Reduced CTLA-4 signaling predisposes to Th2 driven gastric tumorigenesis

Anti-CTLA4 antibodies such as Yervoy, has been used in clinical practice to treat solid tumors. It supposed to work either by augmenting and revitalizing effector T cells function directly or indirectly through temporal silencing of inhibitory Foxp3+ Treg population or both. However, new study from Journal of Experimental Medicine showed that at least in [genetically predisposed] mice reduced CTLA-4 signaling by itself could cause Th2 driven tumorigenic transformation of stomach epithelial tissue.

For this study the authors created transgenic CTLA4 shRNA knockdown (CTLA4KD) mice on the BALB/c × C57BL/6 (B6) mixed genetic background. This they did because it appears that BALB/c but not B6 mice were susceptible developing gastric tumors in this model. CTLA4KD mice showed gastric epithelial transformation by 20w of age. Similarly, month long treatment of newborn BALB/c mice with anti-CTLA4 antibody also led to gastric epithelial transformation.

This tumorigenic transformation was CD4 T cell dependent and effector T cells from CTLA4KD but not from WT mice could mediate it. It indicated that changes in effector T cell composition and functionality were driving de novo inflammatory tumorigenesis.

Interestingly, gastric epithelial transformation were happening even in germ-free CTLA4KD mice lacking microbiota. However, since these mice also harbor increased numbers of inflammatory T cells, in all subsets analyzed such as Th1, Th2, Th17, and independent of microbiota it could indicate that T cells could be responding to antigens from food or environment.

Finally, elimination of canonical T helper cytokines showed that surprisingly neither IFN-γ nor IL-17 but IL-4 deficiency could abolish gastric epithelial transformation under conditions of reduced CTLA-4 activity.

In summary, this study suggests that inherited or clinically-induced reduction of CTLA-4 signaling in predisposed individuals could paradoxically lead to inflammatory tumorigenesis driven by type II immunity.

posted by David Usharauli

Gut microbes fuel effectiveness of anti-CTLA-4 cancer immunotherapy. Part I

Recently two back-to-back papers in Science Express received much media attention. These studies claim that gut microbial flora influence clinical effectiveness of checkpoint inhibitors approved for cancer immunotherapy. I am going to review and provide my analysis of these studies.

First paper I will review came from European team led by Laurence Zitvogel. Of note, her team had another tumor immunology paper just 1 week ago in Science. First notable thing about her publications is the number of names included as authors. Her lab had done this since I first heard about her studies in 2007. Either she is a good collaborator or everyone in her lab is get credit for everyone else's work as a matter of right [definitely good for career].

In this new paper, her team showed that effectiveness of anti-CTLA-4 antibody in cancer immunotherapy was greatly affected by presence of gut microbiota.

For example, they showed that inhibition of cancer growth by anti-CTLA-4 antibody injected in germ-free or antibiotic-treated mice was not as good as in control WT mice. 

Analysis of gut flora revealed that presence of specific set of microbes [B. fragilis, for example] were required to show anti-cancer effectiveness of anti-CTLA-4 antibody.

Now these result are quite preliminary and it is not clear how microbes are enhancing anti-CTLA-4 effectiveness. For example, some microbes can live within growing tumor tissue and anti-CTLA-4 therapy simply unleashes immune response against them destroying tumor tissue in the process. There is a possibility that microbes support presence of cross-reactive T cells required to target tumor antigens when they are unleashed by anti-CTLA-4 immunotherapy. In all, based on this paper we can't say how microbes help anti-CTLA-4 and how much this "help" is meaningful in clinical settings. Maybe second paper will provide some ideas in this regard. Stay tuned for next review. 

David Usharauli   

CTLA-4 deletion during adulthood leads to a paradoxical resistance to autoimmune disease

CTLA-4 is a T cell inhibitory molecule that competes with CD28 for binding CD80/CD86 co-stimulatory molecules on dendritic cells. It is believed that CTLA-4 functions as a "brake" for T cell activation, a function also referred as a "checkpoint" inhibition. Total genetic deficiency of CTLA-4 or just on Foxp3+ regulatory T cells leads to early onset wasting syndrome and fatal autoimmune diseases. This what we knew up to now. 

However, a new study in Journal of Experimental Medicine, has put upside down the whole concept of "checkpoint" inhibition for CTLA-4. Using genetically-modified mouse models, the authors reported that total or Foxp3+ regulatory T cell-specific conditional deficiency of CTLA-4 in adult mice leads to protection rather than acceleration of autoimmune disease.   

