Saturday, June 16, 2018

First-born advantage of early produced CD8 T cells

During immune response to infection or vaccine antigen-specific T cells differentiate into various categories of effector/memory population. This is a stochastic process that follows some not yet fully understood "rules". New study in Cell suggests that one of those rules is a "date of birth" of individual CD8 T cells that participate in immune response. 

For this study the authors used genetically modified mice where T cells could be permanently color-labeled after injection of tamoxifen (CD4 promoter-driven tamoxifen-inducible CRE mice, CD4cre-ERT2). When compared to each other, CD8 T cells produced early on during neonatal stage (day 1-7) harbored high proportion of cells with innate-like phenotype (CD44+/CD122+) than those CD8 T cells produced after day 28.

Similar phenotype was maintained even in mice where both day 1 and day 28 CD8 T cells were produced in the same mouse (following neonatal RFP+ thymus transplantation into YFP+ adult mice and analyzed at indicated time period post transplantation).

Notably, early-born CD8 T cells were characterized by heightened sensitivity to innate cytokines such IL-18 and IL-12 and rapid initial response to cognate antigen (between day 1-7 post-infection).

The authors opined that various categories of effector/memory T cells observed in other studies should be re-interpreted in light of "layered" CD8 T cells wherein CD8 T cells of different "date of birth" are producing different effector/memory T cell population.

It is certainly interesting observation. But there are several missing opportunities in this study. First, due to their innate-like phenotype, the authors should have looked at the role of microbiota in imprinting these features of early-born CD8 T cells. Second, it is not clear and the authors did not examined if biologically, lack of early-born vs. adult-born CD8 T cells, would have modified the host's response to infection. Third, the authors did not analyze whether early-born versus adult-born CD8 T cells differed in their TCR profile (even using transgenic CD8 T cells on WT background is not proper control). Fourth, different effector/memory categories have been produced by injection of a single CD8 T cells so individual CD8 T cells can indeed produce diverse phenotype of effector/memory T cells. 

posted by David Usharauli

Saturday, June 2, 2018

Monocyte-derived cytokines IL-1 / IL-6 contribute to CAR-T cell-induced cytokine-release syndrome and neurotoxicity

This week Nature Medicine published two papers showing in mouse CAR-T model that cytokine-release syndrome (in both studies) and neurotoxicity (in one study only) were primarily driven by IL-1 and IL-6 cytokines released by monocytes following interaction with infused CAR-T cells. 

In the first study led by Michel Sadelain at Sloan Kettering Institute, New York, immunodeficient SCID-beige mice transplanted intra-peritoneally with human B cell tumor cell line and later infused with CD19 CAR-T cells develop cytokine-release syndrome (CRS) that were reversible by anti-IL-6 antibody,  injection.  

Injection of anti-IL-1 antibody, Anakinra, had similar protective effect against CRS-driven mortality without compromising anti-tumor effectiveness. 

However, due to some limitation of their mouse tumor model where CAR-T cells are of human origin and non-T cells such as monocytes are of mouse origin, the authors acknowledged that not all features of CAR-T cell toxicity could be reproduced here since "None of the reported pathologic findings indicative of neuropathology or associated with neurotoxicity (cortical laminar necrosis, hemorrhages, disseminated intravascular coagulation (DIC), gliosis or vasogenic, neurotoxic or interstitial edema) in human patients were observed in any of the mice examined in the present study".   

Fortunately, the second study is more extensive and fills much of holes of the first study. Here, the authors led by Attilio Bondanza at San Raffaele Hospital Scientific Institute, Milano, "transplanted human cord blood (CB) hematopoietic stem and progenitor cells (HSPCs) through intrahepatic injection into sublethally irradiated newborn NSG or triple transgenic NSG (SGM3) mice expressing human stem cell factor, granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-3 and initially profiled lymphohematopoietic reconstitution." Basically, this mouse model, referred as newborn humanized SGM3 (nHuSGM3), had both T cell and non-T cell components of hematopoietic system mostly of human origin. In allogeneic tests, human T cells developed in nHuSGM3 mice showed expected functionality. 

Staining of cells in nHuSGM3 challenged with tumor and CAR-T cells that induced CRS showed that monocytes were producing IL-1 early on, followed by IL-6

Depletion of monocytes/macrophages with liposomal clodronate (LC) could rescue mice from CRS.

Importantly, while both anti-IL-1 and anti-IL-6 antibody injection could significantly reduce CRS, only anti-IL-1 antibody were able to reduce neurotoxicity in nHuSGM3 mice. Similar effect were seen with CD44v6 CAR-T cells as well.

In all, these two studies showed that in addition of anti-IL-6 injection, anti-IL-1 antibody therapy could significantly reduce complications of CAR-T cell immunotherapy in humans. They provided a strong evidence to suggest that monocytes were primarily responsible for CRS and neurotoxicity complications of CAR-T cell therapy. 

However, it is not clear why are monocytes getting activated after CAR-T cell infusion in the first place. There is interaction, at certain level, between CAR-T cells and monocytes, but is it antigen-specific via anti-CD19 CAR-T or endogenous TCR, or is it non-specific, is unknown presently. 

posted by David Usharauli 

Wednesday, May 23, 2018

Flagellin-specific T cells induce colitis by recognizing antigen other than flagellin

This is very interesting study from Journal of Immunology (JI). Here, researcher led by Timothy Hand at the University of Pittsburgh Medical School, showed that CBir1 transgenic T cells, thought to cause gut inflammation by recognizing flagellin expressing microbiota, were in fact specific for non-flagellin antigens

CBir1 transgenic mouse have been used for past 10 years to model human Crohn's disease in mice. CBir1 tetramer+ T cells recognize epitope from flagellin, antigen thought to be a target in Crohn's disease. Interestingly, all of those experiments were done using CBir1 T cells on WT background. This could be an issue because it has been known for some time now that transgenic T cells on WT background could use alternative Vα chain to form TCR with new specificity.

To avoid such limitation, here the authors generated CBir1 mouse on RAG KO background that only expressed transgenic Vα:Vβ chains. Surprisingly, unlike CBir1:WT T cells, CBir1:RagKO T cells when transferred in immunodeficient hosts did not induce colitis, and it was not because CBir1:RagKO T cells differentiate into Foxp3+ T cells. It appears that CBir1:RagKO T cells just did not see flagellin epitope in adoptive host.

In vitro tests showed that both CBir1:WT T cells and CBir1:RagKO T cells could respond to DCs pulsed with flagellin epitope [it would have been better and more relevant here to use DCs pulsed with gut flora component].

Other tests showed that CBir1:WT T cells in gut tissue could express alternative Vα chain to form a completely new TCR specificity together with transgenic Vβ chain such as against Ags derived from Helicobacter (HH1713 tetramer). 

In summary, it appears that CBir1 T cells initiate colitis by recognizing non-flagellin antigen from the gut flora through non-CBir1 TCR and only following gut inflammation and gut leakage do flagellin-specific CBir1 transgenic T cells get activated and participate in overall colitis.

So, what is missing from this study? One, it would have been relevant to transfer in vitro activated CBir1:RagKO T cells or activate them in vivo directly to see if then they could initiate colitis. Second, the authors could have tried monocolonization of germ-free mice to see the source of non-flagellin microbiota. Third, there is inconsistency between Fig. 3B and Fig. 6A with regard of proliferation of  CBir1:WT T cells in response to Vanc-treated samples (in vitro it did not proliferate but in vivo it did).

posted by David Usharauli