea protects rats against autoimmune arthritis by modulating disease-related immune events.

Green tea, a product of the dried leaves of Camellia sinensis, is the most widely consumed beverage in the world. The polyphenolic compounds from green tea (PGT) possess antiinflammatory properties. We investigated whether PGT can afford protection against autoimmune arthritis and also examined the immunological basis of this effect using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA). AA can be induced in Lewis rats (RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis H37Ra (Mtb), and arthritic rats raise a T cell response to the mycobacterial heat-shock protein 65 (Bhsp65). Rats consumed green tea (2-12 g/L) in drinking water for 1-3 wk and then were injected with Mtb to induce disease. Thereafter, they were observed regularly and graded for signs of arthritis. Subgroups of these rats were killed at defined time points and their draining lymph node cells were harvested and tested for T cell proliferative and cytokine responses. Furthermore, the sera collected from these rats were tested for anti-Bhsp65 antibodies. Feeding 8 g/L PGT to Lewis rats for 9 d significantly reduced the severity of arthritis compared with the water-fed controls. Interestingly, PGT-fed rats had a lower concentration of the proinflammatory cytokine interleukin (IL)-17 but a greater concentration of the immunoregulatory cytokine IL-10 than controls. PGT feeding also suppressed the anti-Bhsp65 antibody response. Thus, green tea induced changes in arthritis-related immune responses. We suggest further systematic exploration of dietary supplementation with PGT as an adjunct nutritional strategy for the management of RA.

T cells against the pathogenic and protective epitopes of heat-shock protein 65 are crossreactive and display functional similarity: novel aspect of regulation of autoimmune arthritis.

OBJECTIVE: In autoimmune situations, the outcome of immune response against a disease-related antigen is typically viewed in terms of the balance between the pathogenic versus the protective subsets of antigen-reactive T cells. Using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA), we examined the antigen specificity and the functional attributes of the T cell repertoire directed against defined pathogenic versus protective epitopes of heat-shock protein 65 (hsp65), and determined the immunologic basis of the AA-protective effect of subsets of T cells primed by the pathogenic determinant. METHODS: Lewis (RT.1l) rats were pretreated subcutaneously with the pathogenic epitope 177-191 of mycobacterial hsp65 (B177) in adjuvant (incomplete Freund's adjuvant/complete Freund's adjuvant/CpG) and then immunized with heat-killed M. tuberculosis H37Ra for disease induction. The antigen specificity/crossreactivity of the T cells primed by B177 or the AA-protective determinant 465-479 of the homologous rat hsp65 (R465) was tested by using proliferation assay, cytokine ELISA, tolerance induction, and adoptive transfer. RESULTS: Pretreatment of Lewis rats with the arthritogenic determinant B177 using an immunogenic rather than a tolerogenic regimen affords protection against AA instead of initiation or aggravation of AA. This protective effect of B177 is mediated in part by activation of T cells that are crossreactive with R465. CONCLUSION: Downmodulation of AA by a pathogenic foreign epitope involving T cells crossreactive with a distant, protective self-determinant represents a novel aspect of immune regulation, and suggests further exploration of the use of pathogenic epitopes for the treatment of autoimmune arthritis.

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis.

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.

Prevention of spontaneous arthritis by inhibiting homeostatic expansion of autoreactive CD4+ T cells in the K/BxN mouse model.

OBJECTIVE: K/BxN-transgenic mice are a model of autoimmune arthritis, similar to rheumatoid arthritis. This study was undertaken to determine whether inhibition of lymphopenia-provoked homeostatic expansion can prevent spontaneous development of disease in the K/BxN model. METHODS: To inhibit homeostatic expansion of autoreactive T cells, K/BxN mice with disease in the preclinical stage were adoptively transferred with CD4+ T cells purified from nontransgenic BxN or Thy1.1+ BxN mice. To observe the profile of proliferation of CD4+ T cells derived from the hosts, carboxyfluorescein diacetate succinimidyl ester-labeled autologous CD4+ T cells were cotransferred to K/BxN mice together with BxN CD4+ T cells. Disease onset and progression were scored, and the dynamics and phenotypes of recipient CD4+ T cells were determined by flow cytometry, before and after cell infusion. RESULTS: During the preclinical phase of disease, K/BxN mice exhibited CD4+ T lymphopenia, which was followed by a compensatory expansion of these cells during the early clinical phase. The majority of CD4+ T cells acquired a memory phenotype (CD44(high),CD62L(low),CD25-), which is a hallmark of homeostatically expanding cells. Importantly, K/BxN mice subjected to syngeneic T cell transfer did not develop symptoms of arthritis and also possessed fewer transgenic T cell receptor-encoded Vbeta6+,CD4+ T cells. This effect was associated with decreased proliferation of recipient-derived CD4+ T cells but not with the function of CD25+ T regulatory cells present in donor cells. CONCLUSION: These results provide the first evidence that lymphopenia-associated homeostatic proliferation of autoreactive CD4+ T cells potentiates autoimmune arthritis, and that inhibition of this process protects mice from the development of this pathologic condition.

Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis.

Dimethyl dioctadecyl ammonium bromide (DDA) (C(38)H(80)NBr) is a nonantigenic lipoid material. DDA-induced arthritis (DIA) in the Lewis (LEW) (RT.1(l)) rat is a new experimental model for human rheumatoid arthritis (RA). DIA is a T cell-mediated autoimmune disease. However, the precise self/foreign Ags associated with the disease process in DIA are not yet known. We observed that LEW rats with DIA spontaneously raised a vigorous T cell response both to 65-kDa self (rat) heat shock protein (Rhsp65) and mycobacterial hsp65 (Bhsp65), but not to another arthritis-related Ag, bovine collagen type II. The T cell response to Rhsp65 was focused predominantly on determinant regions 120-134 and 213-227 of the self protein. Interestingly, pretreatment of adult LEW rats using either a mixture of peptides 120-134 and 213-227 of Rhsp65 or a low nonarthritogenic dose of DDA induced protection against subsequent DIA. Intriguingly, the protection induced by the latter was associated with spontaneous priming of T cells specific for peptide 213-227 of Rhsp65. Similarly, LEW rats neonatally tolerized against either Rhsp65 or Bhsp65 were significantly protected from subsequently induced DIA at adult stage, showing the disease-modulating attribute of the hsp65-specific T cells. Taken together, the above findings demonstrate that the hsp65-directed T cell repertoire is of significance in the pathogenesis of autoimmune arthritis induced by nonantigenic DDA. Like other animal models of RA involving hsp65, these first insights into the disease-associated Ags in the DIA model would pave the way for further understanding of the immunological aspects of induction and regulation of RA.

The regulatory C-terminal determinants within mycobacterial heat shock protein 65 are cryptic and cross-reactive with the dominant self homologs: implications for the pathogenesis of autoimmune arthritis.

The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in the Lewis rat (RT.1(l)) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD, but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis.