THE FAUCI FILES, Vol 3(21): CD8 CTLs Don't Need Costimulation

[fred]

THE FAUCI FILES, Vol 3(21): CD8 CTLs Don't Need Costimulation

January 21, 2000

While NIH/NIAID Dictator-Direktor Dr. Anthony "Mussolini" Fauci 
struggles to further the interests of Pharmaceutical Fascism Inc.  
(as well as his own) through his seemingly endless scams, scandals 
and conspiracies of silence, the leak of contrary science has 
recently turned into a flood.

Here we have an article from the February 1, 2000 edition of the 
Journal of Immunology, which provides much more substantial 
evidence that will eventually bring another embarassing end to yet 
another treatment hoax that was authored under Dr.  "Mussolini" 
Fauci's tutelage, particularly by the Boys from the Auschwitz Y2K 
Faggot Experiments headed by Dr. Bruce "The Barbarian" Walker [SEE 
THE FAUCI FILES, Vol 2( 6): "The Hitler Paradigm of the NIH", 
11 May 1999].

Key point: Naive CD8 T-cells do not require costimulation for 
proliferation and differentiation into cytotoxic effector cells. 

By the way, the HIV HAART Cocktails have become so notorious for 
trashing the CD8/CTL cell population and proliferative response 
that, at present, NO studies involving the HAART treatment 
population have been reporting (or perhaps not even collecting) 
this vital, critical and INDISPENSABLE lab measure.

Even a child can see why.

Crooked Murdering Bastards!


W. Fred Shaw
Editor, THE FAUCI FILES
------------------------------

Wang et al. (2000) state: 

  "The results of this experiment argue that given a strong signal 
delivered through the TCR by an authentic ligand, no costimulation 
is required."

  "In addition, mice deficient for the CD28 gene are able to mount 
an efficient CD8+ T cell-mediated cytotoxic response following 
LCMV infection despite the fact that CD4+ helper function and Ab 
class switching are severely impaired in these mice."

  "We have been able to clearly show that naive CD8+ T cells are 
able to be completely stimulated and differentiate into cytolytic 
effectors with only the signal generated from the interaction 
between TCR and peptide/MHC class I complex without apparent 
involvement with costimulatory molecules."

  "This suggests that the rules for CD4+ and CD8+ T cell 
activation may indeed be different. The report that a monomeric 
form of peptide/MHC complex can activate CD8+, but not CD4+, T 
cells, supports this idea."

  "This makes some sense given the role of CD8+ CTL in infectious 
disease, where the ability to respond to infected cells, which are 
typically not professional APC, might well provide an advantage."

===============================

Naive CD8+ T Cells Do Not Require Costimulation for Proliferation 
and Differentiation into Cytotoxic Effector Cells

Bo Wang,* Robert Maile,* Roberta Greenwood,* Edward J. Collins,*1 
and Jeffrey A. Frelinger 2*

Departments of *Microbiology and Immunology and *1 Biochemistry and 
Biophysics, University of North Carolina, Chapel Hill, NC 27599 

The Journal of Immunology, 2000 February 1, 164: 1216-1222.

Abstract: Most current models of T cell activation postulate a 
requirement for two distinct signals. One signal is delivered 
through the TCR by engagement with peptide/MHC complexes, and the 
second is delivered by interaction between costimulatory molecules 
such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC 
tetramers provide an opportunity to test whether naive CD8+ T 
cells can be activated via the signal generated through the TCR-
alpha/beta in the absence of any potential costimulatory 
molecules. Using T cells from two different TCR transgenic mice in 
vitro, we find that TCR engagement by peptide/MHC tetramers is 
sufficient for the activation of naive CD8+ T cells.  Furthermore, 
these T cells proliferate, produce cytokines, and differentiate 
into cytolytic effectors. Under the conditions where anti-CD28 is 
able to enhance proliferation of normal B6 CD4+, CD8+, and TCR 
transgenic CD8+ T cells with anti-CD3, we see no effect of anti-
CD28 on proliferation induced by tetramers. The results of this 
experiment argue that given a strong signal delivered through the 
TCR by an authentic ligand, no costimulation is required. 

