Eureka!!!The Right Immune Response..It is All in the Timing and IL-2 Concentration!!! Melanoma..Jim Breitfeller

So now we have the immune response in full progress. Are we creating the right response?

The difference between CD4+ T-cells and CD8+ T-cells revealed herein must be
interpreted in the context of its significance for the development of an effective
T cell immune response. Within T cell populations, CD4+ T-cells are the primary
source of IL-2 after antigen activation. Consequently, the finding that CD4+ T-cells
cell clonal expansion is only about 25% of the proliferative expansion achieved by
CD8+ T-cells indicates that IL-2 itself is the major parameter retarding clonal
expansion within the total cell population; especially because the rate of T-cell
proliferation is directly dependent on the amount of IL-2 available to the cells. et al Kendall A Smith

The ratio of CD4+ to CD8+ is about 2:1.
Thus, the immunologic relevance of the CD4+ T-cells IL-2 refractory state resides in
its potential as a feedback regulatory control retarding the extent and duration
of IL-2-dependent CD4+ T-cells and CD8+ T cell clonal expansion, ensuring that IL-2-
producing cells ultimately will become limiting.
This interpretation is entirely consistent with observations in lab experiments which indicate that IL-2 rapidly disappears from the culture medium of stimulated T cells.

So what happens if you have excess IL-2 in the Medium?
Studies show that a premature development of CD4+ T-cells IL-2-unresponsiveness arises and the CD4+ T-cells loose their functionality.
This means that premature excess of IL-2 can lead to unresponsiveness of the CD4+T-cells.

Thus, two mechanisms contribute to the depletion of IL-2 from the Medium of rapidly proliferating T cells:

1. The IL-2 internalization and degradation (the uptake) by responding cells
2. The end of CD4+ T-cells + (IL-2 producer) cell proliferative expansion


IL-2 concentration in the surrounding cell environment can regulate the expansion of the CD4+ T-cell population by effecting cell death.
As you keep the IL-2 concentration limiting, you can change the expansion profile if the CD4+T-cells. At low concentrations, the expansion distribution is sharpe and evenly distributive on the time axis. On the hand, at higher or excess concentrations, the distribution becomes skewed, with a longer life cycle.

If the addition of IL-2 is on or before the maximum propagation of the CD4+ T cells, the Tregs population can increase 5-fold in a 96 hour period based on certain growth mediums.
So by increasing the Treg population, you make it that much harder to break the immune tolerance to start an immune response.

So by limiting the excess IL-2 that is introduced into the host at the early expansion phase, you limit the expansion of the CD4+ T-cells. As the Tregs are a subset of the CD4+T-cells, you limit the expansion of the Tregs. By limiting the Tregs and CD4+ T-cells, you limit the CTLA-4 receptors. So blocking the B7 receptors with anti-CTLA-4 antibodies, and limiting the IL-2 in the matrix, you can shift the tolerance balance toward activation.


It is widely accepted that the CD4+ T-cells expand before the CD8+ T-cells.




Activation and Differentiation of (CTLs) Cytotoxic T Lymphocytes

Naïve CD8+ T-cells are referred to as CTL-precursors (CTL-Ps), which are incapable of performing any function other then recognizing the class I MHC-antigen complex on the Tumor cells through the (TCR) T Cell Receptor. For activation, the CTL-P needs at least three signals:

1. Antigen specific signal transmitted by the MHC I peptide/TCR complex for the recognition of the Antigen

2. The Costimulatory signals transmitted by the CD28 receptor and the B7 molecule of the Antigen Presenting Cells (APCs)

3. Cytokine induced signal, IL-2 interaction with the IL-2r (receptor) on the CTL-P leading to activation and differentiation of the CTL-P into effector CTL

The Cytokine IL-2 came from the TH1 cells which means the CTL-P is IL-2 limited and can only be activated by the secreted IL-2 if there is any to be had or by introducing IL-2 through IL-2 therapy. Now you know the reasoning behind using the IL-2 therapy as the second part of the combinatorial Therapy. If there is not enough IL-2 in the host environment, you will only get partial expansion of the CTLs. It may not be enough to eradicate large bulky tumors.

The Interluekin-2 plays another role in this Melanoma Maze. In a study by Janas et al, Il-2 increases the expressions of the perforin and granzyme A, B and C genes in the CD8+ T-cells. This increase expression causes the CD8+ T-cells to mature into Cytoxic T Lymphocytes (CTLs). The exogenous IL-2 is required for the granzyme proteins. CTLs have cytoplasmic granules that contain the proteins perforin and granzymes. A dozen or more perforin molecules insert themselves into the plasma membrane of target cells forming a pore that enables granzymes to enter the cell. Once in the tumor cell, these enzymes are able to breakup (lyse) the cell and destroy it. This is the beginning of the end for the cancer cells. The tumors begin to shrink and the rest is history.

Research suggests that the primary mode of the destruction of the tumors by CTL is by initiating death through the Fas-FasL pathway. Studies have shown that CTLs store Cytotoxic proteins in the form of granules in their cytoplasm. These proteins belong to two categories:
1. Perforins: involved in pore formation
2. Granzymes: responsible for hydrolysis of the cellular products.

Granzymes breaks down the tumors cells just like your detergent enzymes in your laundry detergent.

Immediately following a CTL contact with the tumor cell, the Golgi sacks load with granules and granzymes which create pores to allow the granzymes to enter and destroy the tumors cells.


So what if there is not enough IL-2 produced to supply the overall expansion?

We know the Naïve CD8+ T-cells needs IL-2 for the activation and the differentiation into effector T-cell call Cytotoxic T Lymphocytes (CTLs). We also know IL-2 is needed to upregulate perforin and granzyme A, B and C genes which need to turn the effector cell into a cytotoxic killing machine.

Does the timing of the addition of IL-2 make a difference in the outcome of the immune response?

It surely does. There are a number of research papers that state if the IL-2 is added to early in the expansion phase, you produce non-responsive T-cells. As a matter of fact you could expand the subtype Tregs by a factor 5 under certain conditions. On the other hand, waiting until the expansion is at just passed peak or contracting generates the Cytotoxic T Lymphocytes (CTLs) that we as patients, have been waiting for. That is why Dr. Rosenberg uses IL-2 in his ACT Therapy. It is to generate and maintain the Cytotoxic T Lymphocytes.









Eureka!!!The Right Immune Response..It is All in the Timing and IL-2 Concentration!!! Tumor Shrinkage!!!!
Complete Response!!!!!!


“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”

~Charles Darwin~
Take Care,
Jimmy B