Anti-tumor effect of adoptive CD4 T cells positively correlates with high precursor frequency

It is now well accepted that similar to cytotoxic CD8 T cells, antigen-specific effector CD4 T cells can show a direct anti-tumor effect both in  mice and humans. This concept is still relatively new (actually first paper about it was published only in 2003). Incorporation of CD4 T cells and MHC II + peptides in tumor immunotherapy strategy increases chances of finding tumor-specific antigens (epitopes) relevant for personalized cancer medicine.

In this regard, new paper from journal Immunity is of interest. There, the authors showed that unlike CD8 T cells, intra-clonal competition of adoptive transfer CD4 T cells [at high precursor frequency] improved rather than diminished its anti-tumor effect and productive effector differentiation.  

In this study, the authors transferred different number (10³, 10⁴, 10⁵, 10⁶) of melanoma antigen, TRP-1 specific CD4 T cells into mice implanted with melanoma. Despite significant expansion at low precursor frequency, only high frequency transferred TRP-1 specific CD4 T cells managed to control and eradicate established tumors.

Moreover, the authors showed that in this settings, only TRP-1 specific CD4 T cells from high frequency adoptive transfer hosts underwent productive effector differentiation.

Even addition of anti-PD1 antibody to [low frequency] TRP-1 specific CD4 T cell adoptive transfer hosts failed to rescue their differentiation (increase in IL-21 is a marker of CD4 T cell exhaustion).

In summary, this study suggests that for CD4 T cells intra-clonal competition does not prevent efficient effector differentiation and establishment of productive anti-tumor immunity. It appears that CD4 T cells undergoing significant expansion at low precursor frequency develop "early and checkpoint inhibition-refractory exhaustion" preventing them to participate in effective anti-tumor response. This knowledge should be taken into account when considering adoptive T cell tumor therapy and checkpoint inhibition (according this study, anti-PD1 therapy could potentially accelerate tumor-specific CD4 T cell exhaustion at low precursor frequency).

David Usharauli

Determinants of T cell exhaustion during chronic viral infection and autoimmunity

Typically when we think about ordinary immune response, we imagine several finite steps involving T and B cells, such as antigen recognition and initiation of antigen-specific clonal expansion, control of antigenic spread and elimination of infected cells and finally return to quiescent state and memory establishment.    

This scenario represents what ordinary is called immune response to transient (acute) antigenic stimulation. However, how does host immune system respond to persistent (chronic) antigenic stimulation, for example, chronic viral presence (HIV, HVB, HCV, Malaria, TB) or autoimmune disease (self-antigens)? Are the mechanisms that control immune response similarly activated during acute or chronic antigenic presence?

As you can see many infectious diseases with no effective vaccines fall exactly in the category of chronic infections (HIV, HBV, HCV, Malaria, TB). This is not a random outcome. There should be some biological or immunological underpinning to account for our failure to develop such vaccines. 

This new paper in journal Nature provided some additional results that may help us to better understand mechanisms controlling chronic immune responses. To tell the truth, it is quite difficult-to-digest article with lot of large data set analyses. The paper was under review for more than 1 year and its main finding regarding molecule KAT2B isn't even mention in their abstract. Strange. So we just need to assume that their analyses are done correctly and are statistically valid.

In this paper the authors tried to correlate CD8 T cell exhaustion phenotype with the clinical outcome (flare-free survival) of patients suffering from various autoimmune diseases. Murine chronic LCMV infection-associated CD8 T cell exhaustion phenotype was used as a reference. 

First, the authors noticed that unlike coordinated up-regulation of several inhibitory receptors during murine chronic LCMV infection, CD8 T cell phenotype from patients with autoimmune diseases displayed distinct disease-selective up-regulation of exhaustion-associated inhibitory receptors.

Interestingly, for each patient with autoimmune diseases, CD8 T cell exhaustion phenotype correlated with a favorable prognosis.

Next, in vitro experiments showed that fine balance between incoming co-stimulatory (e.g. CD2) and co-inhibitory (e.g. PD-L1) signals may determine exhaustion phenotype of CD8 T cells during persistent antigenic stimulation (one caveat: anti-CD3, anti-CD28 or anti-CD2 antibody stimulation are not physiological mode of activation, at all).

Finally, the authors showed that level of expression of KAT2B (top-ranked CD4 T cell co-stimulation candidate) could predict (1) favorable response during chronic viral infection and/or positive host response to vaccination, and (2) poor prognosis during autoimmune diseases.

In summary, these results indicate that treatment of chronic viral infection and chronic autoimmune diseases may require activation of opposite receptors.

David Usharauli

Not all CAR-T cells are created equal

CAR-T cell-based adoptive immunotherapy against B cell-derived malignancies shows remarkable effectiveness in clinical trials. But, for some reason, other CAR-T cells, specific for diverse tumor-associated antigens, are not as effective as α-CD19 CAR-T cells. So why is that?

New paper in Nature Medicine may provide some clues to this mystery. The authors, led by Crystal Mackall, showed that unlike α-CD19 CAR-T cells, several CAR-T constructs displayed antigen-independent basal CD3zeta signaling leading to early exhaustion and lack of anti-tumor in vivo effectiveness.   

When comparing two CAR constructs α-CD19 and α-GD2 (sarcoma antigen), the authors showed that while both CAR-T cells displayed similar in vitro cytotoxicity, their in vivo anti-tumor activity against sarcoma cell line double-positive for CD19 and GD2 were vastly different: only α-CD19 CAR-T cells could control tumor growth.  

These results suggested that CAR construct, rather than tumor targets, was responsible for this difference. Indeed, T cells double-transduced with both α-CD19 and α-GD2 CAR constructs displayed diminished in vivo anti-tumor effectiveness against CD19⁺sarcoma (compared to CD19-only CAR-T cells).  

Further analysis showed that unlike CD19 CAR-T cells, GD2 CAR-T cells showed spontaneous, basal CD3zeta activity. Such basal CD3zeta activity were absent in GD2 CAR-T cells with mutations in CD28/CD3 signaling domains indicating that GD2 CAR was responsible for this spontaneous signaling.

Furthermore, the authors showed that spontaneous clustering of GD2 CAR construct (oligomerization of scFv fragments) probably was responsible for basal signaling in GD2 CAR-T cells (and not vice versa).

Finally, the authors showed that substitution of CD28 domain with 4-1BB (CD137) domain in CAR-T construct could partially rescue GD2 CAR-T cells exhaustion phenotype and improve its in vivo anti-tumor effectiveness.

In summary, data provided in this paper indicate that biochemical property of CAR constructs impart their anti-tumor effectiveness. This suggests that design of effective CAR-T construct would take more than just specificity. In my view the issue is that scFv are of BCR origin, but precise nature of BCR signaling is not clear even today. One model suggests that BCR signaling requires their oligomerization and other model suggests exactly opposite: BCR signaling requires disruption of BCR oligomers. But since assembly of TCR singaling complexes may differ from that of BCR signaling complex assembly, simply transplanting BCR domain into T cells may not be as successful as initially thought.

David Usharauli