immunology presentation - "'infectious' transplantation tolerance"

BIMM 19404/13/16

Presented byMargarita Maksimova Nikki Nguyen Fiona Wong

Catherine Terry Jonathan Lim

Organ Transplants

● Transplants can be a life saving measure for all types of diseases.

● Finding an organ donor that “matches” the patient is very difficult

Graft Rejection

● Your immune system can distinguish between self and non-self

● Transplants seen as foreign will be rejected

T-Cell Mediated Graft Rejection

Infected Cell, Foreign Cell or

Tumor Cell

Cytotoxic T CellsLysed Cell

Helper T CellsAntigen Presenting

Cell

T-Cell Receptor Binding is improved by coreceptors CD4 and CD8

Cytotoxic T Lymphocyte Helper T Lymphocyte

Y

Y

Y

Y

Two Types of Antibodies

Non-Depleting Antibodies

Two Types of Antibodies

Depleting Antibodies

YY

Y

Y

YY

YY

YYY

YYY Y

Key Terms

CBA/Ca Mouse ATx CBA/Ca Mouse B10.BR Mouse BALB/c

Graft tolerance with MAb treatment

CBA/Ca MouseB10.BR Mouse CBA/Ca Mouse with B10.BR skin graft

MAbs against CD4 and CD8 are effective immunosuppressants

Y

CD8 T Cell

Y

CD4 T Cell

MAb for CD8

MAb for CD4

Antigen Presenting CellTarget Cell

Result: Lazy T cells that don’t do anything. They just float

around your body.

How treatment of MAb cause life long tolerance?

???

Testing CD4+ Cells in Tolerant Mice

● The CBA/Ca mice used in the following figure was thymectomized (ATxCBA/Ca)

● Concluded that CD4+ cells in tolerant mice prevented naive T cells from breaking tolerance shown in Figure 1 ○ Experimental steps shown in Figure 1:

i. (Figure 1a) T cells in tolerant host were suppressing naive lymphocytes from donor

ii. (Figure 1b) CD4+ T cells from tolerant mice was the key to prevent graft rejection

Figure 1a Original B10.BR graft, no injections of MAbs

Second B10.BR graft, no injections of MAbs

Original B10.BR graft, MAbs injected (T cells depleted) Second B10.BR graft, MAbs injected (T cells depleted)

● Day 0 is when they implemented of first graft and made the T cells in the mice tolerant

● Data suggest that T cells - CD4 and/or CD8 - in the tolerant host cells may be responsible in suppressing new lymphocytes

Figure 1a Visual Explanation

Figure 1b Fifty million tolerant cells added from ATxCBA/Ca

tolerant mice

CD4- cells from ATxCBA/Ca tolerant mice

CD8- cells from ATxCBA/Ca tolerant mice

No tolerant cells were mixed

● Testing external factors to affirm that the tolerance mechanisms were from the splenocytes

● “Test-tube” mice used, no T cells present ● CD4+ T cells from tolerant mice were needed to prevent graft

rejection

Figure 1b Visual Explanation

● The transgenic hCD2+/CBA mouse model was used to do this as all T cells in this mouse line contain human CD2+ receptors which can be targeted by a depleting antibody

● Next they had a model where tolerant T cells of the recipient could be ablated the after injection of new naive cells

Testing hCD2+ Mice for Tolerance

Introduction of tolerance and failure to break tolerance in hCD2+/CBA cells

● The researchers first tested the ability of this model to become tolerant via non-depleting antibodies (Figure 2A)

● Then they tested the tolerance of the mice when new T cells were injected with or without the host T cells ablation (Figure 2B)

Figure 2A

B10.BR graft survival rate for ATx hCD2+/CBA mice

B10.BR graft survival rate for ATx hCD2+/CBA mice treated with depleting anti CD2+ antibodies

BALB/c graft survival rate for ATx hCD2+/CBA mice treated with depleting anti CD2+ antibodies

hCD2+/CBA mice have a functioning immune system and it can be suppressed by treating the mouse with a depleting anti CD2+ antibody

Figure 2B

ATx hCD2+/CBA mice were made tolerant via non-depleting antibodies and given a test B10.BR graft, 2nd grafting done 3 months later to confirm tolerance, this is where this histogram begins