INTRODUCTION

Naive T lymphocytes need to be activated and subsequently 
differentiate into effector cells to perform their immune 
functions. The current model of T cell activation postulates that 
full activation of T lymphocytes requires two signals (1, 2). The 
first signal is postulated to be elicited by the engagement of TCR 
by the peptide/MHC complex. The second, costimulatory signal is 
provided by the interaction between accessory molecules, such as 
CD28, on the surface of T cells (3, 4) and their ligands, such as 
CD80 and CD86, on APCs (5-8). The role of costimulation has been 
studied extensively in vivo and in vitro in the activation of CD4+ 
T cells (9-11). Most studies have shown that a costimulatory 
signal is required for the activation of CD4+ cells. For example, 
in the absence of costimulation, engagement of the TCR on CD4+ T 
cells by Abs to CD3 (12), Abs to TCR (13), or Ags presented by MHC 
class II on APCs (14-16) generally leads to functional 
inactivation.  Blocking the interaction between CD28 and CD80 or 
CD86 molecules in vivo severely inhibits the primary Ab responses 
to T cell-dependent Ags and impairs Ab class switching (17-20). 
These data are consistent with a failure to activate CD4+ Th cells 
in the absence of costimulation. There are many reports that very 
efficient signals via the CD3/TCR complex (for example, by anti-
CD3epsilon alone) are able to overcome the lack of signal 2. 

The data for CD8+ T cells regarding the requirement of 
costimulation for activation are limited and contradictory. In 
vivo studies of tumor rejection have demonstrated that 
nonimmunogenic tumor cells could induce protection against a 
subsequent challenge if the immunizing tumor cells were 
transfected with CD80 (21-25). Treatment of mice with CTLA-4/Ig 
fusion protein designed to interrupt the interaction of CD28 with 
its ligands prolonged the survival of xenogeneic islet transplants 
(26) and cardiac allografts and was found to induce donor-specific 
tolerance (27, 28). 

Other experiments suggest that costimulation is not required for 
CD8 activation. Some CD80/CD86-expressing tumor cells do not 
induce anti-tumor immune responses upon immunization (29-32). 
While CD28-deficient mice infected with vesicular stomatitis virus 
have impaired anti-virus Ab production, their primary CTL response 
against lymphocytic choriomeningitis virus (LCMV)3 is intact (17).  
CD28-deficient mice can still reject allografts (33). The 
disruption of the CD28 gene in NOD mice does not prevent T cell-
mediated destruction of pancreatic -cells (34). Therefore, the 
role of a costimulatory signal in the activation of naive CD8+ 
cells remains inconclusive. 

MHC class I tetramers are molecules comprised of four identical 
class I heavy chains, each bound to 2-microglobulin, and a single 
peptide. The class I heavy chains are biotinylated and added to 
avidin, which forms a stable, but noncovalent, complex. These 
complexes bind to T cells bearing TCRs specific for the 
peptide/MHC complex in the tetramers (35).  We used these class I 
tetramers to determine whether a costimulatory signal is required 
for the activation of naive CD8+ T cells. Naive CD8+ T cells 
isolated from two different TCR transgenic (tg) mice, P14 
(specific for LCMV gp33-41) and HY-TCR, both restricted by H2Db, 
were stimulated with their cognate peptide/MHC tetramers. Our 
results demonstrate that naive CD8+ T cells could be activated by 
the engagement of TCR with only the correct peptide/MHC complexes. 
Following stimulation, these CD8+ T cells proliferated and 
differentiated into functional effector cells. The gp33-specific 
CTL activity of CD8+ cells activated in vitro with gp33/Db 
tetramers was comparable to that of spleen cells isolated from P14 
TCR transgenic mice primed in vivo by LCMV infection. The 
activation of CD8+ T cells by tetramers required engagement of the 
CD8 coreceptor. Addition of anti-CD8 mAb in the cultures inhibited 
the activation of CD8+ T cells induced by tetramers.  The results 
from the present study indicate that engagement of TCR with 
peptide-MHC complex alone provides a sufficient signal for naive 
CD8+ cells to differentiate into cytotoxic effector cells. 

DISCUSSION
In this study we examined the ability of soluble peptide/MHC 
tetramers to activate naive CD8+ T cells in vitro from two 
different TCR tg mice, in the absence of apparent costimulation or 
exogenous growth factors.  The data demonstrate that naive CD8+ T 
cells can be fully activated and differentiate into cytotoxic CD8+ 
T cells without costimulation. When CD8+ T cells were stimulated 
with our tetramers in the presence of anti-CD28 mAbs, no 
enhancement of proliferation (Fig. 9) or CTL activity was observed 
(data not shown). 

Single, naive CD8+ T cells from TCR tg mice were activated with 
soluble peptide/MHC tetramers, and therefore, involvement of any 
other costimulation was excluded. In support of our data, it has 
been shown that CD8+ T cells can be activated by fibroblasts, 
which express the appropriate Ag for T cells, but not CD80/CD86 
molecules (43, 44). In addition, mice deficient for the CD28 gene 
are able to mount an efficient CD8+ T cell-mediated cytotoxic 
response following LCMV infection (17) despite the fact that CD4+ 
helper function and Ab class switching are severely impaired in 
these mice. However, the findings from these studies could not 
rule out the possibility that unknown molecules might act as 
costimulators in the absence of CD28 engagement (11). Cell surface 
expression, cytokine secretion, and cytotoxic T cell assays all 
show that naive CD8+ TCR tg T cells activated by tetramers are 
indistinguishable from T cells activated by peptide-pulsed APC. 