First, the authors have generated mouse model that allowed conditional deletion of total or Foxp3+ Treg-specific CTLA4 (UBC^Cre/ERT2+ Ctla4^fl/fl and Foxp3^{eGFP/Cre/ERT2+} Ctla4^fl/fl).

Next, the authors treated mice with tamoxifen to activate Cre recombinase and delete CTLA4 and challenged mice with myelin self-peptide to induce experimental autoimmune encephalitis (EAE). Surprisingly and contrary to widely held belief, adult mice conditionally deficient for CTLA-4 were highly resistance for EAE induction.

Similar resistance for EAE induction was observed with mice with adult-onset CTLA4 deletion specifically on Foxp3+ regulatory T cells.

To rule out any off-target effect of CTLA-4 deletion, the authors transferred T cells from UBC^Cre/ERT2+ Ctla4^fl/fl into T-deficient hosts and then challenged with tamoxifen and myelin self-peptide. Here too, deletion of CTLA4 on mature T cells protected against EAE. Moreover, adoptive transfer of purified, naive Foxp3- T cells from UBC^Cre/ERT2+ Ctla4^fl/fl mice or transgenic 2D2 T cells specific for myelin self-peptide on UBC^Cre/ERT2+ Ctla4^fl/fl background were capable of inducing EAE, implying that T cells were not inherently incapable of inducing EAE but were actively suppressed by absence of CTLA-4 on Foxp3+ regulatory T cells.

Additional experiments revealed that CTLA-4 deletion in adulthood skewed T cells response towards IL-10 production in UBC^Cre/ERT2+ Ctla4^fl/fl mice.

Finally, the authors showed that CTLA-4 deletion during adulthood did not modify anti-tumor response against MC38 colon adenocarcinoma cells in UBC^Cre/ERT2+ Ctla4^fl/fl mice.

In summary, these results question the assumption that inhibition of CTLA-4 function during adulthood would lead to autoimmune disease. Based on these results, it is unclear how anti-CTLA4 antibody therapy provides benefits during tumor immunotherapy. It maybe the difference between mouse and human immune system or it could be that mechanism of action of anti-CTLA4 antibody is not a "checkpoint inhibition" after all.

David Usharauli

Enigma of the peripheral tolerance: CTLA4+ anergic CD8 T cells

When Sir Burnet first proposed clonal-selection theory of everything to explain how immune system could detect non-self antigens while at the same time avoiding autoimmunity, he probably had no idea that his theory would require major and multiple modifications to accommodate new data.

This new study from journal Science is another piece in the puzzle. This work, led by Shimon Sakaguchi at Osaka University, studied self-reactive CD8 T cells from the healthy humans or from vitiligo patients's blood.

The authors studied self-antigen, melanin-specific CD8 T cells. In T cell proliferation assay, Melan-A specific CD8 T cells, almost undetectable before stimulation, undergo robust, multiple-round proliferation and expand to sizable population. However, in presence of regulatory T cells, Melan-A specific CD8 T cells undergo single-round, abortive proliferation.

Tetramer staining indicated that in presence of regulatory T cells, Melan-A specific CD8 T cells mostly consisted of low-affinity clones and displayed anergic phenotype with low cytokine expression (however, please note that small population of CD8 T cells displayed high affinity binding to tetramers even in presence of regulatory T cells. This suggest to me that regulatory T cells suppress proliferation of these high-affinity Melan-A specific CD8 T cells).

Surface staining showed that anergic CD8 T cells generated in presence of T regs express high levels of CTLA-4 and CCR7.

Direct ex vivo analysis of healthy human CD8 T cells showed that naive CD8 T cells consisted of CTLA4+ and CTLA4- population. Antigen-specific or non-specific stimulation of these two population showed that CTLA4+ naive CD8 T cells were (1) enriched in self-specific CD8 T cells, (2) displayed anergic phenotype (abortive proliferation) and (3) preferentially undergo apoptosis in mixed culture.

In summary, the authors showed that healthy human blood contains two types of "naive" CD8 T cell populations: naive, CTLA4neg functionally-competent CD8 T cells and anergic, CTLA4pos functionally-impaired CD8 T cell population.

It appears, though there is no direct evidence, that T regs control self-antigen specific CTLA4+ anergic CD8 T cell development from high-affinity clones. If the model is accurate, then it is not clear why healthy human blood still contains high affinity, self-antigen specific CTLA4- CD8 T cell population (see Fig. 1b tetramer staining. I wonder whether small population of high-affinity CD8 T cells in Treg culture express CTLA4). 

David Usharauli