Figure 2BNo additional treatment given to mice in this group

50 million hCD2- spleen cells injected into the tolerant hCD2+/CBA mouse at time of second grafting

1st graft survival rate 2nd graft survival rate

50 million hCD2- spleen cells injected into tolerant hCD2+/CBA mouse at time of second grafting

ANDhCD2+ cells ablated with depleting αCD2+ Ab

Figure 2B

Injecting new donor spleen cells (includes T cells) does not break previously induced immunity in the recipient but if the tolerant cells are ablated the new cells will reject the grafts

Table 1

● Mice infused with spleen cells from hCD2- mice at 56 or 63 days after tolerance induction

● hCD2+ selectively depleted after 1 or 2 weeks

Question: What two possible conclusions can be reached from these studies on the coexistence of tolerant cells and naїve spleen cells?

Conclusions from Coexistence Experiments

1. The naїve cells are being tolerized themselves by the tolerant T cells

OR

2. The naїve cells are being killed off by the tolerant T cells

Figure 3A + 3B

Mou

se C

D3

How to Read FACS (Flow cytometry)

Controls:

A: untreated hCD2+/CBA

B: untreated hCD2- littermatehCD2 hCD2

Figure 3CWere the injected cells killed off by tolerant cells?

● Infuse hCD2- cells● Wait 2 weeks● Check hCD2+ (host) versus hCD2- (donor)

After 2 weeks, 26% of CD3+ cells were still donor.● (16.8 / (16.8 + 47.9) = 26%)

Conclusion: Failure to reject B10.BR grafts not from lack of engraftment.

Mou

se C

D3

hCD2

Figure 3DWhat happens if we kill the original tolerant host cells?

● Ablate host hCD2+ cells with MAb● 99% remaining cells were hCD2- donor phenotype● Cells were STILL TOLERANT

Maintained tolerance of old and fresh graft

Conclusion: hCD2- cells infected with tolerance and have

capacity to prevent newly infused cells from rejecting graft.

Mou

se C

D3

hCD2

Potential Caveats

Never injected tolerized T cells into a naive mouse

What if non-tolerant mouse received tolerized CD4 T cells and the skin graft?

Would these CD4 T cells teach the naive mouse to tolerate the graft?

Remaining Questions and Follow-Up

How is this happening?

● Molecular basis of tolerance passing● CD4+ T cells is a broad category

Follow up

● Which cell population “teaches” tolerance?

Take Away● CD4+ T Cells tolerized to a transplant have the ability to inhibit naїve

splenocytes from rejecting a graft. ● New splenocytes can become tolerized themselves and inhibit a new round of

naїve splenocytes from rejecting a graft.

Tolerant T Cell

Naїve Spleen

Cell

Learn to

tolerate!

How does this work? How is tolerance transferred?

New Developments: Infectious Tolerance● The process which tolerance-inducing state is transferred from one cell

population to another● CD4+ T Cells are not limited to helper T cells, but also found on regulatory T

cells● Possible Mechanisms:

○ IL-35○ TGF-β○ Amino acid depleting enzymes

New Developments/Medical Applications

● IL-35 released from Tregs● Induces new generation of a different

population of regulatory T cells● May be a key mediator of infectious

tolerance

New Developments/Medical Applications

● TGF-β is a growth factor that inhibits the proliferation of activated T cells

● TGF-β also inhibits the activation of helper and cytotoxic T cells

● Downregulates activity of immune cells

● Tregs induce the expression of enzymes that consume essential amino acids.

● Without these essential amino acids, T cells may fail to proliferate in the presence of an antigen.

New Developments/Medical Applications

New Developments/Medical Applications

Mechanisms involving regulatory T cells have the potential to mediate infectious tolerance.

How can we use this new knowledge to treat tolerance of a graft?

Human Health and Daily Life

Therapy Now: Lifelong immuno-suppressants needed

Human Application: Non-depleting antibody treatment is detrimental to the adaptive immune system, a huge risk for human health.

Potential Therapy: understand the mechanism of “infectious tolerance” to outsmart the immune system and teach tolerance from one treatment.