Recently, it was shown that TCR ligation with Ab in the absence of 
CD28 ligation results in activation-induced cell death, but in 
these experiments, anti-CD28 prevents cell death (45). Thus, 
although we do not see enhancement of proliferation, it is 
possible that we might see inhibition of cell death. Preliminary 
experiments suggest that most cells stimulated by tetramers 
undergo apoptosis. In this case ligation with anti-CD28 might 
block cell death without enhancing proliferation. 

Delon et al. (46) have demonstrated that Ca2+ mobilization in CD8+ 
T cells can be triggered with soluble peptide/MHC class I complex 
in the absence of a costimulatory signal, and Goldstein et al. 
showed proliferation of ZC allospecific tg mice with soluble 
secreted Ld.  However, this study did not address the 
differentiation of naive CD8+ T cells into effector cells. Two 
other studies have also demonstrated that CD8+ T cells from other 
TCR tg strains are activated and differentiate into functional 
cytotoxic T cells when stimulated with agonist peptide/MHC complex 
without costimulation. However, in these studies, exogenous IL-2 
was required (47, 48). In our study, no exogenous growth factors 
were needed, and our CD8+ T cells produced significant amounts of 
IL-2 as well as IFN-gamma (Fig. 8). One possible explanation for 
the discrepancy between the previous study and ours is that CD8+ T 
cells were positively purified with anti-CD8 mAbs in the former 
study. We have found that CD8+ T cells positively selected with 
anti-CD8 mAb-coupled beads do not proliferate when stimulated with 
tetramers. This is probably due to the blockade of the interaction 
between MHC and CD8 coreceptor (Fig. 6) during culture. 

It has been proposed that the affinity between the TCR and the 
peptide/MHC complex determines the requirement for a costimulatory 
signal in activation of CD8+ T cells (42). Using CD8+ T cells from 
CD28-deficient mice, Bachmann et al. (41) have reported that 
decreasing the affinity of interaction between the TCR and the 
peptide/MHC complex results in an increasing dependence on 
costimulation for CD8+ T cell activation. We have several lines of 
evidence that argue against this interpretation. In our 
experiments, Y4A/Db tetramers caused the equivalent proliferation 
and differentiation of CD8+ T cells, although Y4A/Db required a 
10-fold higher concentration. No added IL-2 was needed, and Y4A 
induced the production of both IFN-gamma and IL-2 from P14 cells, 
which differs from the findings of Bachmann (41). In addition, our 
data (R. Maile and J. A. Frelinger, unpublished observations) show 
that the HY/Db complex has a low affinity for the transgene-
encoded TCR. In this paper we show that the HY/Db tetramer was 
effective at stimulating the T cells, although a higher 
concentration was required than for gp33/Db tetramers. Thus, our 
in vitro results argue that the dependence on costimulation is not 
determined by the affinity of TCR for its ligand. 

Additionally, it is important to note that the use of tetramers 
for the purification of peptide-specific T cells has been 
proposed. Our studies suggest that the binding of the tetramers to 
T cells is not a neutral event, and that investigators should be 
aware of the potential activation of T cells by their binding. 

We have been able to clearly show that naive CD8+ T cells are able 
to be completely stimulated and differentiate into cytolytic 
effectors with only the signal generated from the interaction 
between TCR and peptide/MHC class I complex without apparent 
involvement with costimulatory molecules. This suggests that the 
rules for CD4+ and CD8+ T cell activation may indeed be different. 
The report that a monomeric form of peptide/MHC complex can 
activate CD8+, but not CD4+, T cells (46, 49) (our unpublished 
observations), supports this idea. This makes some sense given the 
role of CD8+ CTL in infectious disease, where the ability to 
respond to infected cells, which are typically not professional 
APC, might well provide an advantage. 

Footnotes
Received for publication June 28, 1999. Accepted for publication 
November 15, 1999. 

1 This work was supported by National Institutes of Health Grants 
AI20288, AI29323, and AI41580. These experiments were initiated 
while one of the authors (J.A.F.) was on leave from the National 
Institute of Arthritis and Infectious Diseases, the laboratory of 
Drs. Jack Bennink and Jonathan Yewdell. 

2 Address correspondence and reprint requests to Dr. Jeffrey A.  
Frelinger, Department of Microbiology and Immunology, University 
of North Carolina, CB#7290 MEJ, Chapel Hill, NC 27599-7290. E-mail 
address: jfrelin at med.unc.edu 

3 Abbreviations used in this paper: LCMV, lymphocytic 
choriomeningitis virus; tg, transgenic.