CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment

Reviewer #1 (Public Review):

The manuscript by Muthana et al. describes the effect of injection of an antibody specific for human CTLA4 conjugated to a cytotoxic molecule (Ipi-DM1) in knock-in mice expressing human CTLA4. The authors show that Ipi-DM1 administration causes a partial decrease (about 50% in absolute number) of mature B cells in blood and bone marrow 9-14 days after the beginning of treatment. Ipi-DM1 also results in a partial decrease in Foxp3+ Tregs (about 40% in absolute number) and a slight increase in activation of conventional T cells (Tconvs) in the blood at D9. Tconv depletion, CTLA4-Ig or anti-TNF mAb partially prevents the effect of ipi-DM1 on B cells. This work is interesting but has the following major limitations:

1- This work could have been of more interest if the Ipi-DM1 molecule would be used in the clinic. As this is not the case, the intimate mechanism of the effect of this molecule in mice is of reduced interest.
2- The fact that a partial deletion of Tregs is associated with activation of Tconvs and a decrease in B cells has been published several times and is therefore not new. According to the authors, their work would be the first to show that activation of Tconvs would lead to B cell depletion. However, this is shown in an indirect way and the mechanisms are not really elucidated. Indeed, this work shows a correlation between an increase in Tconv activation and a decrease in the number of B cells in the blood. The experiments to try to show a causal link are of 2 types: deletion of T cells (Fig 4) and blocking T cell activation with CTLA4-Ig (Fig 5) (neutralization of TNF addresses another question). Neither of these 2 experiments is totally convincing. Indeed, the absence of B cell depletion when T cells are deleted can be explained by other mechanisms than the preservation of B cell destruction by activated T cells. The phenomenon could be explained by B cell recirculation to lymphoid tissues or an effect of massive T cell death for example. The experiment shown in Fig. 5 with Belatacept is more convincing because this time the effect is targeted to activated T cells only. However, the prevention of B cell ablation is only partial. Again, since only blood is analyzed, other mechanisms could explain the B cell loss, such as their recirculation in lymphoid tissues.
3- It is disappointing that only the blood (and sometimes the bone marrow) was studied in this work. The interest of doing experiments in mice is to have access to many tissues such as the spleen, lymph nodes, colon, lung, and liver. To conclude that there is B cell deletion without showing lymphoid organs (where the majority of B cells reside) is insufficient. As discussed above, the drop in B cells in the blood could be due to their recirculation in lymphoid organs. In addition, there is no measurement of functional B cells activity. Do mice treated with Ipi-DM1 have a decreased ability to develop an antibody response following immunization?
4- Although it is difficult to study in vivo, there is not a single evidence of increased B cell death after injection of Ipi-DM1.
5- In most of the experiments, B cells are quantified with the B220 marker alone, but this marker, in some cases, can be expressed by other cells. It would have been preferable to use a marker more specific to B cells such as CD19 for example.

In conclusion, the concept that T cell activation can lead to B cell deletion is interesting but this study shows it only in an indirect and incomplete way.

Reviewer #2 (Public Review):

Despite the fact that CTLA-4 is a critical molecule for inhibiting the immune response, surprisingly individuals with heterozygous CTLA-4 mutations exhibit immunodeficiency, presenting with antibody deficiency secondary to B cell loss. Why the loss of a molecule that regulates T cell activation should lead to B cell loss has remained unclear. In this study, Muthana and colleagues use an anti-CTLA-4 antibody drug conjugate (aCTLA-4 ADC) to delete cells expressing high levels of CTLA-4, and show that this leads to a reduction in B cells. The aCTLA-4 ADC is found to delete a subset of Tregs, leading to hyperactivation of T cells that is associated with B cell depletion. Using blocking antibodies, the authors implicate TNFa in the observed B cell loss.

The reciprocal regulation of T and B cell homeostasis is an important research area. While it has been shown that Treg defects are associated with B cell loss, the mechanisms at play are incompletely understood. CTLA-4 is not normally expressed in B cells so an indirect mechanism of action is assumed. The authors show that the decrease in Treg following aCTLA-4 ADC treatment is associated with activation of T cells, and that B cell loss is blunted if T cells are depleted. A role for both CD4 and CD8 T cells is identified by selective CD4/CD8 depletion. T cells appear to require CD28 costimulation in order to mediate B cell loss, since the response is partially inhibited in the presence of the costimulation blockade drug belatacept (CTLA-4-Ig). Finally, experiments using the anti-TNFa antibody adalimumab suggest a potential role for TNFa in the depletion of B cells.

While the manuscript makes a useful contribution, a number of questions remain. Perhaps most important is the extent to which this model mimics the natural situation in individuals with CTLA-4 mutations (or following CTLA-4-based clinical interventions). aCTLA-4 ADC treatment permits acute deletion of Treg expressing high levels of CTLA-4, whereas in patients the Treg population remains but is specifically impaired in CTLA-4 function. Secondly, although the requirement for T cells to mediate B cell loss is convincingly demonstrated, the incomplete reversal by TNFa blockade suggests additional unidentified factors contribute to this effect. Finally, although the manuscript favours peripheral killing of mature B cells over alterations to B cell lymphopoiesis, one concern is that this may simply reflect the model employed: the short-term (6 day) treatment used here may be too acute to alter B cell development, but this may nevertheless be a feature of prolonged immune dysregulation in humans.

Reviewer #3 (Public Review):

The co-suppressive molecule CTLA-4 has a critical role in the maintenance of peripheral tolerance, primarily by Treg mediated control of the co-stimulatory molecules CD80 and CD86. As stated by the authors, previous studies have found a variety of effects of anti-CTLA-4 antibody treatment or genetic loss of CTLA-4 on B-cells. These include increased B-cell activation and antibody production, autoantibody production, impairment of B-cell production in the bone marrow and loss of peripheral B-cells. In this article Muthana et al use a CTLA-4 humanized mouse model and examine the effects of drug conjugated CTLA-4 on the immune system. They observe a transient loss of B-cells in the blood of the treated mice. They then use a range of immune interventions such as T-cell depletion and blocking antibodies to demonstrate that this effect is dependent on T-cell activation.

Since anti-CTLA-4 immunotherapy is in active clinical use exploration of its effects are welcome, this is helped by the use of a humanized CTLA-4 system which should be considered a strength of the paper. However, currently, the central premise of this paper, that B-cells are depleted, seems underexplored. Direct evidence of T-cell killing of B-cells is never presented, rather it is inferred from the reduced numbers of B-cells in the blood. The status of B-cells in sites that contain a large proportion of B-cells such as the spleen and lymph nodes is not examined. Additionally, no examination of B-cell antibody production is performed.